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Theses and Dissertations Graduate School

1969

Inhibition of Cell Division, Protein Synthesis and Nucleic Acid Inhibition of Cell Division, Protein Synthesis and Nucleic Acid

Synthesis in Escherichia coli W by Tetracycline Antibiotics Synthesis in Escherichia coli W by Tetracycline Antibiotics

George Henry Miller

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Inhibition of Cell D iv i s ion , Prote in Synthes i s

and Nuc leic Ac id Synthes i s in

Escherichia coli W by

Tetracyc l ine Antibiotics

by

George Henry Mi l ler

B . Sc . , Philadelphia College of P harmacy and Science , 196 0

M . Sc . , Phi ladelphia Col lege of Pharmacy and Science , 196 2

Degree of

The s i s

Submitted i n partial ful f i l lment of

the requirements for the

Doc tor of Phi losophy in the Depar tment of

Pharmacy at the Med ical Col lege of Virginia

Richmond , Virg inia

June , 1969

100561

ii

This thesis by Mr . George H. Mil ler is accepted in its

present f orm as satisfying the thesis requirement for the

degree of Doctor of Philosophy

Date : Approved :

. {fle:t. -�' �- . I?. t. J: ... .

. . o.J. .. .It' . . (! ?. � ...... . Dr . Arthur A . Cammarata

Dr . Edward S . Kline

... -� . . �� J.<i 6. r. .... .

.. _(!. 4. . t � . J.? � r: .... . � D . Punch

.Dr . ;� Richard

APPROVED :

iii

Curriculum Vitae

iv

v

Acknowledgments

The author wishes to expre s s h i s apprec i ation and h i s s incere

gratitude to :

Dr . A . N . Martin for providing the opportun ity to study

and learn in a stimulating and plea sant environment and for

h i s counseling , superv i s ion , patience and espec ially h i s

expre ss ions o f confidence during the course of thi s work ;

Dr . A . A . Cammarata and Dr . F . Perade j ordi for the many

stimulating and helpful d i s cus s ion s ;

Dr . S. A . Khal il for h i s help and encouragement durina

the development o f the exper imental procedures;

Luisa C . Mmller for her excellent technical a s s i stance

in performing these experimental procedure s ;

Sung Ho Hahn for h i s a s s i stance in preparing the materials

for the experiments ;

John Andrako Jr . , Willi am Crouthamel , John Gill , Douglas

Martin and Teresa Taylor for the i r help i n the v iable cell

determinations as well as other experiments ;

the members o f my Adv i sory Committee , Dr . E. Kline ,

Dr . Punch and Dr . Richard in addition to Dr . Cammarata and

Dr . Martin for the i r advice and especiali1 for the i r

reading of thi s the s i s ;

the staf f o f the Computer Center , Department o f B i ometry ,

Medical College o f Virginia for the prompt stat i stical

analy s i s o f the experimental data and for the i r a s s i stance

in studying the proposed model of tetracycline act ion;

my parents , Arthur and P r i sc illa , for the i r encouragement

Acknowledgments

and sacr i fices in my behal f ;

my daughter , Claudia , for the inspiration and joy she

provided during this work ;

vi

my wi fe , L i lo , for providing the encouragement to

undertake thi s s tudy and for her patience , understanding and

sacri fices during its complet ion in addi tion to her excel lent

technical a s s i s tance acknowledged above .

TABLE OF CONTENTS

I, I ntroduction

I I . Literature Survey

A. Tetracycline s - Nomenclature , General P roperties

and B iological Activity

A,l, A-Rmng Modi fications

A.2. B-Ring Modi fications

A . 3 . C-Ring Modif ications

A,4, D-Ring Modi fications

A . S . Structure-Activity General i z ations

A,6, Acidity Constants o f the Tetracyclines

B . Tetracycl ine s - Biochemical B a s i s of The ir

Antibiotic Activity

v i i

Page

3

3

s

6

6

7

7

8

1 0

B . l . E f fects o n Rate o f Cell Reproduction 10

B . 2. E f fects on Oxidation and Respirat ion 1 2

B . 3, Ef fects on Protein Synthe s i s in Culture s 13

B . 4 . E f fects on Cell-Free Nitro-Reductase Systems 16

B . S . The Nature o f Cell-Free Protein Synthe s i z ing

Systems- Translation of the Genetic Code

B . S . a . Ribosome s

B . S.b . Mes senger Ribonucle ic Acid

B . S . c. Trans fer Ribonucle ic Ac id

B . S . d . A Model of Cell-Free Prote in Synthe s i s

2 3

24

2 9

31

33

S�ep 1 . - Binding of a 30 S Subunit to mRNA 40

Step 2 . - Formation of the I nitiation Complex 40

Step 3 . - Attachment of the SO S Subunit to

the Initiation Complex

S tep 4.- Bind ing to the Aminoacyl S ite on

the 70 s Ribosome

S tep 5,- Format ion o f the Peptide Bond

S tep 6.- Pul sation ( Movement ) of the

Ribosome

S tep 8 . - Chain Termination and Release

B.6. The E f fects of Tetracycl ines on C e l l - Free

Protein Synthe s i z ing Systems

B.6.a. E f fects of Tetracycl ines in Terms of the

Mode l o f Cel l-Free Protein Synthe sis

Inte rference with S tep I.-

I nterference with S tep 2 . -

I nter ference with Step 3 .-

Interference with Step 4 . -

Interference with S tep 5 . -

I nter ference with S tep 6 . -

I nterference with S tep 8.-

B . 6.b . Binding o f Tetracycl ines to Ribosomes

I I I. Experimental

A . Materials and Equipment

A . l. Tetracy c line Derivatives

A. 2 . Escherichia c o l i

A . 3 . Culture Broths

A . 4. Culture Turbid ity and Colorime try

A . 5. Total C e l l Counts

B . Hethods

viii

Page

4 6

5 0

5 3

5 4

5 4

5 6

5 8

5 9

6 0

6 1

6 2

6 3

6 3

6 3

6 6

6 6

6 6

6 9

7 0

7 0

B . l. E s che richia col i Growth

B . 2. Viable Count Method

B.3 . Total Count Method

B . 4. Nuc leic Acid and Protein Determinations

Nuc leic Acid Determinations

Prote in Determinations

I V . Results and Discuss ion

A . E f fect o f Tetracyc l ines on Rate Constants for

C e l l Div i s ion

B . E f fect o f Tetracyc l ines on Rate Cons tants for

Protein and Nuc l e i c Acid Synthe s i s

c . T ime o f Onset o f Tetracyc l ine I nhibition

D . E f fect o f .Broth pH Change s on Te tracyc l ine

I nhibition

E. Kinetic Mode l s o f the I nhibi tion of Bacterial

Culture s by Tetracy c l ines

V . Summary

VI . B ib l iography

ix

Page

7 1

7 1

7 2

7 2

7 3

7 6

7 9

9 2

1 0 5

111

1 1 9

1 3 6

1 4 0

AA

DPN

DPNH

EDTA

f l , f 2

FMN

FAD

G

ked

kv

L i s t of Abbreviations

ami no acids

ala a lanine

a s p asparagine

cys cysteine

leu leuc i ne

lys lysine

met methionine

phe phenyla lanine

ser ser ine

thr threonine

tyr tyros ine

va l val ine

d iphosphopyr idine nuc leotide

reduced d iphosphopyrid ine nuc leotide

ethylenediaminetetraacetic a c id

X

r ibosoma l proteins which are neces sary for prote in

synthe s i s and which are removed from r ibosome s by

wa shing with NH4C l

f lavin mononuc leotide

flavin adenine d i nucleotide

a supernatant enzyme which cata lyzes the hydrolysis

of guanos i ne - 5 ' - tr iphosphate in the pre sence of

r ibosome s

generat ion rate constant for c e l l d iv i s ion

generation rate constant for cell d iv i s ion a s

determined b y the v iable cell count method

kt

kp

kna

LDH

MDH

na

List of Abbreviations

generation rate constant for c e l l div i s ion as

determined by the tota l c e l l count method

generation rate constant for protein synthes i s

generation rate constant for the synthe s i s o f

nuc leic a c id

xi

the proportiona l i ty constant r elating generation

rate constants to tetracyc l i ne concentration; the

subscripts v , t , p , na i nd icate the type of

generation constant used , the subscript avg

ind icates that a l l g eneration rate constants were

u sed col lectively

lactic ac id dehydrogenas e

mal ic a c i d dehydrog ena se

nuc leic acids

DNA

RNA

mRNA

rRNA

sRNA

tRNA

AAtRNA

met- tRNAf

N-F-met-tRNAf

deoxyr ibonuc leic acid

r i bonuc leic a c id

me s senger r ibonuc leic acid

r ibosoma l r i bonuc l e ic a c id

soluble r i bonuc leic a c id

�ransfer r i bonuuleic acid

aminoacyl transfer r ibonuc leic acid

methionyl transfer r ibonuc l e ic acid

which a llows the addi tion of a

formyl group to the methionyl amine

N-formylmethionyl transfer

r ibonuc leic acid

L

L i st of Abbreviations

met- tRNAm

ATP

CTP

GOP

GMP

GTP

UTP

poly A

poly c

poly G

poly u

methionyl transfer r ibonuc leic

a c id which does not a l low the

addi tion of a formyl group to

the methionyl amine

adenos i ne- 5 ' -tr iphosphate

cyto s ine- 5 ' -triphosphate

guano s i ne- 5 ' -d iphosphate

guanos ine - 5 ' -monophosphate

guanos i ne - 5 ' - tr iphosphate

ur idine - 5'-tr iphosphate

polyadenyl i c acid

polycytidyl i c acid

polyguanyl i c a c id

polyur idylic ac id

x i i

" Nuc leic Acids" the absorbance of an orc inol reaction ( 4 ml . )

Pm

" Proteins "

T

which i s due to the nuc leic acids of E . coli

in a 1 2 . 5 ml . culture sample

puromyc in

the absorbance of a Fol in-Lowry reac tion

( 6 . 5 ml . ) which i s due to the protein of

E . c o l i in a 2 5 ml . culture sample

supernatant prote ins which are nec e s sary for

protein synthe s i s

TCA

e

L i s t of Abbreviations

trichloroacetic a c id

the frac tion of total c e l l s in a culture which

are found to be viable

x i i i

F igure I .

F igure I I .

F igure I I I .

F igure IV .

F igure V .

F igure VI .

F igure V I I .

F igure V I I I .

F igure I X .

F igure X .

F igure XI .

F igure XI I .

F igure X I I I .

L i s t of F igures

The Tetracyc l i ne s

the Ioni zable Groups of Tetracyc l i ne

Ribosome s . Components , Subunits ,

x iv

Page

4

9

Associati on and D i s sociation Reac tions 2 5

The Formation o f Ami noacyl Transfer

Ribonuc leic Ac id

Schema tic D iagram o f the Proposed C lover­

leaf Structure of Transfer Ribonuc leic

Acids

Codon - Anticodon Interaction

A Mode l of C e l l-Free Protein Synthes i s

Ca l ibrat ion Curves for "Nuc l e ic Ac id s "

and " Protei n " Determina t ions

An Exam�le of Viable Cell Genera tion Rate

Curves

An Example o f Tota l Cell Generation Rate

Curves

Examples of the D i f f erence Between Tota l

3 2

3 4

3 5

3 6

7 5

8 0

8 1

and Viable C e l l Generation Rate Curves 9 0

The L inear Dependence o f Generation Rate

Constants , Kv , kp and kna , upon Tetracy-

c l ine Concentration

The Linear Dependence of Generation Rate

Constants , kv , kt , kp and kna , upon

9 3

9-N i tro-dm-do- tetracyc l i ne Concentration 9 4

F igure XIV .

F igure XV .

F igure XVI .

F igure XVI I .

F igure XVI I I .

F igure XIX .

F igure XX .

F igure XXI .

F igure XXI I .

L i s t of F igure s

The Linear Dependence of Generation

Rate Constants , kv , kt , kp and kna '

upon Concentrat ion of 9-D imethylamino­

dm-do-tetracyc l i ne

The L inear Dependence o f Generation

Rate Constants , kv , kt , kp and kna '

upon Concentration of Several

Tetracyc l ines

An Example of Generati on Rate Curves

for Protei n Synthes i s

An Example o f Generation Rate Curves

for Protei n Synthe s i s

An Example of Genera tion Rate Curves

for Nuc leic Ac id Synthe s i s

An Example of Genera t ion Rate Curves

for Nuc leic Ac id Synthe s i s

T ime o f Onset of and Recovery from

Oxytetracyc l ine Inhibition in Peptone

Broth Culture s

T ime of Onset of Oxytetracyc l i ne

Inhibition of Cultures in Dextrose­

Salts Broth

The L inear Dependence of Generation

Ra te Constants upon Concentration of

Zwitter ionic Chlor tetracyc l i ne

XV

Page

9 5

9 6

9 9

1 0 0

1 0 2

1 0 3

1 0 8

1 0 9

1 1 3

L i s t of F igure s

F igure XXI I I . The Effec t of Broth pH on T ime of

Onset of Oxytetracyc l ine Inhibition

F igure XXIV .

F igure XXV .

F igure XXVI .

Propo s ed Mode l of Tetracycline

Inhib i tion of Protein Synthe s i s in

Bac teri a l Cul ture s

A n Unsealed Analog C omputer Program for

the So lution of the D i f ferenti a l

Equations of the Proposed Model o f

Tetracyc l ine Inhibition of Prote i n

Synthes i s i n Bac teri a l Cultur e s

The Continuous Sy stems Mode l i ng Package

Program Used in the Search for Solutions

to the D i f ferential Equations from the

Proposed Mode l of Tetracyc l i ne Inhibition

xv i

Page

1 1 6

1 2 3

1 2 7

o f Pro�e in Synthe s i s in Bac ter i a l Culture s 1 2 9

Table I .

Table I I .

Table I I I .

Table IV .

Table V .

Table VI .

L i s t of Tables

The Tetracyc l ine s

First Order Generation Rate Constants ,

-1 4 sec . x 1 0 , for E . coli W Culture s

Inhibi ted by Var ious Tetracyc l i ne

Antibiotic s , M x 1 0 6

F i r s t Order Generation Rate Cons tants ,

sec . - 1 x 1 0 4 , for E . c o l i W Cultures

Inhibited by Var ious Tetracyc l i ne

Antibiot ic s , � x 1 0 6 , and the Frac tion

of Viable to Tota l Cel l s Present at

the Start , e1 , and at the End , e5 , of

the Exper iments

I nhibitory Rate Constant s , L/M- sec . ,

for Inhibition of E . c o l i W by

Var ious Tetracyc l ine s

F ir s t Order Generat ion Rate C onstants ,

sec . - 1 x 1 0 4 , and Inhibitory Ra te

Con s tants , L/M-sec . , for E . c o l i W in

Var ious Broth Culture s Inhibited by

Setiera l Tetracyc l ine s

The Differential Equat ions from a Model

of Tetracyc l i ne Inhibition o f Prote in

Synthes i s in Bacter ial Cultures

xvi i

Page

6 7

8 2

8 5

9 7

1 1 4

1 2 6

L i s t of Tables

Table V I I . C omp i la tion of Condi t ions Which Were

Employed to Test the Model of

Tetracyc l ine Inhibition of Prote in

Synthes i s i n Bac ter i a l Cultures

xvi i i

Page

1 3 0

I . INTRODUCTION

Tetracyc lines , or igina l ly isolated i n the late 1 9 4 0's

and early 1 9 5 0 ' s from the myce l ium of S treptomyces , have

achieved maj or importance as therapeutic and prophylactic

agents agai ns t a wide range of i nfections i n human and

veter i nary med i c ine . They have a l s o achieved great

importance in agriculture where they are widely used to

promote weight gain in l ivestock . Commerc i a l production ,

duri ng 1 9 5 8 i n the United States a lone was 1 2 0 tons with a

value of approximately $ 1 m i l lion per ton .

The c l inical importance of these compounds has

s t imulated e fforts to def i ne thei r mode of action a s

inhibitor s of bacteri a l reproduction . Antibiotics having

a s many func t i onal groups a s the tetracyclines may have many

modes of action . The problem faci ng the re search worker i s

t o determine the relative contribution of each mode o f action

in a g iven b i ological system a s a function of antibiotic

concentration . I f a l l parts of a biological system are

exposed to an equiva lent antibiotic concentration , one might

expect that the degree of i nhibition of the vari ous

sub-component systems would be propor tional to the relative

stabi lities of the reaction products of the antibiotic and

the sub-component system . The mos t critical reaction from

the s tandpoint of c e l lular reproduct i on would then represent

the point of the pr imary b i ochemic a l les ion or the s i te of

2

action . The type of reaction would be the mode of ac tion for

the biolog ical sys tem . For a g iven biological system i t i s

probable that there i s one c r i tical reaction which i s most

sensitive to the antibiotic , but thi s reac tion may not be the

same for every biolog ical sys tem ( see Sne l l and Cheng ( l ) for

an exce l lent d i scuss ion of the d i f f i culties of def ining a mode

of tetracyc l ine action) . Many investigator s presently feel

that the pr imary b i ochemical l e s ion inf l icted upon susceptible

bac ter ia by tetracyc l ines is a genera l inhibition of protein

synthe s i s ( 2 , 3 , 4 ) . Therefore it was felt that the study of

severa l measures o f inhibi tion , particularly cell reproduc tion

and pro�e in synthe s i s , in the presenc e of several tetracyc l ine s

might be a u seful way o f studying modes of tetracyc l i ne action .

Biological activ i t i e s of a large number of compounds

obtained quantina t ively under identical dond i tions and in a

prec i s e manner are required to estab l i sh structure-activity

relationships . Presently ava i lable activities for tetracyc l ine

antibiotics have been summa r i zed by Barrett ( 5 ) , Boothe ( 6 ) and

Plakunov ( 7 ) . Many of the se activities have been obtained

under cond i tions such that the results par a l l e l c linical

activ i t i e s . Thu s , some compounds may not have achieved

equ i l ibr ium with the test system . Some activities have been

obta i ned for the purpose of s tudyi ng antibiotic r e s i stance

whi l e s t i l l o ther s have been obta ined in widely varying and not

ea s i ly i nterrelatable bio log i ca l te st systems . Consequently ,

quantitative ac tivit�es suitable for s truc ture-activity

relationships have not been reported for most tetracyc l i ne s .

3

The present work was undertaken to obtain activities suitable

for s tructure-activi ty relationships and which are

pertinent to both bacterial reproduction and the proposed

mode of action . The activi ties obtained should inc lude

both clinically active and "inactive " tetracyc l ine antibiotics .

I I . Literature Survey

A . Tetracyc l i ne s - Nomenclature , General Properties and

Biological Activity

The i s olation in 1 9 4 7 o f the broad spectrum antibiotic ,

chlorte tracyc l ine , from Streptomyces aureofac iens was followed

by the dis covery of oxytetracycl ine in 1 950 and tetracycl ine

in 1953 . The c l inical use fulne s s o f the se compounds prompted

the i s olation , synthes i s and characteri z ation of a large number

of derivatives , degradation products and s imilar compounds with

a wide range o f antibiotic properties . The e lucidation of the

s tructure of the tetracycl ine s has been a ma j or achievement in

natural product chemi s try ( 8). The structure o f chlortetracy­

cl ine , e s tab l ished on chemi cal grounds and confirmed by X-ray

analy s i s ( 9 ) , i s shown as ( I ) in Fig . I . The ring numbering

and lettering system shown serve as the bas i s for the

nomenc lature of mos t of the tetracycline s . Several analogs

with altered nuc l e i are also shown in Fig . I .

I.

II ][

F igure I . The Tetracyc l ine s

( I ) 7 -Chlorotetracyl ine

( I I ) diD-do-tetracyc l i ne

( I I I ) 5a ( 6 ) -Anhydrotetracyc l ine

( IV ) 7 -Chloro-Sa ( l la ) -dehydrotetracyc line

(V ) I sotetracyc l i ne

4

5

The s tereochemi s try of the compounds i s depicted in ( l )

of F ig . I . The 4 - d imethylamino-group , the 4a- and Sa-hydrogens ,

the 6-methyl - a nd the l 2 a - hydroxy- are a l l in the a lpha

or i entation . Epimer i zation by general ac id-ba se cata lys i s of

the 4 - d imethylamino -group in neu tr a l solutions occur s rapidly

and g ives r i s e to the so-ca l led 4 -epitetracyc l ines ( 1 0 ) .

Synthetic compound s epimer ic a t both the Sa- and 6a- position

have been reported . The Sa-epitetracyc l i ne i s e s sentially

inac tive whi l e the 6 -deoxy- 6-epite tracycl ines are reported to

be only s l ightly d i f f erent from the norma l 6-deoxy- der ivative

( 6} .

A . l . A-Ring Mod if ications

The 2 -carboxamide-group has been mod if ied in severa l ways .

The 2-acety l - 2-decarboxamido-tetracyc l ines have been prepared

biosynthetically and are e s sent i a l ly i nactive as antibac ter ia l s

( S ) . Dehydration of the carboxamide yields the nitr i le , a

modif ication which i s a l so s a id to result in loss of activity

( S ) . Sever a l compound s which are c l inica l ly useful because of

their i ncreased wa ter solubi l ity have been prepared by employing

the Mannich reaction to form a methylene br idge between the

amide nitrogen and various primary amine s . Hydro lys i s back to

the parent compound is probably respons ible for the ac tivity of

these der ivative s ( 6 ) .

The d imethylamino group at carbon 4 can be epimer i z ed ; it

can be quatern i z ed by treatment with methyl iodide or other

agents and it can be replaced by hydrogen . All of these

compounds are r epor ted to have reduced ant ibacter i a l activity .

The 4 -N-ethyl-methyl-amino a s wel l a s the 4 -N-methyl-amino

der ivative have been prepared and are r eported to pos s e s s

biological activ ity .

6

The �2a-hydroxy-group may be e l iminated by reduction with

z i nc and ammonium hydroxide or by catalytic reduc tion of the

l 2 a - formyloxy der ivative . In these antibiotics nhe phenolic

diketone moiety is conjuga ted with the ketone at carbon l .

Such conjugation i s said to be re spons ible for the loss of

activ i ty by this c ompound . The 4a ( l 2 a ) -anhydrotetracyc l ines

have been prepared by pyrolysi s of the l 2a-acetoxy der ivatives

and are r eported to have minimal antibac ter ial activity . The

l 2 a- f ormyloxy- a nd l 2a-acetoxy- der iva t ives are r eported to

pos se s s activ i ty s o l e ly becau s e of their hydr o ly s i s to the

parent compound s .

A . 2 . B-Ring Modi f ications

5 - Hydroxytetracyc l ine wa s the second tetracyc l ine antibiotic

i solated from Streptomyce s and wa s the f ir s t t o have its

structure d e l i neated ( 8 ) . I t s c l inical u t i l i ty ha s been proven

throughout the last 2 0 year s . The Sa ( l l a ) -dehydrotetracyc l i ne s ,

( IV ) i n F ig . I , are a l so produced biosynthe tica l ly and are

g ener a l ly r egarded a s being inactive ( 6 ) .

A . 3 . C-Ring Modif ications

In s trong a c id solutions , tetracyc l ines are dehydrated to

form anhydro te tracyc line s , ( I I I ) in F ig . I , whi l e in a lka l ine

solutions i sotetracyc line s , ( V ) in F ig . I , are formed . Both

modi f ications r e sult in compounds which are tradi ti ona lly

regarded a s inact ive or a s pos s e s si ng an a l tered spec trum of

7

antibacter ial activ i ty ( 5 , 6 ) . The i sotetracyc l i ne nuc leu s has

such d i f f erent conf orma t ions avai lable to it that its lack of

activity should probably not be u t i l i zed i n s truc ture-ac tivity

relationships . The c linically useful 6 -demethyltetracyc l ine s

are produced biosynthetica l ly , whi le the 6-deoxy-6-epitetra­

cyc l ines are the result o f catalytic hydrog enation reac tions .

Both type s of compound s a s we l l a s the 6-deoxy- 6 -demethyl­

tetracyc l i ne s , ( I I ) i n F ig . I , are potent ant ibac ter ial agents .

In addi tion , the 6 -methylene tetracyc l ines have been prepared

and are a l s o ac tive antibacter ial substanc e s .

A . 4 . D-Ring Modif ications

7 -Chlortetracyc l ine , ( I ) i n F ig . I , and 7 -bromotetracycl i ne

are produced biosynthe t ic a l ly and are among the mo st potent

tetracyc l i ne antibiotic s . The acid stabi l i ty of the

6-d eoxy - 6 -demethyl-te tracyc l ines ha s a l lowed the chemical

substitution of the aromatic D-r ing at positions 7 and 9 and

a number of antibac ter i a l substances with a wide range of

activ i ties have been reported . The ava i labi l i ty of the se

der ivatives made possible our present attempt at quantitative

correlations of struc ture and activ i ty .

A . S . Struc ture-Ac tiv i ty Generali zations

Modif ications of the A-r ing which are not readily rever s ible

by s imple aqueous hydrolys i s lead to drastic reduc tions in

biolog ical activ i ty . The mos t notable exceptions are the

4 -dedimethylamino-tetracyc l ine s which retain about 5 0 % of the

biolog ic a l activ i ty of the ir parent compound . In contras t , the

4 - epitetracyc l ines are e s sentially inac tive . P lakunov ( 7 )

8

s tudied s everal tetracycl ines with altered A-r ing systems a s

well a s several compound s s truc turally analogous t o the A-r ing

of tetracycl ine and concluded that thi s r ing i s the most

important part of the tetracycl ine nucleu s . He f ound that

alterations of it may produce compounds with a d i f f erent

antibac ter ial spec trum which are probably exerting an effec t

by a dif ferent mode of ac tion (v itamin K antagon i sm i s proposed ) .

Modif ications of the substituents at carbons 5 , 6 , 7 and

9 produce der ivatives with quantitatively d i f f erent activities

but do not produce the extreme d i f f erences in activ i ty seen

with A-r ing modif ications . These compound s would seem to offer

the best pos s ibil i ty for structure-act ivity correlations and

Colai z z i , Knevel and Martin ( ll ) have repor ted l imi ted

correlations of biolog ical activity with Hammett- s igma value s

for these der ivatives .

The oxygen func t i ons at pos i t ions 1 0 , ll and 1 2 appear to

be important for biolog ical activ i ty s i nc e alterations of thi s

chromophoric group lead t o very inac tive compounds . The se

grouping s are also one of the mos t l i kely s i te s for metal ion

chelation , a phenomena which ha s always been expec ted to play

a large role mn the activ i ty of the se antibiotic s ( 1 2 ) .

A . 6 . Ac idity Constants of the Tetracycl ines

The normal tetracycl ine nucleus , ( I ) and ( I I ) in F ig . I,

ha s three mea surable pKa value s i n the vic inity of 3 , 7 . 5

and 9 , Table I , see page 61j Stephens et . al . , ( 1 3 ) have

def ined three ioni zable groups , (A ) , ( B ) and ( C ) correspond i ng

to these values a s ind icated in F ig . I I . Thi s a s s ignment o f

8 H@

CH3 OH N(CH3)2

OH

CONH2

OH OH A c

F igure I I. The Ioni zable Groups of Tetracyc l ine

(A ) Tricarbonylmethane

( B ) Ammonium Cation

(C ) Phenolic diketone

9

1 0

the observed pKa values t o spec i f ic functions ha s been the

subject of some controversy ( 1 4 , 1 5 ) , but it is now genera lly

agreed that the f ir s t pKa i s attr ibutable to the

tr icarbonylmethane sys tem (A) , the second to the phenolic

d iketone moiety ( C ) and the third to the ammonium cation ( B )

I t i s recog n i zed that in many compounds con s iderable over lap

may exi s t between the ioni zation of the second and third groups

( 1 6 ) •

B . Tetracyc lines - Biochemica l Ba s i s of their Antibiotic

Act iv i ty

I t i s g ener a l ly a s sumed that a l l the tetracyc l ines have

s im i lar mode s of ac tion . Thi s view i s ba sed upon the

s imilarity of their s truc ture s , the fact that the bac ter iostatic

effects of the mos t active compound s occur at s imi lar

concentrations and the common occurrence of cross-resi stance .

Never thele s s , i nve s tiga tor s have occas iona l ly noted

d i s s imilar i t ie s , part icular ly in the ni tro-reduc ta se system and

in c e l l wal l synthe s i s . Cross-re s i stance i s seen with a great

many other struc tur a l ly d i s s imi lar antibiotics and it probably

does not have any bearing on the mode of bac ter iostatic action .

Also , there i s a large d i f f erence in the concentrations at which

bac ter iosta tic e f fects are seen in some le ss common tetracyc l i ne s .

The resolution o f these problems , undoubtedly , depend s o n the

test system .

B . l . Effect on Rate of C e l l Reproduc tion

The ant ibac ter i a l activ i ty of the tetracyc l ines ha s been

1 1

evaluated i n various media against many pathogenic and

non-pathogenic organ i sms . The data ha s usually been presented

as the minimum concentration of drug necessary to prevent

v i s ible growth dur i ng a spec i f i ed time per iod . These value s ,

the so-c a l led MIC or p value s , have been tabulated by La skin

( 1 7 ) . The wide range o f MIC va lue s obtained for a g iven

tetracyc l ine by d i f f erent i nvestiga tors is the reason why

these activ i t i e s are not useful in making structure-activity

correlations .

The effec t of the antibiotics on the rate of cell d iv i s ion

ha s been studied by turbidimetr ic , viafule plate and tota l cell

count methods . Rates of growth mea sured by turbid imetr ic

method s f orm the bas i s for the biological activities compi led

by Boothe ( 6 ) , Barrett ( 5 ) , P lakunov ( 7 ) and Benbough and

Morr i s son ( 1 8 ) . These methods were c r i t i c i zed by Ha sh et . a l . ,

( 1 9 ) s i nc e they are sensi tive to c e l l volume in addition to

cell number . They c la im that cell vo lume may change during

tetracyc l i ne inhibition . The Coulter Counter can be used to

observe chang e s i n c e l l volume . C e l l vo lume changes , other

than those norma l ly a s sociated with the exponential growth

cyc l e , however , were not seen in cultures inhibi ted with low

tetracyc l ine concentrations ( 2 0 ) . Jones and Morr i sson ( 2 1 )

have pointed out that the ir turbidimetr ic data were correlated

wi th c e l l weight , which to them is the mos t meani ngful and

only exponential growth parameter . They be l ieve that changes

in cell number s are only exponential becau se of exponential

chang e s in cell mas s and that therefore turbidimetr ic measures

1 2

were more meaning fu l . Data to be pre sented later make it c lear

that measurements of culture turbidity and c e l l numbers may be

d i fferent dur ing normal and inhibited c e l l growth .

C iak a nd Hahn ( 2 2 ) , Jone s and Mor r i s son ( 2 1 ) and Garrett

and Brown ( 2 3 ) d emonstrated that exponenti a l growth i s

ma inta ined in cultures conta ining low tetracyc l ine concentrations .

They propo sed k inetic equa tions which expre s sed the dependence

of the growth rate constants upon tetracyc l ine concentration .

Changes in this dependence at higher antibiotic concentrations

have been interpre ted as ind icating a change in the predominant

mode of action .

B . 2 . Effec ts on Oxidation and Re spiration

Eagle and Saz ( 2 4 ) , Snel l and Cheng ( 1 ) as wel l a s Laskin

(1 7 ) r eviewed the effects of the antibiotics on respiration and

on the oxidation of var ious substrates . The pronounced

inhibition in mo st cultures of the se parameter s was one of the

f ir s t areas s tudi ed . Cheng and Sne l l ( 2 5 ) reported 5 0 %

inhibition o f o0 2 in resting E scher ichia coli and

Staphylococcu s aureus by a s l ittle a s 2 x 1 0 - 6 M

oxytetracyc l ine . At the same concentration severa l

tetracyc l ine analogs : 5a ( lla ) -anhydro-5-hydroxy-tetracycline ,

4 -dedimethylam i no-oxytetracycl ine and 2-acety l - 2 -decarboxamido-

oxytetracyc l ine , compounds not normal ly cons idered

bacter i o static , cau s ed inhibitions of 4 6 , 59 and 2 3 % ,

r e spect ively . Crema and Barol i ( 2 6 ) repor t that oxytetracyc l i ne

was a lmo st without e f f ec t on the r espiration of S . curcio when

the organism wa s in the resting state , whi l e a s l ittle a s

1 3

0 . 1 to 1 . 0 mcg . /ml . o f drug inhibited re spiration by 5 0 to 6 0 %

at the beginning of the log pha s e . This observat ion wa s

conf irmed by Wong , Barban and Aj l ( 2 7 ) who r eport 9 0 %

inhibition o f re spiration i n pro l i f erating cultur e s , by 0 . 1 3

mcg . /ml o f oxytetracycl ine .

A large number o f r eport s documenting inhibition of

oxidat ion of compounds involved in carbohydrate metabo l i sm

have been reviewed ( 1 7 , 2 4 ) . The se effects are now thought

to be due to an inhibition of enzyme f ormation or to the

appearance of an add i t iona l mode of ac tion due to the extremely

high concentrations of antibiotic employed . S ince inhibition

o f severa l o f these system s , pyruv ic " hydrogenlya se " , amino

ac id dec arboxy l a s e ,oC-amylase , a lanine dehydrogena se and

polynuc l eotide phosphoryla s e , occur in in v i tro systems wi th

preformed enzyme s , the inhibi tions can not be solely a scribed

to faulty protein synthe s i s . La skin ( 17 ) , however , ha s

conc luded that " in no ins tance i s there compell ing evidence

to sugg e s t that the primary s ite of ac tion is involved among

thes e effects on oxidation and r e spiration . "

B . 3 . Effects on Protein Synthe s i s in Cultur e s

I n l 9 5 0 , Ga le and Paine ( 2 8 ) reported that chlortetracyc l ine

inhibited protein synthe s i s in S . aureus. Thi s s tudy showed that

the antibiotic inhibi ted " c ombined g lutamate " formation , but not

free g lutamate accumu la tion in wa shed c e l l suspens ions . Hahn and

Wi s seman ( 2 9 ) noted that adaptive enzyme f ormation wa s inhibited

in E . c o l i by both chlortetracyc l ine and oxytetracyc l i ne . They

a ttr ibuted this to a general inhibition of protein synthe s i s .

1 4

Ga le and Folkes ( 3 0 ) conf irmed that wa shed suspens ions of

S. aureus do not incorporate g lutamate into protein in the

pre s ence of growth inhibitory concentrations of tetracyc line ;

a 9 0 % inhibition o f rate o f incorporation a t 1 mcg . /ml . of

e i ther chlortetracyc l ine or oxytetracyc line wa s observed .

They a l so noted that inhibition of nuc leic ac id synthe s i s ,

rate of free g lutamate accumu la tion and rate of glucose

fermentation took place at higher antibiotic concentrations

and that the two antibiotic s d i f f ered quantitatively in the ir

effect on thes e parameter s . Park ( 3 1 ) and Sa zykin and Bori sova

( 3 2 ) in shor t communications , conf irmed the preferential

inhibition of protein synthe s i s . Park also noted that

funhibition of c e l l wa l l synthe s i s required higher antibiotic

concentrat i ons , 50 mcg . /ml . Neverthe l es s , Cheng and Snel l

( 1 ) , unaware of Park's r eport , have c la imed that the use of

D , L-Glutamate by Gale and co-worker s had not e l iminated the

pos s ib i li ty of a preferenti a l inhibition of D-Glutamate into

c e l l wal l mater i a l . Ha sh , Wishnick and Miller ( 1 9 ) have

r eported that c e l l s treated with tetracyc l ine show increased

c e l l wal l , hexosamine s and r i bonuc�e ic acid ( RNA) but decreased

protein and deoxyr ibonuc leic ac id ( DNA) as compared with

contr o l c e l l s . S imilarly , the incorporation of c�4 -labeled

lys ine , a lanine and g lutamic a c id into the cytoplasmic protein ,

i s o lated in a manner so that c e l l wa l l protein wa s e l iminated ,

wa s greatly inhibited by tetracyc l ine at 0 . 1 , 0 . 5 and 1 . 0

mcg . /ml . and chlortetracyc l ine at 1 . 0 mcg/ml . Incorporation

of c l 4_a lanine into c e l l wa l l material wa s not inhibited at

1 5

1 . 0 mcg . /mL o f c hlortetracyc l ine , but wa s inhibited at

50 mcg . /ml . ; tetracyc l i ne had no effec t on this incorporation

at 5 0 mcg . /m l . The kinetic s of incorporation of the amino

acids into cytoplasmic prote in revea led that inhibitrnon was

immediate . Mor r i s son et . a l . , ( 1 8 , 2 1 ) provided further

evidence in support of the theory that the ma j or effec t of

the tetracyc l ine s is an impa irment of protein synthe s i s .

v / Cer ny and Haberman (3 3 ) have made kinetic mea surements of the

inhibition of protein and nuc l e ic a c id synthes i s in cultures

o f E . coli C at tetracyc l i ne concentrations of 20 to 5 0

mcg . /ml . They found an immediate c e s sation of protein

s ynthe s i s f o l lowed by a latter , complete inhibition of nuc leic

acid synthe s i s . The s e concentrations are suff ic i ent to cause

c e l l death and it is unfortunate that the se workers d id not

employ addi t iona l lower antibiotic concentrations . Holmes and

W i ld ( 3 4 ) , dur i ng the course of investigatroons of the synthes i s

o f abnorma l r ibosomal RNA , have reported a l s o a t high

chlortetracycl ine concentra tions , results s imi lar to those of

v / Cerny and Haberman . They observed an immediate cessation of

protein synthes i s and continued RNA synthe s i s . Several

addi t iona l tetracyc l ine ana log s have a l so been stud ied and

s imilar f inding s reported ( 3 5 , 3 6 ) . Their mea surements were

completed approximately within the time of one culture

g eneration cycle and they did not observe the inhibition of

muc leic ac id synthes i s which occur s at longer growth time s .

These authors seem to feel that r ibosoma l precur sor RNA

accumu lated under antibiotic treatment i s not only the result

o f an inhibition of protein synthe s i s , but a l so due to a

spec i f ic s t imulatory effect o f the tetracyc line s .

Numerous investigator s have reported that the

16

tetracyc l ine s inhibit the formation of adaptive enzyme s . Sne l l

and Cheng ( l ) a s we l l a s La skin ( 1 7 ) have l i s ted these

inves t igat ions and have empha s i zed the low concentrations of

antibiotic needed to inhibit these phenomena . S�rm et . a l,, ( 3 7 )

i n addi t ion to Hahn and Wis seman ( 2 9 ) , have tr ied to interpret

their data as meaning that tetracyc l i ne caused an impairment of

the over a l l protein synthe s i z ing abi l i ty . These authors showed

that the inhibition o f g lutamic ac id decarboxylation was re lated

to the synthes i s of the enzyme and not to a lack of nec e s sary

co-factor s .

Sne l l and Cheng ( l ) have cautioned against drawing such

general conc lus ions from thi s type of data . They be l ieve that

an inhibition of adaptive enzyme f ormation might not be

suf f ic iently critical to account for bac ter iosta s i s . They were

a l so not sure at that t ime , 1 9 6 1 , that an inhibition of protein

synthe s i s would be able to cause the effects observed in bac ter i a l

culture s . They , however , f e l t that blocking a reaction in the

e lectron transport sys tem or an impairment of c e l l wa l l synthe s i s

wou ld b e suf f ic iently cr i tica l . A n over a l l inhibition of

protein synthes i s is presently thought to be c r itica l enough to

cause bac ter iosta s i s .

B . 4 . Effects on Cel l-Free Nitro-reduc ta se Systems

Ear ly reports ind icated that high concentrations of

chlortetracyc l ine were capable of uncoupling oxidative pho s ­

phorylation in norma l mitochondr ia ( 3 8 ) a s we l l a s i n r a t liver

17

and brain s l ic e s ( 3 9 ) . An interference with orthophosphate

uptake wa s conf irmed in the s tud i e s of Van Meter et . a l . , ( 4 0 ) .

The inhibition by c hlortetracyc line in rat l iver mitochondr ia

at low adenos ine tr iphosphate (ATP ) a nd phosphate concentrations

wa s shown to be reversed by added Mg ions . S imilar inhibi tions

and meta l ion r ever s a l were reported for oxytetracyc l ine and

tetracyc l ine by Brody , Hurwitz and Bain ( 4 1 ) . The se reports

suggested that the inhibition of protein synth e s i s repor ted by

Gal e and Pa ine ( 2 8 ) might be due to di sruption of the e lectron

transport system , a lthough a s imilar inhibition of nuc l e ic a c id

synthe s i s might have been expected . Inhibition wa s postulated

to occur because of interactions with meta l lo-enzyme s known to

be important in these systems . Miura e t . a l . , ( 4 2 ) showed that

oxytetracyc l ine wa s able to r educe incorporation of P3 2 into

a c id soluble phosphorus in intac t r e sting cultures of S. aureus

at growth inhibitory concentrations . However , these

experimental r e sults in add ition to being highly non- spec i f ic ,

could be interpreted a s due to the improper use of controls .

Contr o l c e l l s were a l lowed to enter the log pha se of growth

whi le inhibited c e l l s d id not leave the stationary pha se of

growth . In a r ev i ew of oxidative phosphorylat ion , Brody ( 4 3 )

reached the conc lus ion that the uncoupling caused by the se

antibiotic s wa s non-spec i f ic and was most probably the result

of r emoval of e s sential metal ions. Hunter and Lowry ( 4 4 ) in

a later rev iew d i sagreed with thi s v iewpoint because of the large

Mg ion/tetracyc l ine ratio r equired to rever se the inhibition .

However , they be l i eved that inhibition of these mamma l ian

18

sys tems by tetracyc lines occur s at such high concentrations

as to make unlike ly the postulate that this type of activity

is r esponsible for the compound ' s antibiotic ac tivity

Goma z kov ( 4 5 ) , Santi ( 4 6 ) and Morikawa e t . a l . , ( 4 7 ) have

recently studied thes e inhibitions and a l s o noted that the

high antibiotic concentra tions required for inhibition make

this an unlikely mode of action in bac teria l c e l l s .

The pos sibility sugge s ted by this ear ly work , that

tetracyc line s f unc tion as inhibitor s by chelation of e s s entia l

meta llo-enzyme system s , prompted Sa z and co-worker s to search

for other similar bacteria l systems . Saz and Maramur ( 4 8 )

r eported the inhibition of a c e l l-free nitro-reducta se system

by chlortetracyc line in 1 9 5 3 . The r ever sal of this inhibition

by manganese ions was reported by S a z and S lie ( 4 9 , 5 0 ) .

The se extrac ts were capabl e of mediating the r eduction of

a large number of aryl compounds ( 5 1 ) a s we l l a s the reduc tion

of nitrate to nitrite . The aryl nitro reduction wa s inhibited

by chlortetracyc line , but not oxytetracyc line and neither

compound interfered with nitrate r educ tion .

In addition to chlortetracyc line , sever a l of its

degradation products were active inhibitor s of the reduc ta s e ,

i . e . aureone amide , aureomycinic acid , anhydrotetracyc line ,

isochlortetracyc line and 4 -dedimethylamino-aureomycinic acid .

Several o f these compounds were a l s o ac tive in reducta se

experiments performed with whole c e l l s , a l though they are not

a l l bacteriostatic agents .

1 9

Further s tud i e s ( 5 2 ) with more pur if ied nitro-reductase

systems showed that they r equired ATP , d iphosphopyridine

nuc leotide (DPN ) a nd a d i carboxyl ic ac id a s wel l a s cyste ine

and manganese ions . If r educed d iphosphopyr idine nuc leotide

(DPNH ) wa s u sed in place of DPN and L-ma la te , the r educ tion

of aryl n i tro compounds wa s no long er inhibited by

chlortetracyc l ine .

As a r e su l t of these experiments , S a z and co-worker s

proposed that chlortetracyc l ine wa s an inhibitor of the

formation of DPNH from L-ma late and DPN . They thought that

it accomp l ished this inhibi tion by preventing fur ther

metabol i sm of oxa loacetic a c id , the ma j or product of the

DPN-malate reac tion . I t wa s known tha t oxa loacetic

decarboxyl a s e ha s a manganese ion requirement and this enzyme

wa s the pre sumed s ite of action . The s ite of action wa s

proposed to be spec i f ic for the chlor ine conta ining

tetracyc l ines a s oxytetracyc l ine or tetracyc l ine did not

inhibit the s y stem . The a b i l i ty of the inactive as we l l a s

active chlor ine containing tetracyc l ines t o inhibit the system

made i t a poor choice for the s i te of antibacterial action of

these compound s .

Saz and a s soc iates next d irected their attention to

purif ied extracts obta ined from both sens it ive and r e s i s tant

E . co l i . These more highly pur if ied extrac t s reta i ned some

but not a l l of the or ig ina l nitro-reducta se activ i ty . While

the extracts were no longer able to reduce DPN to DPNH , i . e . ,

they were devoid of mal ic dehydrogenase activity , they were

2 0

s t i l l able t o r educe chloramphenicol o r �-nitrobenzoic acid .

S i nce this r educ tion of p-nitrobenzoic a c id wa s inhibited by

chlortetracyc l ine they were f orced to conc lude that the ir

prev i ously proposed mechan i sm was not correct for the new

r educ tase ( 5 3 ) .

Extracts obta ined from sensit ive c e l l s were shown to be

greatly stimulated by f lavin mononuc leotide and manganese ions

whi l e tho s e from r e s i stant c e l l s were not stimulated . Kinetic

exper iment s demonstrated that the extract s from sens itive

c e l l s ox id i z ed stoichiometr ic amounts of DPNH in the reduction

o f FMN and the produc tion of p-aminobenzoic acid . Saz and

Martinez ( 5 4 ) interpreted the se results to mean that f lavin

and mangane se were loos e ly a s soc iated as co-factor s with

enzymes in sens itive c e l l s while they were more tightly bound

in r e s i s tant c e l l s . Plakunov ( 5 5 ) i n a s tudy o f resi stant

s trains has , however , r eported that the d i f ferences are mor e

l ikely a scr ibable t o d i f f erenc e s in the relative amounts of

FMN and f lav in adenine dinuc leotide ( FAD ) f ound in r e s i s tant

and s en s itive strains of S . aureus .

The hypothes i s that tetracyc l ines inhibit by interfering

with f lavins ha s been s tudied by Yag i e t . a l . ( 5 6 ) . They

showed that the mode of ac tion of the se compound s in the

inhibit ion of D-amino ac id oxida se was by complexation with

FAD . They f ound that chlortetracyc l ine formed highly s table

complexes with r ibof lav i n , FMN and FAD . They a l so showed that

the kinetic s of inhibition of the oxidation of D , L-alanine by

the enzyme-FAD preparation could be explained by a s suming the

rever s ible formation of an inactive chlor tetracyc l ine-FAD

complex .

Continued puri f ication of the reduc tase system by S a z

2 1

and Martinez ( 57 , 5 8 ) has not demonstrated that i t i s the s i te

of bac teri a l res i s tance to chlortetracyc l ine . They i solated

a protein- stab i l i z ed enzyme preparation which catalyzed the

reduc tion of 2 , 6 -dichloropheno lindophenol . This enzyme

preparation wa s inhibited by chlor tetracyc l i ne when prepared

from sensitive but not when prepared from r e s i s tant strains .

However , chlortetracyc l ine inhibited the reduct ion of

ferricyanide and s timulated the reduction of cytochrome c by

extracts prepared from both sens itive and r e s i s tant strains .

V i l lanueva ( 5 9 ) in a study of the nitro-reducta se of extracts

from both sensi t ive and r e s i s tant Nocardia ha s recently

reached the conc lus ion that there is no difference in

reductase activ i ty .

Krfmery and a s sociates ( 6 0 ) have continued work on the

hypothe s i s that development of tetracyc l ine r e s i s tance as we l l

a s a poss ible mode of action l i e s in the e lectron transport

cha i n . They have shown that triphenyltetrazol ium chlor ide

reduction by whole c e l l s of sens i t ive stra ins but not by who le

cells of r e s i s tant strains is inhibi ted by oxytetracyc l ine .

The degree of inhibi t ion var ied markedly with the type of

carbon source in the growth media . Further s tudi e s ( 6 1 ) showed

that malate a nd lacta te dehydrogena s e (MDH , LDH ) activities

dif fered in s en s i t ive and r e s i s tant s trains . Re s i s tant c e l l s

( 6 2 ) were a l so not able t o reduce tel lurite ( K2

Te03

) whi le

2 2

the sensitive stra ins had this abi l i ty , but were inhibited by

oxytetracyc l i ne . Crude c e l l-free preparations which catalyzed

MDH , LDH and N03 reduc tion showed s imi lar dif ferenc e s among

sensitive and resistant s trains . Recently ( 6 3 ) these worker s

have reported that cytochrome oxidas e activity in sensi tive

strains of S . aureus , E . coli and Brucella abortis are

inhibited by short exposure to oxytetracyc l ine . Re si s tant

s tra ins are not affected under the u sual aerobic conditions

but are inhibited only under anaerobic cond itions . The se

worker s be lieve that oxytetracyc l i ne interferes with microbial

enzyme systems involved in elec tron transport and that

resistant s trains have developed a l ternate pathways . Severa l

other worker s bel ieve , however , that resi stance to �etracy­

c lines is a s soc iated with an a l tered permeab i l i ty of the c e l l s

( 6 4 , 6 5 , 6 6 ) .

Colai z z i , Knevel and Mar tin ( 1 1 ) used mammal ian cytochrome

C oxido-reductase system to obta in biolog ical activities for

use in struc ture-ac tivity corre lations . Whi le many of the

active tetracyc l ine s were inhibitor s of this system , severa l

tetracyc l ine s which a r e not considered bac teriostatic were

equ a l ly good inhibitor s . The author s felt that this might

be explained by dif ferences in abi l i ty to permeate c e l l wal l s .

However , S a z and co-worker s ( 5 1 ) have previou s ly noted that

such reasoning wa s not va l id in exp laining difference s in

activity in the ir nitro-reductase system . The high

c oncentrations ( lo - 3 �) of antibiotic nece s sary for inhibition

of this sys tem are typical of results obta ined with mammal ian

system s .

B . 5 . The Nature of C e l l -Free Protein S ynthe s i z i ng

Systems - Translation of the Genetic Code

2 3

The purpose o f thi s section i s to summar i z e the exi sting

knowledge of c e ll-free protein synthe s i z ing systems . In this

way , the reader may have a better under stand ing of the effects

of tetracyc l ines upon the se systems and can judge whether

the se effects are suf f ic ient to account for the inhibition of

growth of whole c e l l s . In add i tion , several recent experiments

that have led to new and detai led mode l s of thi s system wi l l be

d i scus sed .

Trans lation refers to the proc e s s in the synthesis of

proteins by which a particular sequenc e of nuc leotide ba ses

in mes s enger RNA (mRNA) determine a specific sequence of

amino acids in a polypeptide cha i n . Translation i s

accomp l i shed b y the interac tion o f three c la s ses of

macromo lecules : mRNA , the spec i f ied amino ac id cova lently

l i nked to transfer RNA ( tRNA ) and a comp lex r ibonuc leoprotein

cal led a r ibosome . Severa l exc e l lent review ar ticles have

appeared recently which descr ibe exi sting knowledge of the

system in deta i l (67, 68 , 69, 70).

The bas ic components of a s imple c e l l-free protein

synthes i z ing system are :

l . Ribosome s

2 . mRNA

3 . Arninoacyl tRNA (AAtRNA)

4 . Ribosome a s sociated ini tiation factor s , f 1 and f 2

». Non-r ibosome a s soc iated ( supernatant ) enzyme s , T and G

2 4

6 . Guanos ine - 5� Triphosphate ( GTP )

I n addition to these bas ic components , a ce l l - free

system usual ly includes a buf fer , pH 7-8 ; monovalent inorganic

s a l ts ; divalent salts or a polyamine polypeptide ; and a

sul fhydryl reducing agent .

More complex sys tems , or alternate ly more crude systems ,

may generate within the sys tems AAtRNA and these sys tems

require addi tional ly ATP , pyruvate kinase (PK ) , phosphoenol

pyruvate ( PEP ) , amino acids (AA ) , soluble RNA ( sRNA) and

aminoacyl synthetase s . A few experiments have been performed

with systems that generate mRNA s imul taneously and these

require DNA , DNA-dependent RNA polymerase and appropriate

nucleotide s .

B . 5 . a . Ribo somes

Bacterial ribosome s are complex aggregates which cons i s t

of 6 0 - 6 5 % RNA and 3 0 - 3 5 % prote in . They serve a s a template

for the assemb ly of peptide bonds between AAtRNA molecules

held on the ribosomal surface . I n addition to thi s inactive

role , it is thought that peptide synthetase enzyme s as we l l

as initiation enzymes may b e an integral part of the normal

ribosomal surface . Because the s i te of action of tetracycl ines

is presumed to be on the ribosomal surface , a detailed

des c ription of ribosomal structure wi l l be presented .

Ribosomal particles partic ipate in a series of revers ible

as sociation reactions which are i llustrated in Fig . I I I .

The species active ly associated with protein synthe s i s has

been shown to be the 70 S ribosomes ( 71) , which have a

506

36S �

'I� 70S

R I�OM E

t.,. :I

� 2 6 S

C OR E !lOS ssJSsmm S U B UN I T

S PL �0

P�OTE INS( · · �

+

2JS r RNA 6S r R NA +

JJ ) ) � ) � � � ) ) � ) �0 S PRO TE I N S

�_, JOS � C�� S U B Ui T SSJ!fffffff \� J OS f S P L I T P R O T EINS

� ISS r R NA

J O S P RO TE I N S

2 5

Figure I I I . Ribosomes , Components , Subunits , As sociation and

D i s sociation Reactions , Shaded c ircles depic t the ribosoma l

partic les , scrambled l ine s depict the ribosoma l ribonuc leic

acids and s - shaped lines depict the ribosomal proteins for

which the ind icated S values refer to the sedimentation

values of the origina l ribosome subuni ts .

molecular weight of approximately 2 . 7 x 1 06 and a d iameter 0

of about 2 0 0 A . They dimeri z e i n solutions of high Mg ion

concentration in an apparently non-phys iolog ica l reac tion ,

2 6

they a l so undergo an apparent shape change i n EDTA solutions .

The d i s sociation of 7 0 S r ibosome s to 5 0 S and 3 0 S subunits

occur s at Mg ion concentrations s imi lar to those found in

bacteri a ( 7 0 , 7 2 ) . The d i s soc iation of 70 S r ibosomes to

subunits i s inhibited by sulfhydry l blocking agents or

polycationic polyamines a s we l l as high Mg ion concentrations

( 7 3 , 7 4 ) . Moore ( 7 5 ) , in a s tudy of the effects of chemica l

reagents on subunit a s soc iation , ha s conc luded that hydrogen

bond formation e i ther between nuc leotide ba s e s or between a

nuc leotide base and a phosphate group are respons ible for

this a s soc iation .

The 3 0 S submm i t cons i s ts of a s ing le RNA molecule ,

1 6 S rRNA , with a molecular weight of approximately 0 . 6 x 1 06

,

and a t lea st 1 5 spec i f ic prote ins ( 7 6 ) . The 5 0 S subunit

cons i s ts of 2 molecu l e s of RNA , one a 2 3 S rRNA and the other

a 5 S rRNA , as wel l as at leas t 19 spec i f ic prote ins ( 7 7 ) .

The 5 S spec i e s of rRNA wa s f ir st i so lated in 1 9 6 4 by

Ros sett et . a l . ( 7 8 ) and wa s shown to be d i s t inc t from sRNA

which ha s a s imilar sedimentation coe f f ic ient . The entire

nuc leotide sequence of 5 S rRNA from KB carc inoma c e l l s ha s

been determined by Suther land , Carr ier a nd Setlow ( 7 9 ) , and

a l s o that of E . col i , by Brownlee and Sanger ( 8 0 ) and Brownlee ,

Sanger and Barre l l ( 8 1 ) . This r i bosoma l RNA l ike 1 6 S and

2 3 S rRNA conta ins no unusua l nuc leotide s .

2 7

The secondary s tructure of thi s relative ly new rRNA i s

also thought t o b e s i milar t o that of both 1 6 S and 2 3 S rRNA .

I t cons i s ts partly of double stranded hel ical regions and

partly of non-hydrogen bonded s i ngle s trands ( 8 2 ) . Extens ive

s tudi e s on the phy s i cal and chemical properties of rRNA have

been reviewed ( 8 5 ) .

The 5 ' terminal nuc leotide sequences of E . col i 1 6 S and

2 3 S rRNA have been determined by Takanami ( 8 3 , 8 4 ) . These

determinations show that the two rRNA ' s are d i s tinct species .

The prote in components of r ibosome s have been s tudied in

considerable detai l . The large number of prote ins indicated

i n Fig . I I I , have been i solated and s tudied by : end-group

ana lys i s ( 8 6 ) , ultracentri fugation ( 8 7 , 8 8 ) , s tarch gel

electrophore s i s ( 8 6 ) , polyacrylami de gel electrophores i s

( 7 6 , 7 7 , 8 7 , 9 0 , 9 1 ) and chromatography on

carboxyme thylcel lulose ( 9 1 ) and Sephadex ( 9 1 ) They are unique

proteins wi th d i s t inct amino acid analys i s ( 8 9 , 9 1 , 9 2 ) and are

not the result of aggregate s formed during preparation . The

large number o f r ibosomal proteins argues against the theory

that rRNA could serve as a mRNA for their synthe s is . Ribosomal

RNA doe s not contain enough trinucleotide s to synthe s i ze thi s

many large , d i s tinct prote ins ( 7 0 ) .

I t s eems l ikely that ribosomal proteins play d i s tinct

and non- i nterchangeable roles ( 9 3 , 9 4 , 9 5 ) . Thei r functional

spe c i f i c i ty has been inve s tigated by a degradation -

recomb ination technique . In these experiments the proteins are

partially spl i t from the ribosomal subuni ts with C sC l ( Fig . I I I )

2 8

and the l i berated core particles and split prote ins are

separated , isolated and identif ied . Normal r ibosome func tion

can be achieved by mixing the isolated components . Ribosomes

reconstituted in thi s manner but lacking one or more split

proteins have been shown i n some cases to lack one or more ,

but not a l l , r ibosoma l functions ( 9 4 ) . This technique ha s

been u sed to show that streptomycin sensi tivity , resistance

and dependence l i e with the 2 3 S core particle and not with

the split prote ins ( 9 6 , 9 7 ) . Sensitivi ty and resistance have ,

however , been shown to l i e i n the prote in frac tion of the

core partic l e s and not in the rRNA ( 9 8 ) .

EDTA treated r ibosomes conta in a l l known r ibosome

components but probably because they have dif ferent hydro­

dynamic proper ties they lack certa in spec ific ribosoma l functions

( 9 9 ) •

Treatment of r ibo some s with LiC l causes a loss of

protein components s imi lar to that which i s produced by cSC l .

L iC l -r ibosomes are now known to be identical to the particles

found in bacterial culture s inhibited by chloramphenicol ( 1 0 0 ) .

The se r ibosoma l particles are probably the same a s those found

in tetracyc l ine i nhibited cultures ( 3 4 , 3 6 ) . Such r ibosomes

are thought to be norma l precur sor par tic les .

The effect of Mg ion concentration on the a s soc iat ion­

d i s sociation of r i bosomes is outl ined in F ig . I II . I t ha s

recently been shown that Mg ion i s required to neutra l i ze the

phosphate g roups in the r ibosoma l RNA . "Norma l " 7 0 S r ibosome s

ex i s t i n an ionic environment in which the ratio of

2 9

r ibosomal-bound Mg ion to ioni zed r ibosomal pho sphate is 0 . 5

( 7 2 , 1 0 1 , 1 0 2 , 1 0 3 ) . Cationic groups , such a s ca++ , NH4+ ,

spermidine or putr e sc ine , in the proper concentrations are

a l so capable of cata l y z i ng the formation of functiona l

r ibosomes ( 7 2 , 1 0 2 , 1 0 4 , 1 0 5 , 1 0 6 , 1 0 7 ) .

B . 5 . b . Mes senger Ribonuc leic Acid

The existence of a spec ies of rapidly synthe s i zed , unstable

RNA , c a l led mRNA , wa s pred icted in a review by Jacob and

Monod ( 1 0 8 ) in 1 9 6 1 . The demonstration of the exi stence of

such a spec i e s ha s been accomp l i shed by exper iments ba sed on

two phenomena : i t s rapid rate of synthe s i s and turnover a s

compared with rRNA o r sRNA and a l s o the synthe s i s of thi s type

of RNA in phag e - i nf ec ted c e l l s . The pred icted function of mRNA

wa s that i t carr ied the information of the genetic code from

DNA to the r ibo somal template , where it directed the sequence

of add ition of amino acids into protein . Evidence in support

of this predic tion ha s been rev i ewed by S impson ( 1 0 9 ) and a l s o

b y S i nger and Leder ( 1 1 0 ) . Conv inc ing ev idence for t h i s role

has been found in the demonstration of mRNA in peptide­

synthe s i z ing polysome s ( r ibosome-mRNA aggregate s ) ( 1 1 1 ) , in

the synthe s i s of phage proteins of known sequence by bacter ia l

systems d irec ted by phage mRNA ( 1 1 2 ) and in the coinc idence of

amino ac id sequence and nuc leotide sequence of synthetic

polyr ibonuc leotides ( 1 1 3 ) .

Trans lation of the code i s not accomp l i shed by a d irect

interaction of an amino ac id with a spec i f ic tr inuc leotide

sequence ( codon ) of a mRNA , but rather through an intermed iate

3 0

adaptor mo lecule , tRNA , cova lently linked to a n amino acid .

The tRNA conta ins a spec i f ic trinuc leotide sequence ( anticodon)

that recogn i z e s the codon . The codons which direct the

incorporation into protein of particular amino acids have been

determined by studies of the bind ing of AAtRNA ' s to r ibosoma l ­

codon , where the codon wa s e i ther a trinue leotide ( 1 1 4 ) o r a

polyr ibonuc leotide of known sequence ( 1 1 5 ) . S imilar i ly

incorpora tion of AAtRNA ' s into protein in systems directed by

polyr ibonuc leotide s of known s equence ( 1 1 6 ) has a l so been used

to determine the codons which d irect the incorporation of

part icular amino acids . The four common nuc leotide bases

when uti l i z ed in a trinuc leotide code can be arranged in 6 4

poss ible combina tions . S ince there a r e only 2 0 amino acids ,

degenerac ie s might be expected and indeed are found in the

code . The i solation of s everal d i f f erent tRNA ' s capable of

accepting a g iven amino a c id ha s led to the speculation that

there may be a particular tRNA corresponding to each dodon .

D i ff erent leuc ine-tRNA spec i e s have been shown to s elec tively

bind to d if f erent codons ( 1 1 7 ) . However , i t i s unlikely that

this is the c a s e for a l l tRNA molecules ( 1 1 8 ) .

To account for the known degenerac ies in the coae , Crick

has introduced a theory of codon - anticodon pa ir ing , cal led

the " Wobble Hypothe s i s " ( 11 9 ) . This theory states that the

bind ing of the f ir s t two bas e s of the codon ( 5 ' to 3 ' ) with the

appropr iate bases of the anticodon ( 3 ' to 5' ) fol lows s trict

Watson-Crick hydrogen bond ba se pair ing . The interaction of

the third ba se of the codon with the third ba se of the anticodon

3 1

i s not spec i f ic , but ha s certain a l lowable exception s . These

exceptions are not random but may be pred icted by a cons idera ­

tion of the bond d i s tances as soc iated with the non-classical

hydrogen bondi ng of codon to anticodon . The sing le exception

to the known c ode degenerac i e s occurr i ng i n the third ba se of

the codon is in those sequence s capable of cod ing for the

initiator , N-formyl -methiony l - tRNA ( N-F-met-tRNAf ) where

degenerac i e s in the f ir s t ba se of the codon exi st ( 12 0 )

I nterpretation o f this phenomena has not been made .

S i nce i t i s known that hydrogen bondi ng between

trinuc leotides is not suff ic i ently s trong to s tabi l i z e mRNA­

AAtRNA complexes , the s tabl i l i z i ng role of the ribosome must be

empha s i zed .

B . B . c . Transfer Ribonuc leic Ac id

The adaptor mol ecule , tRNA , i s a re latively smal l molecule ,

4 - 5 S , which forms an e ster with activaned ami no acids i n the

pre s ence of amino ac id syntheta s e . Thi s reaction i s a

relatively wel l under stood area of biochemistry ; it i s depicted

in schematic form in F ig . IV and ha s been de scr ibed in severa l

reviews ( 1 2 1 , 1 2 2 ) . That tRNA func tions a s an adaptor molecule

ha s been shown by demonstrating that cysteine-tRNA , chemic a l ly

converted to a lanine - tRNA , leads to the incorporation of a lanine

i n place of cyste ine into protein ( 1 2 3 ) .

The nuc leotide sequence s of at leas t four tRNA mo lecules

have been determined complete ly ( 1 2 4 , 1 2 5 , 1 2 6 , 1 2 7 ) . A common

feature of thes e and a l l other tRNA ' s which have been s tud ied

is a termina l . . . pCpCpA nuc leotide s equence . I t i s thi s part

A 8

D E

+AMP

F igure IV . The Formation of Aminoacyl Transfer Ribonuc leic

Ac id . (A ) Binding of ATP and an amino acid to the enzyme .

( B ) Forma tion o f a n " activated amino ac id . " ( C ) Binding

of tRNA to the enzyme . ( D ) Displacement of the origina l

adenylate bound t o the enzyme b y the termina l adenylate of

the tRNA . ( E ) Forma tion of the e ster , AAtRNA .

3 2

3 3

of the molecule which accepts the amino ac id ( see Fig . IV)

and which is probably involved in the binding of the AAtRNA to

the 5 0 S r ibosomal subunit ( 1 2 8 ) .

I n add i tion to the above mentioned amino acid accepting

s i te , each spec ies of tRNA mus t conta in a region which enables

it to accept , exc lu s ively , the proper amino a c id ( recogni tion

s i te ) and a l so a reg ion which conta ins the appropr iate anticodon

for the amino a c id accepted . Reg ions in tRNA which might contain

these s ites have been predicted on the ba s i s of a c lover - leaf

secondary s truc ture postulated for tRNA . This is shown in a

general manner , fol lowing Ful ler and Hodg son ( 1 2 9 ) in Fig . V .

This structure was f ir s t proposed by Holley et . a l . ( 1 2 4 ) for

yeast ala-tRNA . I t i s ba sed on s tudies of hydrogen bonding and

the results of X-ray d i ffrac tion studies which have shown that

tRNA ' s exi s t in par tly helical and partly non-helical conforma­

tions . A pos s ible codon - anticodon interaction , shown in

F ig . VI ha s been g iven by Ful ler and Hodg son ( 1 2 9 ) .

B . S . d . A Mode l of C e l l-Free Protein Synthe s i s

The sequence of events i n a cell-free protein synthe s i z ing

system is shown in F ig . V I I . They may be outl i ned a s fol lows :

Step 1 . - Binding of a 3 0 S r ibosomal subunit to a mRNA .

Step 2 . - Formation of the ini tiation complex .

a . - Al ignment of the codon for N-F-met- tRNAf on

the r i bosomal surface so that i t may bind to

the anticodon , i . e . , creation of the N-F-met­

tRNAf or peptidyl binding s i te .

AMINO ACID ARM

I' p

�

f c I c I 3' X

�H'\\, ... ,� XTRA ARM

J=t ' / t 7 � ANTICODON ARM

"W0811U" �NTlCOOON BAS[

F igure V . Schematic Diagram o f the Proposed C lover-Leaf

Structure of Transfer Ribonuc leic Ac id s . Hydrogen bond s

are i nd icated a s l ines l i nking the strand s . The number of

nuc leotides i n each s i ng le stranded loop i s indicated

within the loop . X ind icates a nuc leotide which var i e s

with the tRNA spec ies , r i s pseudour idylic a c id , T i s

thymidylic ac id , DMG i s d imethylguanylic acid .

3 4

3'

P E P T I D Y L

T R NA M R N A

A M I N O ACYL

TRNA

5'

F igure V I . Codon - Ant icodon Interaction . Hydrogen

bonds are i ndicated by double l ine s l inking the r ibo­

nuc l e ic ac ids ; depicted by shaded l i ne s . A hel ical

relat ionship i s impl ied between the anticodon loops

of the tRNA ' s and the mRNA . Only the anticodon arms

of the tRNA ' s are shown , see F igure V .

3 5

3 6

Figure V I I . A Model o f Cell-Free Protein Synthe s i s .

Shaded half-circles depict 3 0 S r ibosoma l subunits ,

shaded circles depict 5 0 S ribosomal subunits , a wavy

line depicts mRNA and tRNA ' s are depicted by the

symbo l , � , the s-shaped curve depicts a protein .

3 7

ST E P I . - ST EP 2 A .-

M- F - t.I E T

STE P 2 B.- ST E P 3 . -

ST E P 8. -

F igure VI I .

3 8

b . - Bind i ng o f N-F-met- tRNAf to the r ibosome-codon

in a reac tion which i s s t imula ted by and may

require the initiation factor s and GTP to form

the so-cal led initiation complex .

Step 3 . - Attachment of the 5 0 S subunit to the initiation

complex to form a monosome . Thi s binding s imulna-

neou s ly creates a binding s i te for AAtRNA . The

aminoacyl bidding s ite corre spor.ds to the 3 ' end of

the me s senger . l

Step 4 . - Binding to the aminoacyl s ite on the 7 0 S r ibosome .

E i ther AAtRNA a lone or the complex AAtRNA-GTP-T may

f i l l thi s s ite , where T is a supernatant protein .

Step 5 . - Formation of the peptide bond . N-F-met-AAtRNA would

be the f ir s t peptide formed on the ribosome . Thi s

react ion may b e cata lyzed b y a tightly bound r ibosomal

peptidyl syntheta s e .

S tep 6 . - Pulsation (Movement) of the r ibosome .

a . Remova l of tRNAf from the peptidyl s i te , either

to an exi t s i te or directly runto the surrounding

solution .

1 r f attachment of a 7 0 S r ibosome to mRNA occur s in Step l rather than a 3 0 S subuni t , then both peptidyl and aminoacyl binding s ites may be created at that t ime . Al ignment of the codon may be such that N-F-met- tRNA may bind to either site . I f bind ing to the aminoacyl s i te occur s , then trans location of N-F-met-tRNA is required before the next step . Step 3 is then , of cour se , not nece s sary . Steps 1 to 3 may be repeated to form a polysome with d i fferent r ibosomes attaching to the same mRNA a f ter incorporation of about ten amino acids into a polypeptide .

3 9

b . - A s imul taneous shift o f the mRNA and

N-F-met-AAtRNA on the r ibosome surface so

that a new codon is a l igned under the

aminoacyl s ite and the peptidyl tRNA occupies

the peptidyl s i te .

Step 6 i s accompanied by hydroly s i s of GTP

catalyzed by G , a r ibosome-dependent GTPase .

Step 7 . - Steps 4 , 5 and 6 are repeated to form an increas ing ly

longer peptide cha i n .

Step 8 . - Chain termination and release .

a . - E i ther repetition of Step 4 i s prevented by

a l ignment of a nonsense codon wi th the aminoacyl

s i te or Step 5 is prevented by occupation of the

aminoacyl s ite by a non-func tional sRNA bound by

the nonsense codon . ( Such a nonfunc tional sRNA

which bind s to r ibosome-nonsense codon ha s not

as yet been found ) .

b . - Hydroly s i s of the peptidyl-tRNA on the r ibosome

followed by e j ec tion of the peptide and tRNA or

removal of an intact peptidyl-tRNA from the

r i bosome and subsequent hydroly s i s to peptide and

tRNA .

Several s teps in the proposed mode l have been s tudied in

great deta i l and their nature i s at least partially unders tood .

A lack of spec if ic techniques has prevented thorough inves tigation

of some of the other steps .

4 0

Step l . - B ind ing o f a 3 0 S subunit to mRNA .

Attachment of mRNA to the 3 0 S subunit rather than the

5 0 S subuni t wa s demons trated by Okamoto and Takanami ( 1 3 0 ) .

Bind ing of mRNA to 7 0 S r ibosomes has been presumed to occur

through the attachment to the 3 0 S subunit and not because of

the forma tion of some new s i te which does not exi s t on either

subunit a lone . Moore ( 1 3 1 , 1 3 2 ) has presented evidence that

there i s only one binding s ite for mRNA on 7 0 S ribosomes and

that poly C and poly U compete for this site . The attachement

of mRNA ' s to r ibosome s is thought to invo lve only sing le

s trand ed regions of the mRNA , s ince double s tranded mRNA ' s

do not bind ( 1 3 3 ) . Thi s observation accounts for the strong

bind ing of poly U to r ibosomes since i t ex ists a s a random coil

above 4 ° C . The length of mRNA bound to ribosome s has been

e s t imated to be about 2 7 nuc leotide s . This estimate is ba sed

upon the number of nuc leotide s which are protec ted from

r i bonuc lease hydroly s i s by the ir attachment to r ibosomes . Moore

( 7 5 ) ha s u sed chemical modif ications of r ibosome s and/or mRNA

to study their interac tion . He found that the mo st likely

groups involved in the bind ing were amine groups in the rRNA

and the phes phate backbone of the mRNA .

Step 2 . - Format ion of the initiation complex .

S ince r ibosomes d irec ted by natural mRNA ' s synthe s i z e

comp lete prote ins there mu st b e control mechani sms whereby initial

read ing of mRNA by r ibosomes i s restric ted to a specif ic s i te .

Such control might resu l t from attachment of r ibosomes at a

spec i f ic si te on mRNA . This mechanism would not be suf f ic iently

4 1

strict to a l low proper pha s i ng of the mRNA readout , s ince the

only requirement for binding i s that the mRNA be s i ng l e

s tranded . ( Pha s ing i s the a l ignment of the f irst ba se of a

tr inuc leotide codon whos e trans lation i s des ired on the

r ibosome , so that it spec i f ie s the readout . When a l ignment

is such that the second or third bas e specify the code

readout then the read ing of the mRNA is said to be out of pha se . )

In 1 9 6 1 , Wa l ler ( 1 3 4 ) d i scovered that me thionine wa s the

NH2 -termina l group in at least 4 5 % of a l l E . co l i proteins .

Fol lowing this repor t , Marcker and Sanger ( 1 3 5 ) observed that

one spec ies of met- tRNA could be formylated (met- tRNAf ) while

other spec ies could not be formy lated (met-tRN� ) . A l ikely

f ormyl donor wa s s hown to be N 1 0-Formy l - tetrahydrofolic a c id

( 1 3 6 ) . They showed that formylation occurred only a f ter

formation of the AAtRNA and that an enzyme d i s tinc t from

methionyl synthe ta s e wa s involved . C lark and Marcker ( 1 3 7 )

observed that N-F-met from N-F-met-tRNAf was incorporated into

the NH2 -terminal position of prote ins synthes i zed by a c e ll-free

extract . Later ( 1 3 8 ) they showed tha t c e l l-free extracts

directed by poly UG and capable of formylating met-tRNAf

produced proteins in which more than 5 0 % of the NH2 - terminal

groups were N -F -met . This f inding seemed to explain Wa l ler ' s

early repor t ( 1 3 4 ) .

Adams and Capecchi ( 1 3 9 ) showed tha t N-F-met-ala- ser-a sp-

-phe- thr- was the initial amino acid sequence in R 17 phage

coat protein produced by an extract directed by R 17 me s senger .

S ince the identical s equence without the initial N-F-met wa s

4 2

found i n endogenous coat protein , these worker s proposed

that N-F-met-tRNAf was the univer sal bac ter i a l chain initiator ,

but that in some cases e i ther the termina l N-Formyl or the

N-F-met groups were removed by hydrolysi s . Webster , Eng lehardt

a nd Z inder ( 1 4 0 ) �eported s imilar f inding s . Capecchi ( 1 4 1 ) ,

We i sbach and Red f i e ld ( 1 4 2 ) and Lugay and Aronson ( 1 4 3 ) have

s tudi ed the hydroly s i s of NH2 - termina l formate in cell - free

extracts and have attempted to i solate the respons ible enzyme . �

Vinuela , Sala s and Ochoa ( 14 4 ) found that a l l v iral prote ins

as wel l as the coat proteins synthe s i zed in systems directed

by MS- 2 phage RNA had N-F-met as the NH2 -terminal group .

In add i t ion to demonstrating that N-F-met from

N-F-met-tRNAf wa s the f ir s t amino a c id incorporated into

protein by c e l l -free extract s . C lark and Marcker ( 1 3 7 , 1 3 8 )

proposed that the d i f f iculty in a s s igning a codon to met-tRNA

might be due to the ex i s tence of two dif ferent codons , one

codon for met- tRNAf a nd another for met-tRNAm . Sundararaj an

and Thach ( 1 4 5 ) have estab l i shed that binding of met-tRNAf to

r i bosome s i s best when AUG i s the codon , but other triplets

a l s o code for it at high Mg ion concentrations . Met-tRNAm

i s only coded for by AUG . They showed that N-F-met- tRNAf

wa s c apable of pha s i ng the reading of synthetic mRNA ' s ,

such a s AUGG ( U l n · I n the presence of N-F-met-tRNAf bind ing

of val-tRNA in response to the second triplet , GUU , wa s

s t imu lated . Out o f pha s e bind ing of other AAtRNA ' s in

response to UGG or GGU wa s suppressed . Binding experiments

with other synthetic mRNA' s , inc lud ing several with the AUG

codon not a t the 5 ' termini , gave s imilar results . B inding

4 3

above background d id not occur at low Mg ion concentrations

if the AUG codon wa s located at the 3 ' end of the mRNA .

Thach , Dewey , Brown and Doty ( 14 6 ) reported s imi lar results

when incorporation of amino ac id i nto protein wa s used rather

than r ibosome-codon bindi ng . They noted that if an AUG

codon appeared in a mRNA at a position internal to another

AUG , thi s second codon a lways d irected the incorporation of

met-tRNAm . They felt tha t a l ignment of the AUG codon at the

peptidyl s ite spec i f ied chain initiation by N-F-met-tRNAf ,

whi l e a lignment of the codon at the aminoacy l s i te spec i f i ed

chain continuation with met-tRNAm · Leder and Bur s z tyn ( 1 4 7 )

have confirmed the se f ind ing s on phas ing o f readout by

u s i ng AUG ( U ) n as a mRNA and measur ing the incorporation of

met , val and phe . They a l so showed that N-F-met-tRNA bound

dur i ng binding exper iments wa s not displaced from r ibosomes

by the addition of other AAtRNA ' s . Thi s f indi ng in conj unction

with later results of Nomura , Lowry and Guthr ie ( 1 4 8 ) show that

binding of N -F -met-tRNAf ha s relevance in peptide synthesis .

A solution to the pu z z l ing enigma that c e l l - free extrac ts

d irected by synthetic mRNA ' s requ ire higher Mg ion concentra­

tions than those direc ted by endogenous mRNA was sugge sted by

Clark and Marcker ( 13 7 ) . They noted that met-tRNAf bind s to

r ibosome-codon at much lower Mg ion concentrations than those

which are nec es sary for bind i ng met-tRN� . Thi s observation

was a l s o reported by Sundararaj an and Thach ( 1 4 5 ) . Nakamoto

and Kolakof sky ( 1 4 9 ) have shown that the Mg ion optimum of a

system incorporating phe-tRNA but d irec ted by a mRNA which

4 4

a l lows incorporation of N-F-met- tRNAf wa s greatly lowered by

inc luding the l atter AAtRNA in the reaction mixture .

Kolakof sky and Nakamoto ( 1 5 0 ) studied the Mg ion dependency

of extracts d irec ted by MS- 2 viral RNA . They found a lowered

Mg ion optimum and a large s t imulation of over a l l

incorporation when a formyl donor system wa s added t o the

extract . The synthes i s of coat prote in and a l s o other viral

proteins wa s shown to fol low this pattern .

Clark and Marcker ' s original suppos i tion ( 1 3 7 ) that

N-F-met-tRNAf wa s capable of initiating prote in synthe s i s

because the formamide bond resembl ed a peptide bond wa s shown

to be f a l s e by these same worker s ( 1 3 8 , 1 5 1 ) . They

demonstrated that met- tRNAf as wel l as N-F-met-tRNAf could act

a s an initiator , Leder and Bur s z tyn ( 1 4 7 ) and a l so Sa l a s

et . a l . , ( 1 5 2 ) conf irmed t h i s r e su l t . Leder and Bur s z tyn ( 1 4 7 )

sugges ted that formylat ion might take place af ter attachment

to the r ibosome-codon . They showed that this type of r ibosoma l

formylation wa s pos s ible . Economou and Nakamoto ( 1 5 3 ) have

stud ied the kinetic s of the formylat ion reac tion and conc luded

that the reaction is so fast that met- tRNAf would be formylated

before binding to r ibosomes in any extrac t capable of

performing the reaction . I t mu st , however , be conc luded that

the unique abi l i ty of N-F-met-tRNAf to ini tiate protein

synthes i s l ie s in the tRNAf port ion of the mo lecule and not

in the formamide bond itself .

Incorporation of met- from met-tRNAm into interna l portions

of peptides synthe s i z ed by c e l l-free extracts wa s reported by

Thach et . a l . , ( 1 4 6 ) . Clark and Marcker ( 1 5 1 ) conf irmed thi s

4 5

f indi ng i n systems directed by bac ter iophage RNA , Stanley

et . a l . , ( 1 5 4 ) and Salas et . a l . , ( 1 5 5 ) reached s imi lar

conc lusion s u s i ng synthetic mRNA ' s conta ining the AUG codon .

In summary , these results have been interpreted a s

r e f l ec ting the exi stence of two binding s ites o n the ribo some .

This theory states that N-F-met-tRNAf can unique ly bind to

the peptidyl s i te in response to the AUG codon , whi le

met-tRNAm is bound when the AUG codon is a l igned with the

ami noacyl s i te . Ma tthaei and Voig� ( 1 5 6 ) have demonstrated

the exi stence of bind i ng s ites on the r ibosome-codon with

two d i f ferent Mg ion optima , one of which binds N-F-met-tRNAf

and the other which bind s met-tRNAm .

The reac tiv i ty , in the absence of supernatant factor s ,

of r ibosoma l bound -N-F-met-tRNAf with puromyc in ( Pm) is

cons i stent with the conc lus ion that N-F-met- tRNAf occupies

the peptidyl s ite ( 1 5 1 , 1 5 8 ) . Other r ibosoma l -codon bound

AAtRNA ' s do not have thi s reactivity in the absence of super­

natant . Her shey and Thach ( 1 5 9 ) have repor ted that the Pm

reaction i n their extrac ts , a s we l l a s the forma tion of the

d ipeptide N-F-met-phe wa s strongly stimulated by GTP and

supernatant enzyme s . They propose that N-F-met-tRNAf i s

initia l ly bound t o the aminoacyl si te o f the r ibo some and

is then transferred i n a GTP-dependent step to the peptidyl

site - this ha s been termed the sing l e entry s i te model .

Alternate ly , b i nd i ng may occur d irectly with the peptidyl s i te

but a GTP-dependent chemical activation of the bound met-tRNAf

must then occur before d ipeptide formation - thi s ha s been

4 6

termed the peptidyl s ite activation mod e l .

Stanley et . a l . , ( 1 5 4 ) and Salas et . a l . , ( 1 5 5 ) have

i solated two protei n components , f 1 and f 2 , from salt washings

of E . coli r ibosomes . These proteins are required for the

trans lation of natura l mes seng er s , but not for synthetic mRNA ,

unle s s they conta in the AUG codon . Trans lation of synthetic

mRNA ' s which conta i n AUG i s greatly s t imulated by the se factor s .

This stimulation results from an i ncreased i ncorpora tion of

met-tRNAf but not met-tRNAro . The se f ac tors s timulate

incorporation only at low Mg ion concentrations and do not

promote the bindi ng of other AAtRNA ' s . Ander sen , Bretscher ,

C lark and Marcker ( 1 6 0 ) and Thach , Dewey and Myko la j ewyc z

( 1 6 1 ) and Leder and Nau ( 1 6 2 ) have substantiated these results

and have shown that the binding promoted by thes e f ac tors is

stimulated by addi tion of GTP . I t seems l ikely that in the

presence of the se factors and GTP , N-F-met-tRNAf is bound

e ither directly to , or rapidly transferred to , the peptidyl

s ite . Bind i ng , in their absence , may be to the aminoacyl s ite .

S im i lar f ac tor s have been i so lated by Eisenstadt and

Brawerman ( 1 6 3 , 1 6 4 ) and Revel and Gros ( 1 6 5 , 1 6 6 ) . The se

authors have also noted the pos s ible exi stence of a third

factor , retarded on DEAE-c e l lulose , which may s t imu late or

may be an e ssentia l requirement for the bind i ng of r ibosome s

to mRNA in the presence of DNA .

Step 3 . - Attachment of the 5 0 S subunit to the initiation

complex .

Bind i ng of AAtRNA ' s to 3 0 S subuni t s ha s been s tud ied

by Kaj i , Su zuka and Ka j i ( 1 6 7 ) and Pestka and Nirenberg ( 1 6 8 ) .

4 7

They have f ound that a t high Mg ion concentrations , speci f ic

binding of AAtRNA ' s occur s with one s ite of the subunit .

The add i tion of 5 0 S subunits ( 1 6 9 ) causes a 2 - 3 fold increase

in the observed bind i ng , pre sumably by creating an additiona l

bind i ng area . This may be an extra point of attachment of

the tRNA at the same bind ing s i te or an entirely new binding

s ite . S ince bind i ng in the presence of 5 0 S subunits has

d i f f erent metal ion cofactor requirements ( 1 6 8 ) , it most

like ly ref lec ts a new binding s i te . Matthae i and Milberg

( 1 7 0 ) have interpreted the ir results on the kinetics of AAtRNA

binding in terms of the creation of a new s ite upon addition

of 5 0 S subunits . Igarashi and Ka j i ( 17 1 ) have shown that

phe- tRNA bound to 3 0 S subunit-codon is d i s soc iated by

add i tion of 5 0 S subunits and tha t such prebound AAtRNA ' s are

not incorporated into peptides . This result indicated that

bind i ng of AAtRNA to 3 0 S subunits is non- func tiona l and

extraneous .

Recently , however , Nomura and Lowry ( 1 7 2 ) have

reinvestigated the area . They showed that f - 2 phage RNA

directs the binding of N-F-met-tRNAf to 3 0 S subunits at low

Mg ion concentrations and that such bind ing i s not stimulated

by the add ition of 5 0 S subunits . Thi s bind ing was stimulated

by the initiation factor s , f� and f 2 , and by GTP when

salt-wa shed r ibo soma l subunits were s tud ied . The viral RNA

d id not d ir ec t the binding of phe- , leu- , lys- , tyr- , or

va l -tRNA or met-tRNAro to 3 0 S subunits . These AAtRNA ' s were

bound however to 3 0 S subunits at high Mg ion concentrations ,

4 8

when appropr iate synthetic polyribonuc leotides were used .

This binding wa s s t imulated by the addition of 5 0 s subunits

a s had been prev iously reported ( 1 6 8 , 1 6 9 ) . The se results

ind icated that b i nd ing of N-F-met-tRNAf to 30 S subunits is

d i f f erent from the binding of other AAtRNA ' s . Nomura , Lowry

and Guthrie ( 1 4 8 ) in a s imi lar contrast , have repor ted that

the addi t ion of 5 0 S subunits to N-F-met-tRNAf bound to

3 0 S subunit-codon does not cause d i s soc iation of the bound

AAtRNA ; rather the N-F-met-tRNAf is now bound to 7 0 S

r ibosome s .

Nakada and Kaj i ( 1 7 3 ) had shown earl ier that native ( a s

opposed t o d i s soc iated ) 3 0 S and 5 0 S subunits do not combine

in 1 0 - 2 � Mg ion , but are able to synthe s i z e polyphenyla lanine

in the presence of poly U . The r ibosome found assoc iated

with poly U in these exper iments is a 7 0 S r ibosome . The se

resu lts indicate that mRNA catalyzed the assoc iation of native

subunits . Eisenstadt and Brawerman ( 1 7 4 ) have found s imilar

a s soc iation of native subunits i n the presence of f - 2 phage

RNA and bel ieve such monosomes are more active in prote in

synthe s i s than 7 0 S r ibosome s . They bel ieve that 7 0 S r ibosomes

must d i s soc iate pr ior to partic ipating in peptide synthe s i s .

Mangiarotti and Schles s inger ( 1 7 5 ) developed very gentle

extract ive techniques to s tudy polysome d i s tr ibution in

E . col i . With the se technique s , they showed ( 17 6 ) that E . coli .

do not conta i n free 7 0 S ribosomes but rather have a pool of

d i s sociated 30 S and 50 S subunits . Kaempfer , Me selson and

Ra skas ( 17 7 ) conf irmed the exi s tence of such a subunit pool by

4 9

growing E . c o l i i n heavy and l ight i sotope media . They found

that r ibosomes produced in heavy media d i s soc iated and

reas soc iated in very short t ime periods with r ibosoma l

subunits synthe s i z ed subsequently i n l ight media . Subunits ,

however , once synthes i z ed did not equi librate with rRNA or

r ibosoma l proteins .

Mang iarotti and Schle s s inger ( 1 7 6 ) and Nomura and Lowry

( 17 2 ) were led by their quite d i f f erent exper iments to

propose that protein synthes i s is initiated by attachment of

a 3 0 S subunit to mRNA af ter which a 5 0 S subunit is added .

H i l le , Mi l ler , Iwaki and Wahba ( 1 7 8 ) have conf irmed the

b i nd ing stud i e s of Nomura and Lowry . They a l so showed that

N-F-met-tRNA bound to 30 S subuni t s - codon ( in the presence

o f f 1 , f 2 and GTP ) wa s able to react with Pm when 5 0 S

subunits were added . Ghosh and Khorana ( 17 9 ) have studi ed

the binding of N-F-met-tRNAf to 3 0 S subuni t s in the presence

of poly (AUG) n or poly ( UG ) n . They conf irmed the bind ing ,

the requ irement for initiation f ac tor s and GTP in the bind ing

and were able to i so late the ternary complex by sucrose

dens i ty gradient c entr ifugation . The i solated complex wa s

ab�e to form a d ipeptide , N-F-met-me t , when 5 0 S subunits and

met-tRNAm were added to i t . A s imi l ar complex conta i ning 7 0 S

r ibosomes instead of the 3 0 S subunit wa s a l s o capable of

forming the d ipeptide . They a lso showed that binding of cys- ,

val-tRNA or met-tRNAm to 3 0 S subunits occurred only at high

Mg ion concentration . A complex formed at high Mg ion

concentration with 3 0 S subunits , poly (AUG ) n , N-F-met - tRNAf

5 0

and met-tRNAm wa s a l so i solated . This complex wa s not able

to form a d ipeptide upon the addition of 50 s subunits .

These results support the conclusion that only N-F-met-tRNAf

b i nd s in a func tiona l manner to 3 0 S subunits - codon .

Ohta , Sarkar and Thach ( 1 8 0 ) have observed a lower Mg ion

optimum for GTP - f l - f 2 s t imula ted binding of N-F-met- tRNAf

to 7 0 S r ibosomes ( 5m�) than to 3 0 S subunits ( 8 - 9mM) . They

postulate that this might ind icate that the 7 0 S r ibosome is

attached to codon easier than the 3 0 S subunit . They are

aware , however , that extrac tive procedure s and storage

cond i t ions play a role in r ibosomal Mg ion dependency and that

the d i f f erence in the two optima i s not large .

Step 4 . - Bind ing to the aminoacyl s i te on the 7 0 S

r ibosome .

The bind i ng of AAtRNA ' s to r ibosomes i s a wel l known

phenomena which ha s been reviewed by several author s ( 1 2 8 ,

1 8 1 , 1 8 2 ) . Exper iments designed to test for thi s binding have

been u sed to e stabl ish the genetic code ( 1 1 4 , 1 8 3 , 1 8 4 ) .

Gi lbert ( 7 1 , 1 8 5 ) , Cannon , Krug and Gi lber t ( 1 8 6 ) and Bretscher

( 18 2 ) wer e among the early worker s to establ i sh that AAtRNA ' s

were a s s oc i a ted with ribosome s dur ing protein synthe s i s in

bacter ial extra c t s . Nirenberg and Leder ( 1 1 4 ) showed that

such spec if ic binding occurred in the absence of prote in

synthes i s and developed a convenient a s say method to te st

the b i nd ing . This method , which involves adsorption of the

complex on c e l lulose ester f i lter s , has been widely used in

binding investigations . Igarashi and Ka j i ( 1 7 1 ) showed that

5 1

AAtRNA ' s bound to 7 0 S r ibo somes i n the absence o f prote in

synthes i s were i ncorporated in the NH2 -termina l pos ition of

peptid e s when prote in synthe s i s was initiated . Wettstein and

Noll ( 1 8 7 ) have employed rad ioac tive tRNA to determine that

2 or 3 AAtRNA ' s were bound per r ibosome dur ing protein

synthe s i s . Igara shi and Ka j i ( 17 1 ) have obta ined a s imilar

result with saturation bind ing curves . They a l so showed that

the aminoacyl s ite of 7 0 S r ibosome s wa s more read i ly occupied

than the peptidyl s ite by phe-tRNA in the absence of GTP and

enzyme s . Matthaei and Mi lberg ( 1 7 0 ) obta ined simi lar results

by studying the kinetic s of AAtRNA bind i ng .

Ar l i nghau s , Schaef fer and Schweet ( 1 8 8 ) have demons trated

the exi s tence of two types of AAtRNA binding to rabbit

reticulocyte r ibosome s . One mode is non-enzymatic , non-GTP­

dependent , whi le the other requires the presence of GTP and

enzyme s . The exi s tence of an enzymatic and GTP-dependent

bind ing of an AAtRNA other than N-F-met- tRNA or N-acetyl-phe­

tRNA ( 18 9 ) ha s been demonstrated in E . coli extrac ts , very

recently ( 1 9 0 , 1 9 1 , 1 9 2 ) . Previous fai lures to do so may

ref lect d i f f erenc e s in cha in initiation or may be the result

of inadequate r ibosome pur if ication technique s .

Moldave et . a l . , ( 1 9 3 , 1 9 4 , 1 9 5 ) have isolated complexe s

of the supernatant enzyme , Transferase I , from rat l iver

preparations and showed that thi s enzyme bound GTP and AAtRNA .

They sugge sted that thi s complex wa s involved in the binding

of AAtRNA ' s to r ibosome s . Al lende , Monro and Lippman ( 1 9 6 )

have reported the i solation from E . col i of 2 or 3 prote ins ,

Tu , Ts and G , from the non-r ibosoma l ( supernatant) por tion of

5 2

extracts which are d i stinct from the components o f the AAtRNA

synthe s i z ing system and which are necessary for peptide bond

synthe s i s . Allende and Wei s sbach ( 1 9 7 ) identif ied a complex

s imi lar to that reported by Moldave between GTP and E . coli

initiation fac tor preparations . GDP wa s able to replace

GTP in thi s complexation but ATP , CTP , UTP AND GMP were not

able to compete with GTP . The heat instabi lity and the

chromatographic behavior of the protein invo lved in the

f ormation of the complex suggested that the binding wa s with

the supernatant enzyme T . Al lende , Seed s , Conway and

Wei s sbach ( 1 9 8 ) have now reported that the or igina l initiation

fac tor preparations conta ined trace s of T and that the removal

of this protein caused a los s of bind ing activity . Separately

i so lated T enzyme wa s shown to pos s e s s the origina l

comp lexation a b i l i ty . They i s o lated a T - GTP complex on

cellulose ester f i lters .

Ravel , Shorey and Shive ( 1 9 9 ) i ndependently observed

s imi lar complexation in mixtures of GTP , phe-tRNA and super­

natant factor preparations . They showed tha t GTP wa s initially

hydrolyz ed to GDP by binding to the protein and that following

this s tep , GDP wa s further bound in a complex with AAtRNA .

Gordon ( 2 0 0 ) wa s able to i sola te a complex of GTP which

wa s formed by T in the pre s ence of AAtRNA . Later , he

propo sed a two s tep bind ing scheme of GTP to T, fol lowed by

f ormation of a ternary complex with AAtRNA ( 2 0 1 ) . Lucas-Lenard

and Haenni ( 1 9 1 ) have isolated thi s same ternary complex and

report that it is capable of binding to the ami noacyl s i te

of 7 0 S r ibosome s . When highly pur if ied r ibosome s are

emp loyed , bind ing of thi s complex to r ibosome s is much

greater than bind ing of AAtRNA a lone . GDP i s not able to

subs titute for GTP i n r ibosoma l bind ing , a lthough it forms

a ternary complex with T and AAtRNA . They have also shown

that AAtRNA bound to the aminoacyl s i te in the presence of

T a nd GTP i s able to form a d ipeptide if the peptidyl s i te

ha s previou sly been occupied .

Step 5 . - Formation of the peptide bond .

53

The forma tion of the peptide bond between peptidyl-tRNA

and the cx2 -amine of the AAtRNA i s shown a s g iven by Watson

( 67 ) in F ig . V I I . Monro and Marcker ( 2 0 2 ) and Monro ( 2 0 3 )

have recently shown that N-F-met i s bound to the 5 0 S

subunit when i t i s e ster if ied with the termina l hexanuc leotide

fragment of tRNAf . They demonstrated that N-F-met bound to

5 0 S subunits i n this way i s capable of forming the model

d ipeptide , N-F-met-Pm . This Pm reaction i s not enhanced by

GTP and the b i nd i ng must be carr ied out in ethanolic solution .

The 5 0 S subunits u t i l i zed were as free of initiation and

supernatant enzymes as it is pre sently pos sible to prepare

( 4 X NH4 C l wa shed ) and the Pm reacb�dn cata lyzed by the se

subunits const i tutes good evidence that the peptide synthetase

enzyme is a part of the r ibosome .

Luc a s -Lenard and Haenni ( 1 9 1 ) report that d ipeptide but

not tripeptide formation occurs on 7 0 S r ibosomes ( 4 X NH4C l

wa shed ) between prebound N-Acetyl-phe-tRNA and added phe-tRNA .

Forma tion of thi s d ipeptide i s not enhanced by the supernatant

enzyme G . I n thi s study phe- tRNA wa s added a s the ternary

complex between phe- tRNA , GTP and supernatant enzyme T .

S tep 6 . - Pul sation (Movement ) of the r ibosome .

Wettstein and Noll ( 1 8 7 ) have proposed that their data

indicated that tRNA from the peptidyl s i te wa s transf erred

to a loo sely attached " ex i t " s ite a f ter formation of a

peptide bond . The ir proposal wa s based on the number of

5 4

tRNA molecules found a s socia ted with the r ibosome dur ing

prote in synthe s i s . Fur ther evidence concerning the exi stence

of such a s i te ha s not been pre sented to date . I ndeed ,

evidence that a spec if ic tRNA leaves the r i bosome i s not

ava i lable and probably w i l l not be ava i lable until very highly

pur if ied preparations of tRNA are obtained . Our pre sent

picture of the se steps is ba sed on the proposa l s of Wa tson

( 6 7 ) •

L ipmann e t . a l . , ( 2 0 4 , 2 0 5 ) have proposed tha t the mo st

l ikely func tion of their ribosome-dependent GTPa se , G, is to

cause the pulsation or contractile movement of the r ibosome .

They have shown that one mo lecule of GTP i s hydrolyzed for

each pept ide bond formed ( 2 0 3 ) and Luca s -Lenard and Haenni

( 1 9 1 ) have shown that this GTP hydro lysis occur s af ter

d ipeptide formation .

Step 8 . - Cha i n termina tion and relea se .

I t i s we l l known that cel l-free systems d irected by

natura l mRNA ' s release the ir peptide s into the superna tant

( 7 0 ) whi le systems d irec ted by synthetic homopo lyrner s retain

the produc t peptide on the r ibosome ( 7 1 ) . I t ha s been shown

that nonsense codon s produced in bac teriophage mutants cause

the release of incomplete proteins ( 2 0 6 ) . These codons , UAA

( ochre ) , UAG ( ambre ) , and UGA , lead to termination of peptide

cha in growth in sys tems d irec ted by mRNA ' s from the mutant

s trains or phage s . Synthetic mRNA ' s which contain these

codons a l s o terminate prote in synthe s i s ( 2 0 6 ) . Last et . a l . ,

( 2 0 7 ) have shown that the mRNA AUG-UUU- (A) n synthe s i zes the

peptide met-phe-polylys i ne whi le AUG-UUU-UAA- (A) n which i s

phased t o read the cha in termination codon UAA , d irec ts the

synthe s i s of polylys ine only . S imilar results were obtained

with o ther synthetic mRNA ' s containing termination codons .

These worker s proposed that the absence of met-phe- tRNA in

the supernatant in the exper iment u t i l i z ing AUG-UUU-UAA- (A) n

indicated that hydrolysis of peptidyl-tRNA occurred on the

r ibosome . That i s , the cha i n termination codon , UAA , not

only terminated peptide synthes i s but caused r ibosoma l

hydroly s i s of the completed peptide . Unl ike AAtRNA ' s ( 2 0 8 ) ,

peptidyl -tRNA ' s , e . g . polylysyl-tRNA ( 2 0 9 ) , are re latively

stable in the supernatant of cel l-free systems . Therefore ,

5 5

i t s eems l ikely that hydro lys is o f the ester may occur o n the

r ibosome . Kur iki and Kaj i ( 2 1 0 ) have shown tha t tRNA obta ined

by hydr o ly s i s of peptidyl-tRNA is no longer capable of

accepting new ami no ac id .

Capecchi ( 2 1 1 ) ha s i solated an R 1 7 phage mutant whose

RNA terminates peptide f ormation in its coat protein premature ly .

By contr o l li ng the supply of amino acids to a cel l-free sys tem

d irec ted by thi s mRNA he i so lated a protein , R fac tor , which

5 6

i s neces sary f o r the rej ec tion o f the peptide from r ibosomes .

This R fac tor does not catalyze hydroly s i s of peptidyl -tRNA

i n the absence of r ibosome s . The exi s tence of an R factor

has not been shown in other preparat ions and it is not known

whether it i s of general importance . Bretscher ( 2 1 2 ) has

used Capecchi ' s system to search for a nonsense tRNA . Such

a tRNA i s one which would be c apable of interacting with

the nonsense codon and thereby cause cha in termination . He

demons trated that cha in termination occur s in systems which

are uti l i z ing highly pur i f i ed tRNA ' s and proposes that i t is

very unl ikely that such a nonsense tRNA exists .

B . 6 . The Effect of Tetracyc l ines on C e l l-Free Protein

Synthes i z i ng Sys tems

In an ear ly s tudy of the mode of chloramphenicol ac tion

Rendi and Ochoa ( 2 1 3 ) mentioned , without suppor ting data ,

that oxytetracyc l ine i nhibited amino acid incorporation in

their cell-free system . They were u s i ng a c e l l - free system

directed by synthetic polyr ibonuc leotide s . Franklin ( 2 1 4 )

reported exper iments which showed that chlortetracyc l ine

i nhibited leuc ine incorporation in rat l iver and in E . coli

cell-free systems . Inhibition of the system derived from

E . coli wa s greater than that observed with the sys tem from

rat l iver . C hlortetracyc l ine caused a 9 0 % inhibition of

leuc i ne incorporation in the E . coli system at a concentration

of 4 X l o - 4� . Chlortetracycline d id not affect the formation

of leu-tRNA by e i ther extract but did prevent incorporation

of preformed leu-tRNA into protein . S imilar results were

obtained with oxytetracyc l i ne and tetracyc l ine . Laskin and

Chan ( 2 1 5 ) have shown that c e l l-free systems d irected by

5 7

poly U were i nhibi ted equa l l y b y tetracyc l i ne , chlortetra­

cyc l ine , oxytetracyc l ine and 6 -demethy l - 7 -chlortetracyc l ine

but that 4 -dedimethylamino - 5 -hydroxytetracyc l i ne was only

a bout 1/1 0 as potent . They a l so reported that tetracyc l ine

d i d not affect the formation of phe-tRNA . Suarez and Nathans

( 2 1 6 ) and Day ( 2 17 ) have repor ted s imi lar i nhibi tions of

phe-tRNA i ncorporation . Suarez and Nathans ( 2 1 6 ) reported

that inhibition wa s rever sed by the addi tion of r ibosome s -

poly U but not by the add i tion of phe-tRNA or supernatant

extract . Hierowski ( 2 18 ) has shown that lys ine , proline and

phenylalanine i ncorporation i s inhibited in systems d irec ted

by synthetic polyr ibonuc leotide s . Okamoto and Mi zuno ( 2 1 9 )

and Maxwe l l ( 2 2 0 ) have reported inhibition of cel l-free

sys tems in which incorporation of a lgal protein hydrolyzate

wa s d irec ted by endogenous mRNA . Rif i no e t . a l . , ( 2 2 1 ) have

reported that s ever a l tetracyc l i ne der ivatives are ac tive

inhibitor s of poly U d irected phenylalanine incorporation but

noted that i sochlortetracyc l i ne , an inactive antibacter ial ,

wa s an inhibitor of endog enous mRNA d irected incorporation .

Because of thes e report s i t has been as sumed that tetra­

cycl ines cause a genera l inhibition of amino acid i ncorporation

by c e l l - free systems , Tur ley , Thomas and Sne l l ( 2 2 2 ) have

recently i nvestigated the incorporation of 2 0 d i ff erent amino

acids i nto protei n by a c e l l -free system from E . coli d irected

by endogenous mRNA . They found that i ncorporation of 18 of

these amino acids was inhibited at low leve ls of oxytetracy­

c l ine . Cys te ine and tryptophan incorporation were not

inhibited at low leve l s of oxytetracycl ine and the ir

58

incorporation was s t imul ated at high leve l s of oxytetracyc line .

The authors postulated that this s timulation may be due to

induced codon misreading or that the se amino ac ids might be

incorporated into prote in by a non-ribosomal system .

B . 6 . a . E f fects o f Tetracyc l ines in terms o f the mode l

o f Ce l l-Free Protein Synthe s i s

I nterfe rence with Step l . - B inding o f a 3 0 S subuni t

t o mRNA .

Hierowsk i ( 2 1 8 ) has reported that the inhibition by

tetracycl ines of the incorporation of AAtRNA ' s into prote in

is more pronounced in sys tems in which polyribonuc leotides

serve as the me ssenger than i n sys tems directed by endogenous

mRNA . She noted that tetracyc l ine did not change the Mg ion

optima of these system . Polypeptide synthe s i s directed by

poly C in the presence of chlortetracycl ine ( 4 5� �) was inhibited

by 7 5 % . Les s inhibition was observed when poly U ( 7 0 % ) , poly A

( 5 6 % ) , poly U§A1 ( 5 0 % ) or poly UC ( 2 5 % ) were used as me s sengers .

I t was mentioned that the binding of poly U to ribosomes was

not a ffected by tetracy c l ine . Suare z and Nathans ( 2 1 6 )

f · d h · 1 · · · 1 4c 1 con l rme t l S resu t ln an experlment us lng -po y U .

Connamacher and Mandel ( 2 3 3 ) have also reported that tetracycl ine

did not prevent the formation of ribosome - codon complexe s .

They showed that tetracycl ine was , however , ab le to bind to

poly U in EDTA smlution s . Day ( 2 1 7 ) has reported binding to

5 9

poly U and poly A i n the absence o f Mg ion a s wel l a s a t

severa l Mg ion concentrations . Kohn ( 2 2 4 ) has reported that

metal ions are nec e s sary for binding to DNA . Maxwel l ( 2 2 5 )

ha s recently shown that cultures o f B . megater ium inhibited

by tetracyc l ine are able to form polysome s . The se results

constitute a body o f evidence that tetracyc lines do not inter­

f ere with the attachment of mRNA ' s to r ibosome s , but that the

par ticular mRNA used in a c e l l-free system does af fect the

degree of inhibition produced by tetracyc l ine s .

I nter f erence with Step 2 . - Formation of the initiation

complex .

Exper iments des igned to test the hypothes i s that

tetracyc l ine inhibits the f ormation of the initiation complex

have not been performed . However , exper iments have been

performed which sugge s t that tetracyc line does not interfere

with binding to the peptidyl s ite on 7 0 S r ibosomes .

Gotte sman ( 2 2 6 ) ha s recently stud ied the binding of polylysyl-

tRNA to r ibosome s . I t i s be l i eved that thi s peptidyl- tRNA

e ither b i nd s d irectly to or i s rapidly trans located to the

peptidyl s ite on 7 0 S r ibosome s . Ribosoma l bound polylysyl­

tRNA i s c apable of reacting with puromyc in ( Pm ) to form

polylysyl-Pm ( 2 2 6 , 2 2 7 ) and thi s produc t i s released from the

r ibosome under certain c ircumstance s . Chlortetracyc l ine does

not prevent the bind i ng of this peptidyl- tRNA tm r ibosome s

( 2 2 6 , 2 2 8 , 2 2 9 ) nor does i t prevent the reac tion or release

of bound polylysyl -tRNA with puromyc in . I f r ibosomes with

prebound polylysyl-tRNA are mixed with lys-tRNA in the absence

6 0

o f GTP and superna tant enzyme s , a s ing le lys ine is added to

the polylysyl on the r ibosome ( 1 9 1 ) . Tetracyc l ine prevents

thi s add i tion whi le chloramphenicol does not prevent thi s

s ing le peptide bond formation . S imi larly , r ibosome s containing

prebound N•acetyl-phe-tRNA ( a presumed initiator of poly u d irected systems ) react with Pm and release N-acetyl-phe-Pm

into the supernatant . Chlortetracyc l ine doe s not prevent

e i ther the reac tion o£ the relea s e . Chlortetracyc l ine ,

however , does par t i a l ly prevent the addi tion of phe-tRNA to

r ibosomes conta ining prebound N-acetyl-phe-tRNA . The phe- tRNA

which i s bound to the ribosomes i s able to f orm a d ipeptide

with the N-acetyl-phe-tRNA .

The above results are consi s tent with the general idea

that tetr acyc l ines do not af fect AAtRNA bind i ng to the peptidyl

s ite but rather they inhibi t bind i ng of AAtRNA ' s to the

aminoacyl s ite . D irect evidence for this type of inhibition

is pres ented in the f o l lowing sections .

Interf erence with S tep 3 . - Attachment of 5 0 S subunits

to the Initiation complex .

Thi s s tep ha s not been studied direc tly , but Day ( 2 17 )

ha s shown that i so lated 3 0 S subunits containing bound

tetracyc l ine are able to f orm 7 0 S r ibosomes upon the add i tion

of 5 0 S subunit s .

Suzuka , Kaj i and Kaj i ( 1 6 9 ) and Vazquez and Monro ( 2 3 0 )

have shown that tetracyc l ine prevents the binding o f AAtRNA ' s

to 3 0 S subunit s - codon and a l so prevents the s timulation

of bind i ng observed when 5 0 S subunits are added . Binding of

6 1

AAtRNA ' s t o 3 0 S subuni t s - codon ha s now been shown t o be

non-functiona l unless the AAtRNA i s N-F-met-tRNAf . I t is

gowever , generally agreed that the stimulat ion of binding

observed when 5 0 S subunits are added i s due to the creation

of the ami noacy l s ite .

I t seems unlikely that tetracyc l ines interfere with

step 3 if i ndeed such a s tep is i nvolved in prote in synthesis .

I nterference with Step 4 . - Bind i ng to the aminoacyl s i te

of r ibosome s .

Hierowski ( 2 18 ) u s ed sucrose den s i ty gradient centr ifuga­

t ion to show that binding of phe-tRNA to 70 S r ibosomes-codon

i s partially inhibited ( 6 0 % ) by chlortetracyc l ine . The se

stud i e s were conduc ted at 1 2 mM Mg ion and it is probable that

phe-tRNA i s c apable of enter ing the peptidyl si te as wel l a s

the ami noacyl s ite under the se c ircums tances . Suare z and

Nathans ( 2 1 6 ) have shown that the bind ing of N-acetyl-phe-

tRNA at 2 0 mM Mg ion is inhibited by chlortetracyc l ine . The

maximum inhibition obta i nable at very high tetracycline

concentrat ions wa s 5 0 % . C lark and Chang ( 2 3 1 ) observed s imi lar

i nhibi t ion ( 4 0 % ) of AAtRNA binding to rabbit reticulocyte

r ibosomes i n the presence of the transfer enzyme s . They a l so

showed that tetracyc l ine d id not i nterfere with the puromyc in

reaction in systems synthes i z ing protein . Vazque z and Monro

( 2 3 ) have noted inhibi t ion of poly A directed lys- tRNA ( 5 1 to

6 1 % ) a s wel l a s poly U d irected phe-tRNA binding ( 3 8 to 5 2 % ) .

Because of these reports , i t ha s been genera l ly as sumed

that tetracyc l ine s prevent binding of AAtRNA ' s to only one

s ite on the r ibosome - codon . S i nce these experiments were

carr ied out under condi tions which favor binding to the

ami noacyl s ite , these i nve stigators have proposed that this

6 2

i s the s ite of tetracyc l ine action . The recent reports of

Gottesman ( 2 2 6 ) and Lucas-Lenard and Haenni ( 1 9 1 ) , mentioned

ear l ier , have substantiated thi s conc lus ion . I t should be

noted , however , that Wolfe and Hahn ( 2 3 2 ) were only able to

demons trate a very sma l l inhibition of phe-tRNA binding to

r ibosome s - poly U by sucrese dens i ty gradient centrifugation .

S imilar ly , La skin ( 1 6 ) ha s s tated that he wa s only able to

demonstrate a sma l l inhibition ( 3 0 % ) of thi s binding in

exper iments util i z i ng the technique of c e l lulose ester

f i ltration . He emphas i zed the poor quantitative nature of

these experiments . I t would seem , that the conc lusive

experiment ha s not as yet been performed .

I nterference with Step 5 . - Formation of the peptide

bond .

I n the pre sence of tetracyc l ine , the abi l i ty of prebound

polylysyl-tRNA to react with Pm has been demonstrated by

Gotte sman ( 2 2 6 ) . Thi s result and the abi l i ty 0f prebound

N-acety l-phe-tRNA to react with either Pm or bound phe- tRNA

in the presence of tetracyc l i ne , demonstrated by Luca s-Lenard

and Haenni ( 1 9 1 ) , i nd icates that s tep 5 is not inhibited .

6 3

I nterference with S tep 6 . - Pul sation (Movement) of the

Ribosome .

S i nce i t i s thought that release of Pm - peptides occurs

by norma l ribosomal pul s ation and thi s is not inhibited by

te tracy c l ine , i t is unl ikely that te tracyclines inhibi t this

s tep in protei n synthe s i s .

I nterference with Step 8 . - Chain termination and release .

E f fects o f tetracycl ine on thi s step have not been studied .

B . 6 . b . Binding o f Tetracycl ines to Ribosomes . -

The pre sumed mode of tetracyc line action es tablished by

these exper iments i s that of a d i rect competition with

AAtRNA ' s for the aminoacyl b inding s i te on 70 S ribosomes . I t

i s unl ikely that tetracy c l ine inhibi tion i s due to a n indirect

e f fect caused by binding of tetracycline to some other ribo­

s omal s ite , s ince a l l other ribosomal functions appear normal .

Direct binding to the aminoacyl s i te has , of course , not

been demons trated . Connamacher and Mandel ( 2 2 3 ) have , however ,

shown by equ i l ibrium dialys is and by sucrose dens i ty gradient

centri fugation , that tetracycl ine is bound to 70 S E . coli

and B . cereus ribosome s . When experiments were conducted at

low Mg ion concentrations , the tetracycl ine was largely

associated with the 3 0 S subuni t . Ribosome s prepared from

preincubated extracts , so that endogenous mRNA activity was

low , showed a reduced abi l i ty to bind tetracyc l ine . Las t ,

I z ak i and Sne l l ( 2 3 3 ) were unable to show any binding of

tetracycl i ne to a ribosome sediment produced by centri fugation

in sucrose . Thi s failure may be due to a l ack of endogenous

6 4

mRNA in their preparations or to the ir sed imentation technique

which may require s tronger binding than bind i ng demonstrated

by sucro se density grad ient c entr ifugation . Day ( 2 3 4 ) ha s

substantiated the f i nd ing s of Connamacher and Mandel ( 2 2 3 )

that tetracyc l ine i s a s sociated with the 7 0 S r ibosomes

dur i ng sucrose density gradient centr ifugation . He showed

that tetracyc l ine can bind to e ither 3 0 S or 5 0 S subunits .

Tetracyc l i ne , that i s bound to e ither of the se subunits i s

able t o d i s soc iate and rebind t o the other subunit if i t i s

added . A l l o f the sucros e den s i ty gradient centrifugations

repor ted by Day �ere conducted on r i bosome - tetracyc l ine

migtures that had been subj ected to extensive d ialysis pr ior

to centr ifugation . Day calculated that approximately 1 mole

of tetracyc l i ne is a s sociated with 1 mole of 70 S ribosome s

and that 1 mole of tetracyc l ine i s a s sociated with 2 mo les

of e i ther 30 S or 50 S subunits . He a l so showed that ribosome s

contai ning bound tetracyc l ine were not able to bind phe-tRNA

or incorporate phe-tRNA i nto prote in af ter addition of

supernatant enzyme s . Day ( 2 3 5 ) later showed that the cellu lose

ester f i l tration technique a l so indicated that tetracyc l i ne

bound to r�bosome s .

Recently , Maxwel l ( 2 3 6 ) has shown that large quantities

of tetracyc l ine are found a s sociated with the sediment of

und i a ly z ed r ibosome - tetracycl ine mixtures a f ter high speed

centr i fuga tion . He found about 3 0 0 molecules of rever sibly

a s s oc iated tetracyc l ine per 70 S r ibosome . I t i s d i f f icult

to say if the tetracycl ine a s soc iated with the r ibosomes

under these c ircums tanc e s is the result of binding or of

entrapment . However , if the se sediments were subj ected to

dialysis or an addi tional centr ifugation through sucrose

6 5

and then binding s tudied by sucrose dens ity grad ient

centr ifugation , a sma l l amount of tetracycl ine remained with

the polysome s or r ibosome s . Sedimentation at low Mg ion

concentrations revea led that mos t of the tetracyc l ine was

a s sociated with the 3 0 S subunits .

In the l ight of these experiments , i t seems l ikely that

tetracyc l ine does bind to r i bosome s , probably the 3 0 S

subuni t , and that such binding i s capable of preventing

attachment of AAtRNA ' s to the aminoacyl s ite .

I I I . EXPERIMENTAL

A . Mater i a l s and Equipment

A . 1 . Tetracyc l i ne Der ivativ e s

6 6

The antibiotic s used i n this study were the generous

g if t s of Dr . James H . Boothe , Leder le Laborator ies , Pear l

River , N . Y . , and Dr . Char les R . Stephens , Charles Pfi zer

Laborator i e s , Groton , Conn . . They have been identif ied by

their ultraviolet absorption spectra . The wave lengths of

several absorption maxima and the absorptiv i ty at these

maxima are g iven i n Table I . The spectra were obtained from

solutions buf f ered a t pH 6 . 6 5 with phosphate buffer ( 0 . 2 M) .

Solutions of the samples of 7 -amino - 6-demethyl-6 -deoxy­

tetracyc l ine and 9-nitro- 6 -demethy l - 6-deoxy-tetracyc line had

" shoulder s " in their absorption spectra at approximately

4 0 0 mp . Thes e solutions were separable into two spots by

thin layer s i l ica gel chr omatography . The solvent system for

this chromatography was a mixture ( 2 : 2 : 3 : 3 ) of phosphate

buf f er ( 0 . 2 �' pH 6 . 6 5 ) , ethanol , butanol and ethyl acetate .

It seems l i kely that the se samples contain trace quantities

of 5a ( 6 ) -anhydrote tracyc l i ne s , however , they were used without

further puri f ication .

A . 2 . Escherichia coli

E scher ichia c o l i W wa s obtai ned as a frozen pa ste . 2 I t

was cultured over several growth cyc les in broth and on agar

plate s . An i solate from a s ing le colony was used to inoculate

2Genera l B iochemica ls , Chagrin Fal l s , Ohio .

Tab le I . - The Tetracyc l ines .

Tetracyc l ine Ana loga pKa Va luesb Ultraviolet Abs orpt ion Spectrac

max .'1 m}l Abs orpt ivity x lo-4

7 -N itro-dm-do-tetracyc l ine 360 , 2 6 5 l . 3 , 1 . 6

7 -Chloro-dm-tetracyc line 3 . 3 , 7 . 3 , 9 . 3 ( 2 3 7 ) 3 6 7 , 2 7 5 1 . 4 , 1 . 5

7 -Chlorotetracyc l ine 3 . 3 , 7 . 4 , 9 . 3 ( 2 3 7 ) 368 , 2 7 5 l . l , 1 . 4

Tetracycl ine 3 . 3 , 7 . 7 , 9 . 5 ( 2 3 7 ) 3 58 , 2 7 5 1 . 5 , 1 . 5

5 -Hydroxytetracyc line 3 . 6 , 7 . 4 , 9 . 1 ( 2 38 ) 3 5 5 , 2 7 5 1 . 4 , 1 . 5

7 -Amino-dm-do-tetracyc l ine 400 , 340 , 2 7 8 0 . 4 , l . l ,

9-Amino-dm-do-tetracycl ine 344 , 2 8 2 l . 3 , 1 . 2

drn-do-tetracyc l ine 3 . 5 , 7 . 9 , 9 . 7 ( 17 ) 348 , 2 7 3 l . 3 , 1 . 7

a . dm-do- is 6-demethy l-6-deoxy - , while dm- is 6-demthyl-6-epihydroxy- . b . Numbers in parenthes is indicate l iterature referenc e . c . Values g iven were obta ined in 0 . 2 M phosphate bu f fer , pH 6 . 6 5 �

a Mode l DK�2 Ratio Recording Double Beam Spectrophotometer , Beckman Instruments , Palo Alto , Cal i f . was used .

1 . 4

0'\ -...)

Tab le I . - ( c ent . )

Tetracyc l ine Ana log pKa Va lues

7 -Brorno-drn-do-tetracyc l ine

9-Nitro-drn-do-tetracyc l ine

4 -Dedirnethy l arnino-tetracyc l ine 5 . 97 , 8 . 56 ( 1 3 )

9 -D irnethy larnino-drn-do-tetracyc l ine

5a ( 6 ) -Anhydrotetracyc l ine

1 2 a-Deoxytetracyc l ine

Tetracyc l ine rnethiod ide 3 . 56 , 7 . 80 ( 1 3 )

5a ( l la ) -Dehydroc nlorotetracyc l ine

7 -Chloro- i s otetracyc l ine 3 . 9 , 6 . 7 , 7 . 9 ( 2 3 8 )

2 -Cyano- 2 -decarboxarnidotetracJClme

Ultraviolet Absorpt ion Spectra

max . rnu Absorpt ivity x lo-4

3 50 , 2 7 2 0 . 7 ' 1 . 4

4 1 5 , 3 54 ' 2 8 5 0 . 4 , 0 . 7 , 1 . 1

360 , 2 7 2 l . l , 1 . 3

34 6 , 280 0 . 8 , 0 . 7

4 2 5 , 2 68 1 . 2 , 5 . 0

4 5 0 , 3 3 0 , 2 6 5 0 . 2 , l . 3 , 1 . 7

3 5 0 , 300 , 2 7 5 l . 6 , 1 . 4 , 1 . 6

408 , 2 7 0 1 . 1 , 2 . 1

344 , 288 0 . 3 , 1 . 7

360 , 2 7 5 0 . 9 , 1 . 0

0"1 (X)

a nutrient agar s lant . This s l ant was s tored at 3 ° C and

maintained by monthly trans fers . I t served as the source

of the tes t organ i s m . For experiments in dextrose-salts

broth another s lant was prepared from organisms which had

grown through several cycles in this broth .

A . 3 . Culture Broths

6 9

The culture broth used for activity determinations was

a high peptone media containing in a volume of l L . , 40 g .

casein hydrolysate ( GB I # 2 0 ) 2 , 1 5 g . yeast autolysate ( GBI # 1 2 0 ) 2 ,

3 g . K2 H P043 and 0 . 7 g . KH2 Po4 . 3 The pH o f thi s broth was

constant throughout the experiments at 6 . 6 5 . The divalent

cation content of the broth was estimated to be equivalent

to about l o - 3 M calcium ion by means of a divalent cation

e lectrode . 4 For exper iments at pH 6 . 2 or 7 . 4 , the broth

contained in addition to the peptones either 0 . 5 g . K2HP04 and

2 . 5 g . KH2Po4 or 2 1 . 8 5 g . K2 HP04 and 1 . 7 g . KH2P04 , respective ly .

S imilar divalent cation concentrations were observed in the se broths .

The dextrose-salts broth ( pH 6 . 7 ) contained in each l iter ,

1 0 g . dextrose , 3 g . (NH4 ) 2 so4 , 6 g . K2HP04 and 8 g . KH2P04 .

A l l broths were f i l tered through ce l lulose ester membranes 5

( 0 . 2 2 p dia . pores ) and autoclaved prior to use .

3Al l unspeci fied chemicals were analytical reagent grade .

4orion Re search I nc . , Cambridge , Mas s . 0 2 1 3 9

5Mi l l ipore Corporation , Bed ford , Mas s .

A . 4 . Cul ture Turbidity and Colorimetry

A d i f f raction grating color imeter6 equipped with a red

sensitive phototube in conj unction with matched l/2 inch

square cuvettes was used . Culture turbidity was measured

at 6 5 0 mp . Absorbance o f orcinol reaction mixture s was

measured at 6 5 0 mp whi l e that of Folin-Lowry reactions was

measured at 7 5 0 mp .

A . 5 . Total Ce l l Counts

An electronic particle counter7 was used . The counter

was equipped with a 3 0 p orifice and two threshold pulse

70

height analy zers . Ins trument s ettings whi ch gave satis factory

results were :

1/ Aperture Current , 0 . 7 0 7 ; 1 /Amp l i fi c ation , l/4 ;

Matching Switch , 3 2 H ; Gain , 1 0 0 ; Lower Thre shold , 1 0 and

Upper Thre shold , o f f . These settings were es tab l ished by s i ze

frequency analy s i s of latex suspens ions o f known particle s i ze

as wel l as suspensions of E . col i W .

Cel l s were counted in a s a l ine - formal dehyde solution

which had been f i l tered to remove particulate contamination .

Background counts in thi s fluid were 2 0 0 to 6 0 0 partic les

per 50 l ambda . The formaldehyde concentration in the counting

fluid was e i ther 0 . 2 or 1 % . The h i gher concentration gave

constant values for total ce l l s over a 2 4 hour period whi l e

6 spectronic 2 0 , Bausch and Lomb , Roches te r , N . Y .

7coulter Counter Mode l B , Coulter E lectronics , Inc . Hialeah , F l a .

the lower concentration a l lowed some growth ( 1 to 5 % ) in

the f ir s t ten minutes a f ter samples were removed from the

culture .

B . Methods

B . 1 . Escherichia c o l i Growth

7 1

A broth culture was a l lowed t o grow for 1 2 hour s at

3 7 . 5 °C . A d i lution of this cul ture , whose growth was

measured turbidimetr ica l ly , wa s a l lowed to grow into the log

phase . When a concentration of 1 x 1 0 8 organisms/ml . wa s

obtai ned , 2 ml . samples were used to i noculate nine replicate

1 0 0 ml . volume s of broth conta ined in loosely capped 5 0 0 ml .

f la sks . The f la s k s were sfuaken i n a constant temperature

water bath8 at 3 7 . 5 ° C . Freshly prepared antibiotic solutions

were added i n 1 ml . volumes to 8 of the 9 repl icate cultures

af ter 2 , 7 0 0 s econds of growth .

Exper iments d e s igned to s tudy time of mnset of inhibition

were performed with culture s of e ither 2 0 0 ml . per 1 L . f la sk

or 3 5 0 ml . per 2 L . f la s k .

B . 2 . Viable Count Method

One ml . sampl e s of the cultures were obta ined at 9 0 0 or

1 , 8 0 0 s econd i nterva l s . A minimum of 8 samples was obtained

from each culture . Sample s were d iluted with ster i l e 0 . 9 %

sal ine so lution so that 1 0 0 to 2 0 0 organisms/ml . would result .

One ml . of this aaspension wa s pipetted onto each of 5 agar

plate s . Agar plates had previou sly been made by add i ng

8American Instrument Company , S i lver Spring , Md .

approximately 1 5 ml . of s terile mel ted Peptone-Casein Agar

U . S . P . 9 to s terile plastic petri dishes ( 1 0 0 x 15 mm . ) .

After spreading the bacterial suspension over the surface of

the plate , approximately 5 ml . o f warm agar was added . When

the agar had solidified the p l ates were incubated at 3 7 . 5 ° C .

The colonies which were visible a fter 3 6 to 4 8 hours of

incubation were counted with the aid o f an e lectronic

reg i s ter . 1 0 The mean number of colonies v i s ible on the

5 plate s from e ach s ample was used to calculate the number

o f v i ab le organisms in the original culture .

B . 3 . Total Count Method

One ml . culture s amples were obtained at the same time

7 2

a s the s ample s ased for viab le c e l l counts . They were placed

in· bottles containing appropriate volume s of the s al ine -

formaldehyde counting f luid so that a concentration o f 1 0 , 0 0 0

to 3 0 , 0 0 0 organisms per 5 0 l ambda was obtained . The mean o f

.4 counts per s amp l e was corrected for background noise , but

not coincidence and used to calculate the total number of cel l s

i n the original culture .

B . 4 . Nuc leic Acid and the - P rotein Determinations

Appropriate volumes of cul ture ( 0 . 5 to 25 ml . ) were f i l tered

under vacuum through 25 mm . diameter cel lulose e s ter membranes

( 0 . 2 2 )1 di a . pores ) which had previous ly been treated to

extract detergent . Culture s amples were obtained at times

s imi lar to those at which viable and total cel l counts were made .

9 Fisher Scienti fic , Fairlawn , N . J . l O 0 0 1 C I t t 0 0 0 f f 1 N Y Amerl can Optlca ompany , ns rumen DlVlS lon , Bu a o , . .

7 3

I t wa s necessary t o r emove the detergent impregnated

in these membranes ( 2 3 9 ) s ince i t reacted with the orc i nol

reagent a nd gave high blank value s , if not removed . Detergent

extra c t ion was accomp l i shed by suspendi ng f i lters in cold

d i s t i l led water ( 4 L . per 1 0 0 f i lter s ) and then boi l i ng the

suspens ion for 1 5 m i nute s . Thi s proc e s s was repeated 3 t imes

w i th f r e sh cold water . F i l ters treated i n thi s manner were

s tored i n d i s t il led water for t ime s no longer than 4 8 hour s .

These f il ter s were able to r e t a i n E . coli qua n t i ta tive ly and

gave m i n imal a nd constant blank reactions with the orc inol

reagent .

Af ter culture samples were collec ted on the f ilter s ,

the r e t a i ned bacter i a wer e wa shed with 0 . 9 % s a l i ne solution

under v acuum . The washed s amples were prec ipita ted by

layer i ng , without v acuum , 2 ml . of t richloracetic acid (TCA)

over the f il ter s for 5 minute s . TCA soluble material was

r emoved by app l ic a tion of vacuum a nd 2 ml . of 9 5 % ethanol

l ayered over the f i lters for 5 m i nu te s . After remova l of the

ethanolic extrac t , the f i lters with retained nuc leic acids

and proteins wer e placed i n screw cap test tubes . Two ml . of

5 % TCA was added a nd the tubes were placed i n a boi l i ng water

bath for twe nty minutes .

Nuc leic Ac id DeterminatieftB

One ml . a liquot s of the hot-TCA soluble mater ial were

r emoved from the cooled tube s . Three ml . of the orc i no l

reagent wer e added a nd after heat i ng i n a boi li ng water bath

for 2 0 m i nutes , the absorption at 6 5 0 � of the cooled solution

was mea sured . This absorption wa s corrected for that

produced when an equal volume of s teri le broth wa s appl ied

7 4

to the f ilter s instead o f culture . I t was neces sary to prepare

separate blank reactions for each lot of f il ter s and each lot

of broth . The orc i no l reagent wa s pur if ied , mixed and used

accordi ng to the method s of Schneider ( 2 4 0 ) . It i s a

solution of recrysta l l i z ed orc inol ( 0 . 6 6 % ) , FeC l 3 ( 0 . 3 3 % ) and

5% TCA in concentrated hydrochloric acid .

The quantity reported a s " Nuc leic Ac id s " i s the corrected

absorbance due to a 1 2 . 5 ml . culture sampl e in an orc inol

reac tion volume of 4 ml . S ince only the logari thm of a

quantity proportional to nuc leic a c id concentration was needed

to calculate g eneration rate constants the absorbance was

used directly rather than a proportiona l quantity obtained

by r eference to a s ta ndard curve . A s tandard curve for the

a s say procedure was prepared by f i lter i ng var iou s volume s of

cultur e . An example is shown in F ig . VI I I . I t and other

standard curves prepared with E . c o l i extr ac ts were l inear

throughout the tested rang e s . Standard curve s were also

prepared with highly polymer i zed yea s t RNAl l and also with

s a lmon sperm DNAl l . They were found to be l inear throughout

the tested conc entration range (as high as 6 0 mcg . /ml . of

e ither RNA or DNA) . The RNA had an absorptivity of 0 . 0 2 3 em .

2/mcg . and a negative i ntercept of 0 . 0 4 2 whi le the DNA had

an absorptivity of 0 . 0 0 4 cm . 2/mcg . Mixture s of DNA and RNA

produced absorbanc es equivalent to those ca lculated from the

l lcalbiochem , Box 5 4 2 8 2 , Los Angel e s , California

7 5

g., 0 0 . 6-Lf) r--

0 0 . 5 -

g., 0 0 . 4 -Lf) 1.0

• 0 . 3 -

+l cU

Q) 0 . 2- • •NUCLE IC AC IDS•

() !=! 0 •PROTEI N S • cU ..0 D . l-)...! 0 Ul ..0 �

l 2 3 4 5

Volume o f C e l l s F i l tered , ml .

F igure VI I I . Cal ibration Curves for " Nuc l e ic Ac id s "

and " Prote i n " Determinations . The amounts o f

"Nucleic Ac ids " o r " Protein" found b y the a s says

are l inear ly related to the amount of culture

f i l tered .

7 6

sum o f the two Beer ' s law equations .

Maa �e and K j eldgaard ( 2 4 0 ) have estimated the DNA : RNA

ratio i n c e l l s a s a function of growth rate . They f ind this

ratio to be about 1 : 1 0 in rapidly dividing c e l l s . While in

s lowly d ividing c e l l s the ratio of DNA : RNA was reduced to

about 1 : 4 . U s ing the se f igur e s and the absorptivities for

yeas t RNA and salmon sperm DNA g iven above one can ca lculate

that the contribution of DNA to the quantity cal led " Nuc leic

Acids " in thi s work rang e s from about 2 to 5 % . S ince thi s

i s the case it might have been more proper t o report the

results a s " Ribonuc leic Acid s " rather than " Nuc leic Ac ids "

but s ince there i s a contribution to thi s quantity by DNA in

thes e exper iments the more g eneral term wa s preferred .

Another reason for preferring this term i s that in tetracyc l ine

i nhibited c e l l s the DNA : RNA ratio might be dif ferent than

that found in norma l c e l l s . v � The results of Cerny and

Habermann ( 3 3 ) , however , indicate that thi s ratio does not

change very muc h .

Prote in Determinations

The TCA solution was completely removed from the tube s

containing the f ilter s , by vacuum aspiration . The f i lter s

with retained protein precipitate were covered with 1 . 5 ml�

of a trypsin solution- ( 1 8 ) 10 mcg . /ml in 0 . 0 0 5 � NH40H and

0 . 0 5 � (NH4 ) 2C03 - . The suspended f i l ter s were stirred

vigorou s l y and a llowed to digest for 2 hours ( the same results

were obtained af ter a s much a s 48 hour s of digestion) . I t wa s

7 7

nece s sary t o utili z e tryps in digestion rather than s trong

a lka l i , because the c e llulose e s ter membrane s were hydro­

l y z ed and gave very high b la nk reactions in the pre sence of

s trong alka l i . The resulting solution wa s u sed a s a sample

for protein determination by the Folin-Lowry method . The

r eagents were added directly to the tubes and the absorbance

of the colored solutions mea sured at 7 5 0 rnp . B lank reac tions

were prepared by f i l ter ing s teri le broth and u sed to correct

the absorbance of the samp le s . The Fol in-Lowry reagents were

prepared , standardi z ed and used according to the methods of

Layne ( 24 2 ) . A solution of cupric sulfate ( 0 . 0 1% ) , sodium

tartrate ( 0 . 0 2 % ) and sodium carbonate in 0 . 1 N NaOH was added

to the prote i n solution ( 5 ml . per tube ) and a f ter 10 minutes ,

0 . 5 m l . of 1 . 0 N Folin-C iocal teu phenol reagentl 2 was added .

The quantity reported as " Prote i n " i s the corrected

absorbance due to a 2 5 ml . sample of culture in a f inal

reaction volume of 6 . 5 m l . A s in the previous ly mentioned

case of " Nuc leic Ac ids " absorbance wa s used directly rather

than a proportional quant i ty obtained from a calibration

curve . A standard curve for the a s say procedure wa s prepared

by f il ter ing various volnmes of E . coli culture s . An example

is shown in F ig . V I I I . I t and other s tandard curves prepared

with E . coli extract s were linear throughout the tested range s .

I t i s , however , we l l known ( 2 4 2 ) that the Folin-Lowry reaction

1 2wi l l Scientif ic I nc . , Baltimor e , Md . 2 1 2 2 4

7 8

-�oe s not a lway s fol low Beer ' s law . Standard curves were also

prepared with bovi ne serum albuminl l and pancreatic trypsin . ll

Adherence to Beer ' s law wa s observed i n the case of the a lbumin

i n the tested concentration range (as high as 45 mcg . /ml . )

whi l e Beer ' s law was only followed for tryp s in concentrations

of l e s s than 2 5 mcg . /ml . The a lbumin had an absorptivity of

0 . 0 0 8 cm . 2/mcg . whi l e the initial absorptivi ty of the trypsin

was 0 . 03 4 cm . 2jmcg . The dec i s ion to uti l i z e the absorbance

of Folin-Lowry reaction mixtures d irectly in the calculation

of the term cal l ed " Protein" wa s based , therefore , f ir stly

on the fact that the avai lable standard curves with E . coli

extracted by the exper imental method were l inear and secondly

on the rea l i zation that conversion of absorbance to concentra­

tion of some s tandard protei n would be val id only if the

s tandard protei n were the same , in respect to non-linear i ty of

absorbance and absorptiv i ty , as the experimental ly measured

proteins . Such standard proteins were not avai lable .

IV . RESULTS AND DISCUSS ION .

A . E ffect o f Tetracy c l ines on Rate Cons tants for Ce l l

Divis ion .

I t has previou s l y been s hown ( 2 2 ) that the e f fect o f

7 9

low concentrations o f tetracy c l ine on an exponentially dividing

culture is to cause a reduction i n the generation rate constant ,

that i s :

[ N ] Eq . ( 1 )

When the t ime , t , i s greater than 3 0 minute s after ant ibiotic

add i t ion and where [N° ] is the concentration o f cells in the

culture 3 0 minutes a fter antibiotic add i tion , N is the con­

centration of ce l l s at any later time , ked is the generation

rate cons tant for ce l l d iv i s ion in the presence of antibiotic

and k�d is the generation rate cons tant i n the absence o f

antibiot i c . Generation rate con s tants f o r c e l l divis ion ,

ked ' o f cultures i nh i b i ted by 1 8 tetracycl ines have been

measured by v i able , kv , and by tota l , kt , c e l l count methods .

Typical results o f the s e exper iments are shown in Fig . I X and

F i g . X . These exper iments can a l l be quanti tative ly de scr ibed

by Eq . ( 1 ) . Generation rate constants obtained from the f i t

o f t h i s exper imental data t o Eq . ( 1 ) as we l l as data t o be

presented l ater are given in Tables I I and I I I . Te sts for

the f i t of e xperimental data to Eq . ( 1 ) were found to be highly

s igni f icant in cases where data obtained at time s greater than

30 minutes but l e s s than 4 hours after antibiotic addit ion

were used .

8 0

'J, -

6 -.--l E -......

\D 5 - • I 0 .--l

:><: 4 -UJ E UJ • +

-� 3 - �ICH,Ia s:: 0-(lj � co� tJ> 0 0 )..j 0

2 - AN TIBIOTIC COliC . •

Q) W/L X 10 o.oo 0 s::

...:I 2.34 • 1 - 4.87 •

7.01 ... • . 34 •

l 2 3 4

T ime , hr .

F igure I X . An Example of Viable Cell Generation Rate

Curve s . The logarithm of the number of viable cells a s

a func tion of time of growth in culture s with var ious

concentrations of 9-Nitro-dm-do-tetracyc l ine . The

s lope s , kv , are g iven in Table I I I .

6 -

,.., 5 -E1 ""-.

\!) I 0 ,.., 4 -X rn + E1 rn 3 -

���·

·rl c

OHHa Cll 01 0 0

lo-l 0 2 - AH TIBIOTIC CONC. ___.

M/L )( 1) 6 Q) 0.00 0

c H 2.34 ...

l- 4.87 e 7.01 A 8. 34 •

l 2 3 4

Time , hr .

F igure X . An Example of Tota l C e l l Generat ion Rate

Curve s . The logar i thm of the total number of c e l l s a s

a func tion o f time of growth in cultur e s with var ious

concentrations of 9 -N itro-dm-do-tetracyc l i ne . The

s lopes , kt , are g iven in Table I I I .

8 1

Tab le I I . - F irst Order Generat ion Rate C onstants ,

- 1 4 sec . x 10 , for E . c o l i W Cu ltures Inh ibited by

var ious Tetracyc l ine Ant ib iotic s , M x 106 .

M

Tetracyc l ine

0 . 000 5 . 57 5 . 8 5

0 . 599 3 . 80 4 . 4 6

0 . 898 3 . 2 3 2 . 97

1 . 198 2 . 27 1 . 96

1 . 4 9 7 1 . 4 9 1 . 7 6

1 . 7 96 1 . 10 1 . 2 5

7 -Chlorotetracyc l ine

0 . 000 5 . 4 1 5 . 94

0 . 24 6 4 . 8 2 4 . 5 1

0 . 4 9 2 3 . 48 3 . 3 3

0 . 7 88 2 . 3 3 2 . 17

0 . 98 5 2 . 16 1 . 84

1 . 2 3 1 0 . 5 2 0 . 9 3

8 2

5 . 07

3 . 8 5

2 . 89

2 . 10

1 . 4 5

1 . 2 7

5 . 80

4 . 97

3 . 5 3

2 . 14

2 . 16

0 . 5 1

Tab le I I . - ( c ont . ) 8 3

M kv kp kna -, 5 -Hydroxy tetracyc l ine

0 . 000 5 . 7 5a 5 . 7 9 5 . 97

0 . 5 3 7 4 . 7 1 3 . 4 9 3 . 7 5

0 . 87 8 4 . 04 3 . 4 7

0 . 87 8 3 . 7 5b

1 . 07 3 2 . 2 5 2 . 24 2 . 14

1 . 3 7 6 2 . 2 3 2 . 24 2 . 4 0

1 . 3 7 6 2 . 2 0

7 -Chloro-dm-tetracyc l inec

0 . 000 5 . 4 2 5 . 0 2 5 . 90

0 . 2 2 1 4 . 99 4 . 4 1 5 . 2 6

0 . 44 1 3 . 37 3 . 0 5 3 . 68

0 . 7 06 1 . 4 6 1 . 1 3 1 . s o

0 . 88 2 0 . 5 5 0 . 68 1 . 06

dm-do-tetracyc lined

0 . 000 5 . 89 5 . 0 5 5 . 2 5

0 . 97 8 4 . 58 4 . 5 5 4 . 7 7

1 . 9 5 5 3 . 6 5 3 . 4 2 3 . 50

2 . 97 2 2 . 7 6 2 . 57 2 . 4 7

3 . 9 10 1 . 4 6 1 . 5 7 1 . 6 5

4 . 888 0 . 84 1 . 07 0 . 97

a . An average va lue , determined on severa l d i fferent days .

b . Values of kt which were used in tne c a lcu lat ion of the v iab le inh ibitory rate c onstant , k� .

c . dm- is 6-demethy l-6-ep ihydroxy- . d . dm-do- is 6-demethy l-6-deoxy- .

Tab le I I . - (cont . )

M e kp kt k n a

7 -Bromo-dm-do-tetracyc l ined

0 . 000 5 . 8 9 5 . 4 1 5 . 8 9

2 . 4 68 4 . 2 9 3 . 7 8 3 . 5 2

4 . 9 38 2 . 7 0 2 . 59 2 . 2 9

7 . 406 l . 26 l . s o l . s o

9 . 87 6 0 . 8 5 0 . 5 5

7 -Ch loro- i s otetracyc l ine

0 . 000 5 . 7 1 5 . 2 6 5 . 40

16 3 . 4 9 5 . 8 1 4 . 74 5 . 3 9

3 2 2 . 30 5 . 7 1 4 . 86 5 . 5 3

l 2a-Deoxytetracyc l ine

0 . 000 5 . 7 1 5 . 26 5 . 40

7 9 . 14 3 . 90 3 . 6 9 3 . 59

1 5 6 . 0 3 l . 59 l . 57 0 . 9 5

Sa ( l la ) -Dehydrochlortetracyc l ine

0 . 000 5 . 7 1 5 . 26 5 . 40

1 5 0 . 68 5 . 2 3 4 . 26 4 . 5 5

2 97 . 06 4 . 64 4 . 0 3 4 . 66

2 -Cyano-2 -decarboxam ido-tetracyc l ine

0 . 000 5 . 7 1 5 . 2 6 5 . 40

3 3 . 16 5 . 94 4 . 92 5 . 44

1 28 . 88 5 . 88 4 . 6 3 4 . 7 2

e . Va lue s o f kt were obta ined with 0 . 2 % HCHO in the d i lut ing s olut ion , see Experimenta l .

8 4

Table I II . - F irst Order Generat ion Rate Constants , sec- 1 x 104 , for E . c o l i w .

Cu ltures Inhibited by Tetracyc l ines , M X 106 , and the Fract ion of Viable to

Tota l C e l l s Present at the Start , el , and at the End , e5 of the Experiments .

kv ka

kp k b b M t na e l e5

7 -Amino-dm-do-tetracyc l inec

0 . 000 5 . 84 5 . 87 5 . 4 6 5 . 9 3 0 . 84 0 . 8 1

0 . 4 9 3 4 . 9 3 4 . 80 4 . 3 2 4 . 7 2 0 . 80 0 . 96

0 . 98 5 3 . 7 1 3 . 8 3 3 . 36 3 . 66 0 . 7 5 0 . 6 3

1 . 4 7 8 2 . 98 3 . 0 5 2 . 74 2 . 7 7 0 . 68 0 . 62

l . 97 1 2 . 4 1 2 . 4 1 l . 96 2 . 02 0 . 7 2 0 . 7 1

a . Va lues of kt · we re obta ined with 0 . 2 % HCHO in the d i luting s olut ion , see Exper imenta l .

b . e was c alculated from Eq . ( l ) us ing the va lues ked and N° , obtained by regress ion ana lys is of the orig ina l data .

c . dm-do- is 6-demethy l-6-deoxy- .

co lJl

Tab le I I I . - ( c on t . )

M kv ka t

9-Amino-dm-do-tetracyc l ineC

0 . 000 5 . 84 5 . 87

0 . 474 5 . 27 5 . 27

0 . 94 8 4 . 06 4 . 59

1 . 4 2 1 3 . 7 9 3 . 7 3

1 . 89 5 3 . 06 -

5a ( 6 ) -Anhydrotetracyc l ine

0 . 000 5 . 82 5 . 6 2

4 . 4 7 7 5 . 00 5 . 4 6

8 . 9 5 3 4 . 38 4 . 67

1 3 . 4 30 4 . 12 3 . 88

17 . 907 - 3 . 32

kp kn a

5 . 4 6 5 . 9 3

4 . 6 1 4 . 86

3 . 9 1 4 . 08

3 . 38 3 . 44

3 . 02 3 . 17

5 . 40 5 . 90

4 . 70 4 . 90

3 . 7 9 3 . 87

3 . 2 5 3 . 4 5

2 . 56 2 . 7 7

e£

0 . 84

0 . 74

0 . 87

0 . 7 2

0 . 9 1

0 . 69

0 . 7 9

0 . 64

eg

0 . 8 1

0 . 7 4

0 . 40

0 . 80

1 . 2 1

0 . 3 5

0 . 52

0 . 90

OJ 0'\

Table I II . - ( c ont . )

M kv k� 7 -Nitro-dm-do-tetracyc l inec

0 . 000 5 . 8 2 5 . 62

0 . 1 1 1 5 . 68 5 . 50

0 . 2 2 2 4 . 20 4 . 7 5

0 . 3 3 3 3 . 17 3 . 8 2

0 . 444 2 . 4 5 2 . 8 2

9 -Nitro-dm-do-tetracyc l inec

0 . 000 6 . 10 5 . 86

2 . 3 3 7 4 . 7 7 5 . 37

4 . 67 4 3 . 9 3 4 . 11

7 . 0 1 1 2 . 9 3 2 . 96

9 . 348 l . 9 5 2 . 02

kp kna

5 . 4 0 5 . 90

4 . 86 5 . 44

3 . 88 4 . 32

3 . 1 1 3 . 5 2

2 . 19 2 . 59

5 . 8 1 6 . 14

5 . 07 5 . 1 5

3 . 48 3 . 54

2 . 8 2 2 . 55

l . 9 2 1 . 8 5

e�

0 . 9 1

0 . 67

0 . 74

0 . 7 2

1 . 00

0 . 74

0 . 97

0 . 68

0 . 8 2

0 . 69

e�

l . 2 1

0 . 8 7

0 . 3 3

0 . 28

0 . 6 1

1 . 0 5

0 . 4 1

0 . 5 3

0 . 7 7

0 . 62

00 -..J

Table I I I . - ( c ont . )

M kv It� kp kna �f .eb 5

4 - Dedimethy lamino-tetracyc l ine

0 . 000 6 . 10 5 . 86 5 . 8 1 6 . 14 0 . 74 1 . 0 5

3 . 0 30 5 . 2 7 5 . 39 4 . 6 3 5 . 0 1 0 . 7 9 0 . 67

6 . 060 4 . 3 2 4 . 50 3 . 96 4 . 3 0 0 . 8 0 0 . 53

9 . 091 3 . 4 3 3 . 80 3 . 58 3 . 5 1 0 . 86 0 . 50

1 2 . 12 1 3 . 17 3 . 12 2 . 97 3 . 04 0 . 6 5 0 . 7 0

9-Dimethylamino-dm-do-tetracyc l inec

0 . 000 6 . 2 5 6 . 09 5 . 4 5 5 . 4 0 0 . 66 0 . 8 3

3 . 4 6 7 5 . 60 5 . 7 1 5 . 0 1 5 . 00 0 . 87 0 . 74

6 . 9 3 5 4 . 32 4 . 69 3 . 92 3 . 99 0 . 7 0 0 . 4 1

10 . 40 2 3 . 34 3 . 4 7 3 . 0 9 3 . 5 1 0 . 60 0 . 50

1 3 . 87 0 2 . 8 3 2 . 94 2 . 4 0 2 . 56 0 . 74 0 . 6 3

Tetracyc l ine methiod ide

0 . 00 6 . 2 5 6 . 09 5 . 4 5 5 . 4 0 0 . 66 0 . 8 3

6 3 . 4 8 4 . 87 5 . 14 4 . 0 3 4 . 2 6 0 . 8 3 0 . 56

1 2 6 . 9 5 3 . 02 3 . 39 2 . 7 8 2 . 9 1 0 . I l 0 . 4 2 OJ OJ

Generation rate constants for cell divis ion have been �

obtained by two d i f ferent methods , by viable counts , kv , and

by tetal c e l l counts , kt . I t can be seen in Table I I I that

rate constants for mos t cultures obtained by the two methods

are apparently identical , within experimental error .

However , i n these cultures the concentrations o f cel l s

a t any time as determined b y the two methods were quite

d i f fe rent . This i s shown in Fig . XI for cultures inhibi ted

by 7 - amino - 6 - demethyl - 6 -deoxy-te tracycl ine . S imilar results

were obtained with the other tetracycl ines tes ted by both

methods . The fraction of viable ce l l s to total cel l s , e ,

8 9

would be 1 i f there was no d i f ference between the concentration

of c e l l s measured by the two methods . This f raction , � ' has

been calculated f rom the regre s s ion f i t of the experimental

data to Eq . ( 1 ) for the tetracy c l ines tested by the two

method s . Values o f e at two cul ture growth time s : e1 , 9 0 0

s econds a f ter antibiotic addi tion and 85 , 1 5 , 3 0 0 seconds

a fter antibiotic addition , are given in Table I I I . I t

should be noted that values o f e calculated in this manner

are very s en s i tive to sma l l d i f ference s in generation rate

constants . Despite thi s sens i t ivity , € remains relatively

cons tant in mos t cultures throughout the time o f antibiotic

i nhibi t ion . I n those cases where i t does vary , i t doe s not

appear to be con s i s tently related to antibiotic concentration .

The average value of e in these cultures i s about 0 . 6 . At

higher antibiotic concentrations when generation rate cons tants

obtained by the viable count method are negative , thi s fraction

1

6

..-; 8 ......_ I.D I 0 ..-;

� en 8 en ·ri � cU 0> � 2 0 ......,

Q) � ...:<

F igure XI .

+

��· ANTBOTIC CONe. COUIT METHOD

lot/L X 106 TOTAL VIABLE o.oo 0 ... o.ee e A 1 .9 7 • 0

Time , hr .

Examples of the Difference Between Total

and Viable C e l l Generation Rate Curve s . The logarithm

9 0

o f either the total number o r the number of v iable cells

a s a func tion of t ime of growth in culture s with var ious

concentrations of 7 -Amino-dm-do-tetracyc l i ne . The

s lope s , kt or kv , are g iven in Table I I I .

9 1

has value s o f 0 . 1 o r 0 . 0 1 . I t i s not pos s ible in the present

work to conclude w i th certainty that the tetracycl ines are

acting solely a s growth inhibitors and not as both

bacteriostatic and bactericidal agents . However , i f

there i s a n antibiotic induced cell death , i t is not large .

Rather i t seems mos t l ikely that the d i f ference in cell

concentrations obtained by the two me thods i s caused by the

format ion o f s ingle colonies by more than one viable cell .

I nclusion o f a sur factant in the viable cell di luting solution

did not , howeve r , greatly a ffect this fraction . The use of

the higher formaldehyde concentration in the di luting fluid

for total counts made thi s fraction , e , more cons tant .

Garrett and Brown ( 2 2 ) and Garrett , Mi ller and Brown

( 2 4 3 ) have observed that generation rate constants for cell

divi s ion obtained in the presence o f tetracycl ine obey the

re lationship :

i f f kcQ>O Eq . ( 2 )

I whe re ked may be e i ther kv or kt , ked i s the inhibitory rate

cons tant for ce l l divis ion and [ Te l is the antib iotic

concentration . This equation was obtained by simpl i f ication

of a more general relationship :

Eq . ( 3 )

where K i s a cons tant . The der ivation o f the se equations

will be pre sented in a later section of this the s is .

The generation rate cons tants obtained in the pre sent

work can be described by Eq . ( 2 ) but not by Eq . ( 3 ) . Typ ical

' , ,

examples o f the f i t o f experimental data to thi s expre s s ion _ ,

are shown in Figures XI I , X I I I , XIV and XV . Values o f the

I inhibi tory rate con s tan t , ked ' obtained by app l ication o f

regres s ion analy s i s t o the experimentally determined

generation rate con s tants and the antibiotic concentrations

in the cultures are given in Table IV . Thes e values are

e s timates of the activi ty of the tetracycl ines which should

be sui table for use in s tructure- activity relationships .

B . E f fect o f Tetracyc l ines on Rate Constants for

Protein and Nuc l e i c Aci d Synthe s i s .

I t was mentioned i n the l i terature survey that many

inves tigators have observed that tetracyclines cause an

inhibition of protein synthe s i s in bacterial culture s . The

results of two typical experiments in which the rates of

9 2

prote i n synthe s i s i n E . col i cultures inhibited by tetracyclines

were s tudied a re shown in Fig . XVI and XVI I . The se results

can be described by an equation s imilar to Eq . ( 1 ) :

[ P ] = [ P O ] ekPt , Eq . ( 4 )

when the time , t , i s greater than 3 0 minutes a fter antibiotic

addition and where [ P0 ] i s the concentration o f prote in in

the ce lls 30 minutes a fter antibiotic addi tion , [ P ] i s the

protein concentration in the cel l s at any later time and k p

i s the generation rate cons tant for prote i n synthes i s in

presence of antib iotic . The generation rate cons tant for

prote in synthe s i s in the absence of antibiotic , k� , i s

identical with the generation rate constant f o r cel l division

in the absence o f antib iotic , k�d · Value s o f kp are g iven in

<:!' 0 .--l :><

.--l I

() (j) Ul

� Ul -!-) � m -!-) Ul � 0 u

(j) -!-) m 0::

� 0

· ri -!-) m H (j) � (j) 0

9 3

+ 6 - �D'h o-

COt.Ha 5 - OH O H O 0

4 - 0 VIABLE Ci"NUCLEIC ACIDS"

3 - 4"PROTEIN"

2 -

1 -

1 . 0 2 . 0

Tetracyc line , M/L X 1 0 6

F igure X I I . The Linear Dependence of Generation Rate

Constants , kv , kp a nd kna , upon Tetracyc l ine Concentra­

t ions . The line shown i s the average inhibitory

rate constant , kivg ' it and va lues of k� , k� and k�a

are g iven in Table IV .

""' 0 .--i X

.--i I

() <lJ UJ

UJ 4-l c: rcl +l UJ c: 0 u

<lJ +l rcl p::;

c: 0

·ri +l rcl )...! <lJ c: <lJ �

6 -

5 -

4 -

3 -

2 -

1 -

5 . 0

+ om�·-o-

0/f COMi OH 0 HO 0 1

• TOTAL c V I �BLE

0 -NucLEIC ACIDS• A �ROTE IN•

1 0 . 0

9 -Nitro-dm-do-tetracyc l ine , M/L x 1 0 6

F igure X I I I . The Linear Dependence o f Generation Rate

9 4

Constants , kv , kt , kp and kna , upon 9-Nitro-dm-do

tetracyc l i ne concentration . The slope of the line shown

i s the average inhibitory rate constant , klvg , i t and

values o f k� , kf_ , k� and kAa. are g iven in Table IV .

""' 0 .--i �

.--i I

u (J) (/)

(/) +J s:: m +J (/) s:: 0 u

(J) +J m �

s:: 0

·rl +J m H (J) s:: (J) (.9

6 -

5 -

4 -

3 -

2 -

1 -

+ NHICt\l1

C H, �o-" :-... H N CON ct( ai 0 H O

Ha

• TO TA L c VI ABLE

:0 •N UC L E IC A C I D S

•

A •

P R O T E I N•

5 . 0 1 0 . 0

9-Dimethylamino-dm-do-tetracyc l ine , M/L x 1 0 6

F igure XIV . The L inear Dependence of Generation Rate

Constant s , kv , kt , kp and kna , upon Concentration of

9-Dimethylamino-dm-do-tetracyc l ine . The s lope of the

l ine s hown is the average inhibitory rate constant ,

k�vg ' i t and values of k� , kf , k� and k�a are g iven

in Table IV .

9 5

""' 0 .-I X

.-I I

u Q) Ul

Ul .jJ c ctl .jJ Ul c 0 u Q) .jJ ctl p:: c 0 ·ri .jJ ctl � Q) c Q) t9

6- · CONH2 HO 0

R, Ra Ra 5 - • H H NOa " PROTEIN"

0 H H H VIABLE 4 - 0 H ltia H TOTAL

A OH CL "NUCLEICAOOS'

3 -

2 -

1 -

5 . 0 1 0 . 0

Antibiotic Concentration , M/L x 10 6

F igure XV . The Linear Dependence of Generation Rate

Con s tants , kv , kt , kp and kna ' upon Concentration of

s everal Tetracyc l ine s . The s lopes of the l ine s shown

are the average inhibitory rate constants , k�vg ' they

and va lue s of k3 , k£ , k� and k�a for each antibiotic

are l i s ted in Table IV .

96

Table IV . - Inhibitory Rate Cons tants , 1./M- sec . , for Inhibition o f E . coli W by

Various Tetracyc l ine s .

Tetracycl ine Analoga -ki -ki -ki -ki -ki b v t p na avg

7 -Ni tro-dm-do-tetracyc l ine 8 3 3 . 5 1 6 5 6 . 7 6 7 3 5 . 6 8 7 6 9 . 1 9 7 4 8 . 7 8 * *

7 -Chloro-dm-te tracyc l ine 5 9 1 . 8 6 - 4 6 0 . 2 5 5 3 6 . 4 8 5 1 7 . 2 1

7-Ch lorotetracyc l ine 3 8 7 . 7 7 - 3 9 6 . 1 4 4 2 0 . 2 0 4 0 1 . 3 7

Tetracyc l ine 2 6 7 . 8 4 - 2 7 9 . 9 7 2 6 7 . 9 5 2 7 1 . 5 0

5 -Hydroxytetracy c l ine 2 7 5 . 1 9 - 2 5 4 . 7 9 2 5 1 . 8 3 2 5 7 . 1 5

7-Amino-dm-do- tetracycl ine 1 7 9 . 0 1 1 7 5 . 9 0 1 7 4 . 0 8 1 9 7 . 9 5 1 8 1 . 7 3 * *

a . dm-do- i s 6 -demethy l - 6 -deoxy- , whi le dm- i s 6 -demethyl - 6 -epihydroxy- . b . Tested by ana lys is of covariance to determine i f s ign i f icant d i f ferences

exi sted between the inhib itory rate constant determined by us ing a l l values of genera­tion rate constants col lective ly , ki

v , and ind ividual inhibitory rate constants determined separately . The symbo l � �ndicates sign i f i cant d i fferences at P) 0 . 9 5 , whi l e * * indicates s igni f icant a t P> 0 . 9 7 5 . S e e f o r example , S tee l , P. . and Torrie , 0 . , " Principles and P rocedure s of S tatistic s " , McGraw H i l l Book Co . , I nc . , New York , N . Y . , ( 1 9 6 0 ) pp . 1 7 4 .

1.0 -...)

Tab l e I V . - ( cont . )

Tetracyc l ine a n a l o g

9 - Amino-dm-do-te tracyc l ine

dm- do- tetracy c l ine

7 - Bromo- dm-do - t e tracyc l ine

- k i v

1 5 5 . 5 3

1 0 3 . 6 2

9 - N i tro - dm-do- tetracy c l ine 4 3 . 4 0

4 - Dedime thy l amino - te tracyc l ine 2 5 . 3 9

9 - D imethy l amino-dm-do- t e tracyc l ine 2 6 . 2 2

S a (6 ) -Anhydrote t r a cyc l ine 1 2 . 7 5

1 2 a - Deoxytetracyc l ine

Te tracyc l ine me thiodide 2 . 5 4

S a ( l l a ) -Dehydrochlorote tracyc l ine

7 - Ch loro- i s ot e t racyc l ine

2 - Cyano - 2 -decarboxamido - te tracyc l i ne

- k i t

1 5 1 . 2 3

6 2 . 6 8

4 3 . 1 5

2 3 . 2 9

2 4 . 6 2

l 3 . 81

2 . 6 3

2 . 1 2

0 . 3 6

- k i p

1 2 8 . 82

86 . 7 0

4 6 . 0 9

4 2 . 9 1

2 2 . 2 4

2 3 . l l

1 5 . 9 1

3 . 85

l . 9 6

0 . 2 5

- k i n a

1 4 6 . 1 3

9 2 . 9 3

5 1 . 4 2

4 7 . 86

2 5 . 4 5

2 0 . 7 3

1 7 . 2 3

2 . 3 7

2 . 1 0

0 . 4 2

no a c t iv i ty obs e rved

no a c t ivi ty obs e rved

- k i avg

b

1 4 5 . 0 3 *

9 4 . 4 2 *

5 1 . 7 9

4 4 . 3 3

2 4 . 0 9

2 3 . 6 7 * *

1 5 . 4 8* *

2 . 6 2 c

2 . 1 8C

0 . 3 4 c

c . I n s u f f i c i ent d a t a ava i l ab l e for t e s tinq by a n a l y s i s o f cova r i ance .

I.D co

9 9

+ 2 -

+ 1 -

.:: . .., 0 -Q) +l 0 H P< +

,. , � 1 - w���· .......

Q) O.N CONHa .:: 0 H

- 2 - AN TIBIOTIC CONe. MIL X 10

6

0.00 0 2 .34 •

- 3 - 4.67 Q 7.01 A 9 .34 •

l 2 3 4

Time , hr .

F igure XVI . An Example of Generation Rate Curves for

Protein Synthe s i s . The logar ithm o f the " Protein"

f ound in E . coli cultures a s a function of time of

growth i n the pre sence of various concentrations of

9 -N i tro-dm-do-tetracycl ine . The s lope s , kp , are g iven

in Table I I I .

Q) s::

H

+3

+ I

0

-I

·2

·3

0:/� ��

2

OH 0 HO 0 ANTIBIOTIC COHC.

MIL XI08 0.00 0 0. 1 1 • 0.22 Q 0.33 A 0.44 •

3

T ime , hr .

4 4

F igure XVI I . An Example of Generation Rate Curves for

Protein Synthe s i s . The logarithm of the " Protein "

f ound i n E . c o l i cultures a s a func tion o f time of

growth i n the pr esence of var iou s concentrations of

7 -Nitro-dm-do-tetracyc l ine . The slope s , kp , are g iven

in Table I I I .

1 0 0

Tab le s I I and I I I for the 1 8 antibiotics s tudied . I t can be

seen that the se value s are e i ther identical with or are

very close to the values of kv or kt observed in any given

culture .

1 0 1

�eneration r ate cons tants for prote in synthe s i s obtained

by regres s ion analy s i s from Eq . ( 4 ) are in agreement with an

equation s imilar to Eq . ( 2 ) :

Eq . ( 5 )

where k� i s the inhibi tory rate constant for protein synthe s i s .

Values o f kp do not f i t an equation s imilar to Eq . ( 3 ) . Fig .

XII , XI I I , XIV and XV are examples o f the fit of values o f kp

to Eq . ( 5 ) . Values o f the inhibi tory rate cons tants for

protein synthe s i s are given in Tab le I V .

The results o f two typical experiments i n which the rate s

o f nuc leic acid synthe s i s in culture s inhibited by tetracycl ines

were s tudied are shown in Figures XVI I I and XI X . The se

experiments did not show an increased rate o f nucleic acid

synthes i s in inhibi ted cul tures as sugges ted by several

inve stigators ( 1 8 , 3 0 , 3 6 ) but rather showed a decreased

amount o f nucleic acids corre lated to the observed inhib ition

of cel l divis ion . The data from these exper iments a l so f i t

a n expre s s ion s imilar t o Eq . ( l ) :

[ NA] = [ NA0 ] eknat , i f f kga>kna� Eq . ( 6 )

when the time , t , is greater than 3 0 minutes after antibiotic

addition and where [NA0 ] i s the concentration o f nucleic ac ids

in the cells 30 minutes after antibiotic addition , [NA] i s

the nuc leic a c i d concentration in the ce l l s a t any later time

1 0 2

+ 2 -

•

= + 1 -Ul

'0 · rl u � 0-u · rl Q) r-i + u - 1 - NHk;H,J1 ::l

���NHa z

--' H 0

Q) - 2 - A N TlBIOTIC COHC. c

...:l W,I\. X 6 10

0.00 0

- 3 - 2 .34 ... 4.67 8 7 .0i A 9 .34 •

1 2 3 4

T ime , hr .

F igure XVI I I . An Example of Generation Rate Curves for

Nuc leic Ac id Synthe s i s . The logar i thm o f " Nuc l e ic Ac ids "

f ound in E . c o l i culture s a s a function of time of

growth in the pre sence of various concentrations of

9 -N i tro-dm-do-tetracyc l ine . The slope s , kna , are g iven

in Table I I I .

= Ul

'"d ·ri u � u

·ri (J)

� u ::l z

+3

+ I

0

-I

-2

0

2

• +

W

N�

� 0

ANllBIOTIC CONC.

Mil.. . )CI06 0.00 .-.- 0

J

0.98 - · 1 .96 � e 2 .9 7 · A 3.9 1 Q 4 -89 � .

T ime , hr .

•

F igure XIX . An Example of Generation Rate Curves for

1 0 3

Nuc leic Ac id Synthe s i s . The logar i thm o f "Nuc leic Ac ids "

found i n E . c o l i culture s a s a function of time o f

growth i n the pre sence of various concentratQons o f

diD-do-tetracyc l ine . The s lopes , kna ' are g iven in

Table I I I .

and kna i s the generation rate cons tant for nucleic acid in

the presence o f antibiotic . The generation rate constant

1 0 4

for nucleic ac id s ynthes i s i n the absence of antibiotic , kga ,

i s identical to the generation rate cons tant for ce ll divis ion

in the absence of antibiotic , k�d · Generation rate constants

obtained from the f i t of experimental data to Eq . ( 6 ) are

given in Tables I I and I I I . I t can be seen that these values

are e i ther i dentical to or very s imi lar to the values o f

kv , kt o r kp observed in any given culture .

Generation rate cons tants obtained from Eq . ( 6 ) are

in excellent agreement with an equation s imi lar to Eq . ( 2 } :

Eq . ( 7 )

whe re k i i s the inhibito ry rate constant for nucleic acid na

synthe s i s . Figure s XI I , XI I I , XIV and XV are examples of the

f i t o f va lue s o f kna to Eq . ( 7 ) . Values of the inhibitory

rate cons tants derived from Eq . ( 7 ) are l i s ted in Table I V .

Value s o f kna do n o t f i t a n equation s imilar t o Eq . ( 3 ) .

As mentioned above , the values o f kv , kt , kp and kna

for any given culture are e i ther identical or very s imi l ar .

There fore , values of the respective inhibitory rate constants ,

I I I I d b . ' 1 � h kv , k t , k or kna ' would be expecte to e slml ar . n e ave p

tes ted the hypothe s i s that the se constants are experimentally

ind i s tingui shable and find no s tatistically s igni f i cant

dif ferences between the individual values and an average

inhibitory rate constant , k�vg ' for 7 of the antibioti cs .

Stati s tically s igni ficant d i f ferences were found , Table I V ,

for the 6 o the r antibiotics tes ted . Suff icient data was not

l O S

ava i lable t o test the other antibiotics i n this fashion . This

s imple analy s i s of covari ance te s t did not consider the error

invo lved in the determination of the individual generation

rate constants . I t seems l ikely that within present experimental

accuracy the 4 generation rate cons tants , viable and total cell

divis ion , protein and nuc l e i c acid synthe s i s , are the same

for a l l antibiotics . That i s a one-to-one relationship , balanced

growth , is found among the se parameters not only in contro l

cul tures but also in antibiotic inhibited culture s .

I f balanced growth i s indeed maintained in tetracycline

inhibited cultures then the average inhibito ry rate constant ,

k�vg in Table I V , would be the bes t measure o f tetracycline

activity to use in s tructure-activi ty relationships .

C . Time o f Onset o f Tetracyc l ine Inhibition of Whole

Cell Culture s .

The fact that inhibited cultures exhibit paral lel rates

o f cell divis ion , prote in and nuc l e i c acid synthe s i s is not

in accord w i th the interpretation of previous experiments

( 1 8 , 3 0 , 3 4 , 3 6 ) . These experiments were e i ther s ingle

point measurements or kinetic experiments which were carried

out only during t ime s immediately preceeding antibiotic

addition . The se expe riments reported e i ther decreased

protei n/ce l l and increased nuc l e i c acid/ce l l or inhibition

of rate o f prote in synthes i s and s timulation o f rate o f

nucleic acid s ynthe s i s . V /

However , Cerny and Habermann ( 3 3 )

in a kinetic experiment covering several generations showed

parallel rates o f protein and nucleic acid synthe s i s with

different t ime s of onset of action . The i r work was done at

antibiotic concentrations 1 5 times higher than those

presently employed , which caused complete inhibition and

undoubtedly cell death .

The agreement of exper imental data with Eq . ( L ) , Eq .

1 0 6

( $ ) o r Eq . ( ¢ ) wa s only observed for data obta ined approxima tely

3 0 minutes af ter antibiotic addi tion . Dur ing thi s initial

t ime per iod cultures appeared to maintain growth rates s imi lar

to tho s e observed in control s . Thi s can be observed in the

exper iments shown in F igures IX , X , XI , XVI , XVI I , XVI I I and

XIX . S imi lar observations were made in the other experiments

not shown . Figure XX shows an experiment des igned to document

this observa tion . I t can ea s i ly be seen that the onset of

inhibition for a l l parameter s is cons iderably beyond the time

of antibiotic addi tion . Viable plate counts , not shown ,

par a l l e led tho se obta ined by the total count method . Thi s

exper iment ha s been reproduced at a dif ferent oxytetracyc l ine

conc entration with s imi lar results . In addi tion , Fig . XX

shows that the time of onset of recovery from inhibition

upon d i lution of cultures into antibiotic free media is not

immed iate e i ther , but a l so exhibits a lag time . I t was

thought that both inhibition of and recovery of protein syn­

the s i s occurred s l ightly before the other parameters are aff ected .

In order to test the observation on time of onset of inhibition

an exper iment wa s performed in a dextrose-salts broth where

the rate of c e l l d iv i s ion in control cultures wa s 1/2 that in

the high peptone broth . Thi s s ing le exper iment , Fig . XXI ,

sugg e s t s that prote in synthe s i s measured by the Fo lin-Lowry

technique and a l so c e l l ma ss as measured by turbidity might be

1 0 7

F igure XX . T ime o f O n s e t o f and Recovery F r om Oxytetracy­

c l ine Inhibition i n Peptone Broth C u l tur e s . Genera t i o n rate

curves for tota l c e l l s , nuc l e i c a c id synthe s i s a nd pro t e i n

synthe s i s i n t h e pre s ence o f 1 . 3 8 x l o - 6 � 5 -Hydroxytetra ­

cycl i ne and a one - to - e l even d i lu t i on i n to a n t i b i o t i c -

free med ia of the i nh i b i ted c u l tur e . The gener a t i on r a te

curve f o r pro t e i n synthe s i s has been transpo s ed by sub­

trac t i ng 0 . 5 f r om a c tu a l v a l u e s of Lne " Pr o t e i n . " The

genera tion rate c o n s ta n t s , sec . - 1 x 1 0 4 , f o r the und i luted

c u l tur e s are g iven i n Table II exc ept f or v a lu e s obta i ned by

measur i ng cu lture tur b i d i ty ( no t s hown ) whi c h were 5 . 6 3 a nd

2 . 1 9 before and a f ter a n t i b i o t i c add i t i on , r e spec t iv e ly . The

values of the generation r a t e c o n s t a n t s in the d i luted c u l ture

wer e k t 5 . 4 3 ; kp = 5 . 0 9 ; kna = 4 . 6 1 ; kv ( not s hown ) = 5 . 3 3

and for c u l ture turb i d i ty = 4 . 7 6 .

The t ime o f a n t i b i o t i c add i t i o n wa s 4 , 4 7 0 sec . and the

t ime o f d i lution wa s 1 0 , 8 5 0 sec . The t im e s o f onset o f

inh i b i t io n and the t im e s o f o n s e t o f r ecovery c a lc u l ated a s

the inter s e c t i on o f the regr e s s ion l i ne s , r e spec t i v e l y i n

sec . , wer e : f or protein synthe s i s , 6 , 2 0 0 a n d 1 1 , 8 0 0 ; f or

turbid ity , 6 , 4 0 0 and 1 1 , 5 0 0 ; f o r nuc l e i c a c i d synthe s i s ,

6 , 4 0 0 and 1 2 , 4 0 0 ; and for c e l l d iv i s ion - tota l 7 , 1 0 0 a nd

1 2 , 5 0 0 and viable , 6 , 5 0 0 a nd 1 2 , 2 0 0 .

l O B

DILUTION

+4

/ r /'

r OXYTETD�yt - LINE 1.38X 106 MIL

!

l

0 TOTAL A "NUCLEJCACICS" e "PROTEIN"

-4

T ime , hr .

F igure XX .

F igure XXI . Time of Onset of Oxytetracyc line Inhibition of

Cultures in Dextrose-Sa lts Broth . Generation rate curves

1 0 9

for nuc leic acid synthe si s , protein synthes i s and culture

turbidity in the presence of 1 . 1 6 x 1 0- 6� 5-Hydroxytetra­

cycline . The generation rate curve for nuc leic acid

synthesis has been transposed by adding 2 . 0 to the actual

value of lne "Nucleic Ac ids . " Values of generation rate

constants , sec . -1 x 10 4 , in the absence and presence of

antibiotic respectively , were : kna = 2 . 1 3 and 0 . 5 6 ; kp = 2 . 6 2

and 0 . 3 7 ; kt ( not shown ) = 2 . 4 4 and 0 . 1 2 ; and for turbidity

2 . 5 4 and 0 . 3 7 .

The time of antibiotic addition was 8 , 9 0 0 sec . The

times of onset of inhibition , sec . , ca lculated a s the inter­

cept of the regre s sion lines were : for protein synthesi s ,

9 , 9 0 0 , for turbidity , 1 0 , 3 0 0 ; for cell division , 1 0 , 7 5 0 ; and

for nuc leic acid synthesis , 1 4 , 2 0 0 .

1 1 0

+3

+t

+I

s:: 0 . o ·.-i .j.) 1\l )...! .j.) s:: Q) u s:: 0 u �

Q) s:: ..:I A "Nucc.£Jc /lCD/I' • "Fitol'EIN"

e lUt8lliTY

- ·

2 4 8 10

Time , hr .

F igure XXI .

1 1 1

inhibited sooner than nuc leic acid synthe s i s . Cell divis ion ,

not shown , was inhibited at an intermediate time . The exis tence

of the s e d i f ferent l ag times explains the dis crepancy between

parallel equ i l ibrium inhibition rate s observed in thi s work

and non-parallel amounts of nucleic acid and prote in per cell

reported by o thers ( 1 8 , 3 0 , 3 4 , 3 6 ) . Experiments per formed

in cul ture med i a whi ch do not al low rapid rates of cell

divis i on would be expecte d to accentuate thi s d i f ference in

times o f onse t o f inhibi tion .

Thi s o rder o f inhibition and recovery is cons i stent with

the hypothe s i zed primary inhibi tion o f prote in synthes i s

fol lowed b y a secondary , indirect inhibition of cell divis ion

and nucleic acid synthe s i s due to a lack of prote in .

D . E f fe c t o f Broth pH Changes on Tetracyc line-Inhibited

Culture s .

Jones and Morris son ( 2 0 ) and Benbough and Horris son ( 1 7 )

have reported that when the pH o f culture media o f known free

metal ion concentration i s varied , the inhibition produced

is proportional only to the " mo lecular " , presumab ly zwitterionic ,

species o f several tetracyclines . Connamache r , Mande l and

Hahn ( 6 6 ) have recently reported data on the uptake o f

tetracycline in B . cereus cultures at pH 6 . 7 and 7 . 3 and

at two Mg ion concentrations which are in qual itative agree­

ment with the hypothe s i s o f Jones and Horr i s son .

Because the s l ow growth rate characteristics o f E . coli

w in dextro s e -s a lts broth made it unlikely that the

tetracyc l ines would be suffic iently stable it was not

pos sible to ver i fy these results for other te tracyclines in

1 1 2

a media o f known free metal ion concentration . However ,

experiments in peptone broth are con s i s tent with these results

at two broth pH values where free metal ion concentration would

be expected to be constant . Figure XXI I shows a plot o f

Eq . ( 2 ) f o r experiments at p H 6 . 2 and 6 . 6 5 . The concentration

o f tetracycl ine is adj us ted to reflect the concentration of

zwitterionic form rather than the total concentration .

Experiments at a broth pH o f 7 . 4 , where a much lower free

me tal ion concentration would be expected because of the

higher HP04- concentration , do not f i t the s ingle l ine

plotted in Fig . XXI I . Activities obtained with 5 a ( 6 ) ­

anhydrotetracycline ( pH 6 . 2 and pH 6 . 6 5 ) and 4 - dedimethyl­

amino-tetracycline ( pH 6 . 6 5 and pH 7 . 4 ) do not f i t such a

s imple concept e i ther , Table V . The se tetracycl ines have ,

however , unusual pKa ' s .

I n addition to the growth-rate dependent d i fference in

time of onset o f inhibi tion o f the various culture parameters ,

there i s a lag time observed before any parameter is inhibited ,

Fig . XXI . Two general explanations are pos s ible for thi s

l a g time . Firs t , a finite time may b e required t o es tablish

equ i l ibrium concentrations within the cell s . Second , antibiotic

may permeate the cell and arrive at the receptor s i te but

require finite time to exert a measurable e f fect .

Bes ides causing a change in the degree o f equ i l ibrium

inhibition produced by a given total antibiotic concentration ,

broth pH changes are also capable o f changing the growth-rate

independent lag time for onset o f inhibition . Figure XXI I I

shows an experiment identical to that pre sented previously ,

\0 0 H X

..-il u

u Q) Ul

Ul +l .:: m +l Ul .:: 0 u Q) +l m p::; .:: 0 ·rl +l m H Q) .:: Q) (j

6 -

3 -

4 -

3 -

2 -

1 -

l .,H 6 .65 .,H 6.1S

... A •NUCLEIC ACIDS•

�� ' • 0 •PROTE IN•

• VIABLE

c TOTA L oe D

0 . 5 1 . 0

[Chlortetracyc l ine ] , M/L x 1 0 6

1 + Ka/ [ H+ ]

F igure XXI I . The Linear Dependence of Generation Rate

1 1 3

Constants upon Concentration o f Zwitter ionic Chlortetra-

cyc l ine . Constants were obta ined at two broth pH value s ,

6 . 1 5 and 6 . 6 5 . Values of generation rate constants

obta ined at a higher broth pH value , 7 . 4 , and , therefore ,

a higher HP04 = concentration do not f it thi s s ingle line .

The s lope of the line shown i s 4 7 0 L/M- sec .

1 1 4

Table V . - F irst Order Generat ion Rate Cons tant s , sec- 1 x 104 ,

and Inhibitory Rate C onstant s , L/M-s ec . , f or E . c o l i w in

Various Broth C u l tures Inhib ited by Several Tetracyc l ines .

7 -Chlorotetracyc l ine , peptone broth , pH 6 . 2

0 . 000 5 . 56 5 . 50 4 . 88

0 .. ll.92 4 . 9 5 4 . 55 4 . 66

0 . 384 4 . 06 3 . 50 3 . 38

0 . 57 5 3 . 0 1 2 . 69 2 . 7 1

0 . 7 6 7 2 . 10 1 . 78 1 . 86

-4 6 1 . 1 1 -484 . 15 -4 17 . 52 -4 54 . 28**

Sa ( 6 ) -Anhydrotetracyc l ine , peptone brot h , pH 6 . 2

0 . 00 5 . 56 5 . 50 4 . 88

4 . 67 4 . 7 1 4 . 68 3 . 90

9 . 34 3 . 94 3 . 2 3 3 . 4 5

14 . 0 1 3 . 35 2 . 57 2 . 92

18 . 68 1 . 7 3 1 . 99 2 . 3 5

- 1 9 . 27 - 19 . 54 - 1 2 . 9 5 - 1 7 . 2 5 **

* * S ig n i f icant d i f ferences between inh ib itory rate constants determ ined by analy s is of c ovar ianc e as expla ined in Tab le IV .

Table v . - ( cont . )

106 M kt kp kna k i avg

4 -Ded imethy l amino-tetracyc l in e , peptone broth , pH 7 . 4

0 . 00 4 . 57 5 . 2 2 4 . 7 6

3 . 34 3 . 87 5 . 0 1 3 . 96

6 . 6 5 3 . 3 3 3 . 7 3 3 . 48

9 . 96 2 . 7 1 3 . 0 3 3 . 00

1 3 . 24 2 . 0 9 2 . 1 3 2 . 54

k i - 1 8 . 48 - 24 . 7 0 - 16 . 3 2 - 1 9 . 8 3 **

7 -Chlorotetracyc l in e , pH 6 . 7 , dextrose-sa lts broth

0 . 000 2 . 39 2 . 30 2 . 18

0 . 16 3 2 . 07 1 . 92 2 . 0 1

0 . 32 7 0 . 97 0 . 84 0 . 7 9

0 . 4 90 0 . 39 0 . 51 0 . 50

0 . 6 5 5 0 . 2 2 0 . 38 0 . 37

k i - 368 . 34 - 3 1 5 . 8 1 - 3 12 . 59 - 3 3 2 . 2 5

* * S ign i f icant d i f ferences between inhib itory rate constants dete rmined by ana lys is of c ovar iance as exp la ined in Table IV .

1 1 5

F igure XXI I I . The E f f e c t of Broth pH on Time o f O n s e t of

1 1 6

Oxytetracy c l ine I nhib i t i on . Genera t ion r a t e curve s , done on

s epara te days a t two peptone broth pH v a l ue s , f o r c e l l

d i v i s ion- b y the to t a l c ount me thod ana f or c u l ture

turb i d i ty in the presence of two d i f f er e n t c oncentr a t ions o f

5 - Hydroxy t e tr a c yc l i ne . The g ener a t i on r a t e curve s f o r c e l l

d iv i s ion have been transposed b y subtr a c t i ng 3 . 0 f r om the

a c tual value o f Lne ( orga n i sms/ml . x l o - 6 ) . The g ener a t i on

rate constant s , sec . - 1 x 1 0 4 , i n the absence and pr e senc e

of antibiot i c , re spec tively , f o r c e l l d i v i s io n , were 5 . 1 6

and 2 . 2 0 a t pH 6 . 6 5 and 4 . 7 5 and 2 . 3 0 a t pH 7 . 4 , and f o r

turb i d i ty wer e , 5 . 6 3 and 2 . 1 9 a t pH 6 . 6 5 a n d 5 . 5 6 a n d 2 . 2 1

a t ph 7 . 4 .

The t ime s of a n t i b i o t i c add i t ion were 4 , 4 7 0 s ec . a t ph

6 . 6 5 and 5 , 4 0 5 sec . at pH 7 . 4 . The t im e s o f o n s e t of

inhibi tion , sec . , c a l c u la ted a s the inte r s ec t i o n o f the

reg r e s s ion l i nes were f o r c e l l d iv i s ion 7 , 1 0 0 and 6 , 2 0 0 and

for turbid i ty 6 , 4 0 0 and 5 , 9 0 0 a t pH 6 . 6 5 a nd 7 . 4 , r e s pec t i v e ly .

1 1 7

+ 3 -

+ 2 -

+ 1 -

s:: 0 0 -·rl .jJ C1l H - 1 -.jJ s:: Q) u - 2 -s:: 0 u ---'

Q) - 3 -

s:: ...:l - 4 - ,.H 6.65 ,H 7.4

0 • TOTAL

"' • TURBIDtTY - 5 -

- 6 -

1 2 3 4

T ime , hr .

F igure XXI I I .

1 1 8

Fig . XX , except that the pH o f the broth was 7 . 4 instead o f

6 . 7 . The lag for onset o f inhibition o f protein synthesis

( me as ured a s cel l mas s ) and ce l l divis ion are seen to be

shortened by two-thirds . The equil ibrium amount o f inhibition

achieved by two d i f fe rent total oxytetracycl ine concentrations

was the s ame i n both experiments . I f the hypothes i s o f Jones

and Mor r i s son ( 2 0 ) i s correct , then the concentration of

zwitterioni c species present in the broth mus t be the same in

both experiments . The nature o f the initial inhibi tory process

mus t be quantitative ly d i fferent i n these experiments . I t

seems mos t l ikely that the proces s o f permeation into the

cel l s is a f fecte d by the pH change and that thi s is the proces s

respons ible for the growth-rate i ndependent lag time observed

in the s e experiments .

Arima and I z ak i ( 6 4 , 2 4 4 , 2 4 5 ) have s tudied permeation

o f oxytetracy c line in sens i tive and res i s tant cells at high

antibiotic concentrations and found that i t depends upon an

energy requ iring proce s s . Franklin ( 6 5 ) has veri fied a

glucose-dependent uptake at low concentrations o f tetracycline

and chlortetracy c l ine . The condition o f the cells in these

cultures i s so d i f ferent from the cel l s in the present work

that it is hard to make meaningful comparisons . However , i t

i s c l e a r that the abi li ty to permeate the ce l l s is a n important

factor in antibiotic activity , whether thi s proces s is an

inactive or an active di ffus ion . E i ther proces s might be

expected to be sensitive to the broth pH .

E . K inetic Mode l s for Inh ibition o f Bacterial Cultures

by Tetracyc l i ne s .

S tudi e s of the k inetics o f inhibi tion o f ce l l growth

are capabl e of provi ding in formation of a type other than

1 1 9

the estimate s o f rel ative potencies o r the estimates o f order

of onset of inhibition which have been mentioned above .

Kinetic s tudies can provide information which is useful in

testing proposed mode l s of antibiotic activity . Any proposed

mode l of tetracy c line activity should be consistent with

several results of the present study .

The se are :

a . Exponential growth i s maintained in cultures inhibited

by l ow concentrations o f tetracyc l ines ; see Eq . ( 1 ) , ( 4 ) and

( 6 ) • b . The generation rate constants for ce l l divi s ion ,

prote in and nuc leic acid synthes i s are the same in any given

tetracyc line-inh i b i ted culture , kcd�O .

c , Rate cons tants for inhibited cultures are l inearly

dependent upon the f i r s t power of the antibiotic concentration ;

see Eq . ( 2 ) , ( 5 ) and ( 7 ) .

d . Ant ib io t i c inhibition i s reversed by di lution into

antibiotic free med i a , Fig . XX .

I n addi tion , it seemed l ikely that :

1 . P rote in synthes i s i s inhibi ted be fore ce l l divis ion or

nucl e i c ac id synthe s i s .

2 . Permeation o f antibiotic into ce l l s i s important and

might be responsible for the l ag in time of onset of inhibition .

A s imple mode l which des cribes the kinetics o f ce ll

div$s i on observed in E . coli culture s inhibited by low

tetracycl ine concentrations has been proposed by Garrett ,

Mil ler and Brown ( 2 4 3 ) . I t assumes equ i l ibrium ( K )

between free E . co l i ( Ef ) and tetracycline ( T c ) t o form

inhibited E . coli ( Ei ) :

1 2 0

E f + T c .J<..t: Ei Eqj ( 8 )

and i t a l s o a s s ume s that the rate o f generation o f total

E . coli ( E t ) i s proportional to the concentration of free

E . c o l i 'f

Eq . ( 9 )

When the fraction of inhibited E . coli ( � ) i s calculated

from Eq . ( 8 ) :

� E · K [ T e l l Eq . Et 1 + K [ Te l

and when s ub s t i tuted i n Eq . ( 9 } , one obtains :

d ( Et ) = k�d ( l - � ) Et = k�d ( l - �K:..:........::...[ T:._c:....;l:..__ ) l + K [Te l a t

S ince it i s found experimental ly that ,

d E t Eq . ked

( Et ) d t

by comb ining Eq . ( ll } and Eq . ( 1 2 ) ' one obtains :

ked kgd l - K [TcJ Eq .

l + K [Te l

f E ( 3 ) When K [Tc ] i s much which is a rearrangement o q . ·

( 1 0 )

Eq . ( l l }

( 1 2 )

( 1 3 )

less than 1 , a " l imiting case " , then Eq . ( 1 3 ) i s equivalent to

Eq . ( 2 ) , where k; . d . = k� . d .

K .

Thi s simp l e kinetic mode l is con s istent with re sults

outlined as a, c arrl d.. I t does not consider the observations

outl ined in b , 1 and 2 . Consequently , i t does not lead to

1 2 1

Eq . ( 2 ) i n a biochemi cally meaningful fashion . Garrett et . al .

( 2 4 3 ) have tried to exp l ain why this l in;iting case of Eq , ( 1 3 )

i s obs erved during tetracyc l ine inhibi tion by postulating

that there are d i f ferences between rates of protein synthes i s

and the rates o f c e l l divis ion in inhibited cultures . The

present report has shown that this is mos t probab ly not true ,

result b , and i t would be helpful i f another hypothes is were

advanced to explain thi s adherence to the l imiting case o f

E q . ( 1 3 ) .

A model cons i stent with the results outl ined above has

been sought w i thin the proposed b i ochemical mode of action .

This mode l a s s umes that tetracyclines bind to the aminoacyl

site of ribosome s and thereby prevent the binding of AAtRNA .

The evidence for this mode of action has been presented in

the l i te rature survey . I f the usual type o f enzyme- inhibitor

kinetics is f o l l owed it would be expect�d that thi s mode o f

action would generate a n inverse re lationship between rate o f

protein synthe s i s and inhibitor concentration . However , a

growing E . coli culture i s not the normal in vitro test systero .

Such a culture w i l l not have constant concentrations o f s ubs trate

or enzyme s . It was thought , therefore , that a mode l which

a ll owed the se concentrations to vary with the ti�e of growth

and a l s o the inhibitor concentration , might lead to results

s imi l ar to that observed in the experimental cultures .

1 2 2

Conditions have not been found such that the proposed

model generates outputs which agree with the exper imental

data . The model wi l l be pre sented , however , s i nce it or a

s imple var iation of i t may be con s i s tent with the exper imenta l

observations when other conditions are tr ied .

The model , F ig . XXIV , i s pre sented i n terms of some

of the reactions i nvo lved in protein synthes i s in a bacter ial

culture . The s implest model invo lving the lea s t number of

reactions ha s been pos tulated as a starting point . Reac tants

are expres s ed in terms o f concentration i n bhe tota l culture .

The model s tates that there i s a rever s ible pa s sage of

tetracyc l ines from out s ide the c e l l s ( Tc ' ) into the c e l l s

( Tc ) , Eq . ( 14 ) . T h i s s tep ha s been ignored i n initial model s .

Tetracyc l ine s inside the c e l l s bind to r ibosome-RNA ( R ) and

form a complex ( R • Tc ) which i s not capable o f synthe s i z ing

protein , Eq . ( 1 5 ) . Norma l r i bo some s-mRNA complexes are , of

cour se , c apable of bind i ng sub s trate ( S ) , AAtRNA and forming

a peptide bond ( P ) on the r ibosoma l sur face , Eq . ( 1 6 ) .

Peptide bond f orma tion frees the aminoacyl s ite of the

r ibosome-mRNA complex and a l lows either tetracyc l i ne or AAtRNA

to bind .

Two steps , Eq . ( 17 ) and ( 1 8 ) , have been i nc orporated

i nto the mode l with the expectation that they might lead to

results d i f fe�ent from ordinary enzyme kinetic s . The f ir s t

o f these steps i s that substrate i s synthe s i z ed from substrate

precur sors ( S ' ) i n a reaction which ha s been pre sumed to

proceed at a rate proportiona l to the concentration of prote in

1 2 3

Tc ' K1 < Tc

Ka R + Tc � R ·Tc �

R + s & R · S k , , � p + R I k 8 .. s · C,P,

s

p Ca \ R

P;. * 'b ( R + R · S + R ·Tc ) + � p

F igure XXIV . Propo s ed Mode l o f Tetracyc l i ne Inhib i t ion of Pro-

t e i n S y n th e s i s i n Bacter i a l Cu l tur e s . Tc ' is the c oncentr a t i on

o f t e t r a c y c l ine i n the cu l ture bro t h , Tc i s the c oncentrat ion

w i th i n the c e l l s , R is r i bo s om e s - mRNA - peptidy l tRNA , S i s

AAtRNA , P i s t h e p e p t i d e bond c o n c e n tr a t i o n , S ' a r e AAtRNA pre-

cur s or s , S ' are AAtRNA d egradation produc t s , P t is the tot a l

pro t e i n c onc e ntra t i o n w i t h i n the c e l l s , q wa s 0 . 4 and oC wa s 2 0 .

1 2 4

i n the cul ture . That i s , the rate o f this synthes i s step has

been pre s umed to be proportional to P . The proportionality

constant uti l i zec , c 1 , i s a pseudorate cons tant and contains

in addi tion to the concentrations of the precursors , the

fraction of the total number of peptide bonds in the culture

whi ch are part of the enzymes cataly z ing this reaction , i . e .

aminoacyl synthetas es . S ubs trate i s also lost , non-enzymatically ,

in thi s mode l by hydrolys i s , Eq . ( 17) . The second d i f ferentiating

feature of thi s mode l is that it postulates that a fraction

o f the product peptide bonds are ribosomal precursor proteins

and that these prote ins give rise to new ribosomes , Eq . ( 1 8 ) .

The constant , c 2 , uti l i zed in thi s step i s also a pseudo-rate

constant which contains the concentration of rRNA in the

culture as we l l as the fraction of total peptide bonds which

are ribo s oma l peptides . However , the as sumption that c 1 or c2 are cons tants , which has been employed , is probably

not valid . The concentration of tRNA or rRNA per ce l l mis;ht

be re lative ly cons tant , but the concentration of the se species

in the culture is undoubtedly not constant . They are probab ly

increas ing at the s ame rate as the number of ce l l s in the

cul ture . Perh aps there are o f f setting change s in other subs trate

or ribosomal precur sors and there fore , thi s approach , because

of i ts s imp l i c i ty , was tr ied f i rs t .

The concentration o f tota l prote in ( Pt

) present i n the

culture has been a s s umed to be the sum of the ribosoma l

and non-r ibosomal prote in , Eq . ( 1 9 ) . For the se calculat ions

it was a s s umed that ribosomes were 4 0 % protein ( q ) and that

1 2 5

there are 2 0 peptide bonds in the average E . coli protein pc) .

The di f ferential equations , Eq . ( 2 0 ) through ( 2 6 ) ,

derived from thi s mode l are g iven in Table VI . They are too

complex to be solved by ordinary integration . They do

however , lead to a s imple express ion for the rate of total

prote in synthe s i s :

0/oe q ) [ P ] Eq . ( 2 7 ) d t

Thi s equation s tre s s e s the important role played by oc and

q in determining the nature of the Pt output in the model .

The above mode l i s based upon the concentration of the

various reactants in the total culture . I f one were to write

a s imi lar mode l based upon the concentrations per ce ll one

might be j us t i f ied in a s s uming a s teady-state in R . S 1 in

which case :

[ R · s I l [ S I l Eq . ( 2 8 )

+ [ S I l

where the s uperscript ( I ) i s used to de signate concentrations

per individual ce ll rather than in the total culture and Rt is the concentration of a l l forms of r ibosome s within the cel l .

While thi s equation cannot b e substituted into Eq . ( 2 7 ) , i t

ind icates that the model might b e tes ted under two general

conditions , when s is greater than Km and when S is less than

S ince ordinary integration methods are not use ful , the

model ha s been te s ted by analog s imulation . An unsealed

an�log computer program is given in Fig . XXV . The mode l is

too big ( too many integrations ) for the avai lable analog

Tab le V I . - The Different ial Equ a t i on s from a Mode l of

Tetracyc l in e Inhibit ion o f P rote in Synthes is in

Bacter ial Cu lture s .

d Tc 1 d t

d Tc d t

d R · Tc d t

d R d t

d s d t

d R •S d t

d p d t

k_ 1 (Tc )

k l (TC I � k_ 2 [R · Tc )

k 2 ( R ] (TC )

k_2 (R · TC ]

k_ 3 [R · S ]

k 3 [R ] [ S ]

k1

(Tc 1 )

k_ 1 (Tc ) - k2 (R ] (Tc ] +

k_ 2 [R · TC )

k2 [ R ] [TC ] + k4 [R · S ]

k 3 [R ] [ S ] + c 2 [P ]

+

1 2 6

Eq . ( 2 0 )

Eq . ( 2 1)

Eq . ( 2 2 )

Eq . ( 2 3 )

Eq . ( 24 )

Eq . ( 2 5 )

Eq . ( 2 6 )

F igure XXV . An Unsealed Ana log Computer Program for

the Solution of the D i f f erential Equa tions of the

Propos ed Mode l of Tetracyc l i ne Inhibition of Protein

Synthes i s i n Bac ter ial Culture s .

1 2 7

computer1 3 and could only be part i a l ly tes ted with this

ins trument . I n add i tion , mos t analog computers are only

able to perform accurate calculations over about 2 log

cycle s . The s e d i f f iculties were overcome by us ing the

I nternational Bus iness Machines 1 1 3 0 digital computerl 4

with associ ated Continuous Sys tems Mode ling Package . The

actual program uti l i zed i s given , in F i g . XXVI , in ordinary

1 2 8

analog computer symbo l s . E i ghty analog e lements were avai lable

for use wi th thi s package and the output i s accurate over

several log cycle s .

S ince the value s for initial concentrations o f reactants

and rate constants which were tried did not lead to outputs

which agreed with the e xperimental data , the va lues will

not be de s cribed in detai l . The value s tried are l i s ted in

Table VI I . When Km was greater than S , no conditions could

be found where the output was s imilar to experimental data ,

even when Tc ' and Tc were zero . Whi l e i t was pos s ible to

obtain an exponential increase o f total prote in synthe s i s ,

ba lanced growth was not maintained . That i s , the ratio

of ( S ) to ( R) was not constant . These experiments were

unfortunately performed before the need to uti l i z e a value

o f � larger than l was rea l i zed . Inspection o f the results

reveals that thi s would not have changed the results o f the se

experiments , however , other condi tions might be found where

1 3 . TR 2 0 , Elec tron ic Assoc iates Inc . , Long Branch , N . J .

1 4 . We are grateful to Mrs . L i l l i an Kornhaber o f the Department o f B i ometry , Medical Col lege of Virginia , for performing the se calculations .

F igure XXVI . The Continuous Systems Modeling Package

Prog ram Used in the Search for Solutions to the

D i f f erential Equations from the Proposed Mode l of

Tetracyc l ine Inhibition of Prote in Synthe s i s in

Bac ter i a l Cultur e s .

1 2 9

Table VII . - C omp i l a t ion of C ond i t i on s Which Were Emp loyed to Test the Mode l of

Tetracyc l ine Inhib it ion of Prot e in Synthe s is in Bacter i a l C u lture s .

Ident ity # so Ro Po t k3 k_ 3 k4 � cl c 2 k

7

A 3 . 7 50 0 . 07 5 0 . 0 3 0 . 02 0 . 004 1 . 2 6 0 . 20 1 . 4 1 . 0 0 . 008

B 3 . 7 50 0 . 07 5 0 . 0 3 0 . 02 0 . 004 1 . 2 6 0 . 2 0 1 . 4 1 . 1 0 . 008

c 0 . 37 5 0 . 07 5 0 . 0 3 0 . 02 0 . 004 1 . 2 60 . 20 1 . 4 1 . 0 0 . 008

D 0 . 37 5 0 . 07 5 0 . 0 3 0 . 10 0 . 02 0 6 . 0 60 . 20 7 . 0 5 . 0 0 . 04 0

E 3 . 7 50 0 . 7 50 0 . 30 0 . 10 0 . 02 0 6 . 0 60 . 20 7 . 0 5 . 0 0 . 04 0

F 0 . 37 5 0 . 07 5 0 . 0 3 1 . 00 0 . 02 0 6 . 0 6 . 0 2 7 . 0 5 . 0 0 . 040

G 1 0 . 37 5 0 . 07 5 0 . 0 3 1 . 00 0 . 200 6 . 0 6 . 20 7 . 0 5 . 0 , 0 . 04 0 2 5 . 5 , 3 6 . 0 , 4 6 . 5

H 1 0 . 37 5 0 . 07 5 0 . 0 3 1 . 00 0 . 200 6 . 0 6 . [20 10 . 0 , 6 . 0 0 . 040 2 1 5 . 0

I 1 0 . 37 5 0 . 07 5 0 . 0 3 0 . 10 0 . 02 0 6 . 0 60 . 20 1 . 6 5 , 0 . 5 0 . 040 2 1 . 7 5 , 3 1 . 8 5

J 1 0 . 37 5 0 . 07 5 0 . 0 3 100 . 00 20 . 000 6 . 0 0 . 26 2 . 0 , 0 . 5 0 . 004 2 2 . 4 , 3 2 . 5 , f-'

w 4 3 . 0 0

Tab l e V I I . - ( cont . )

I . D . # so Ro R • S0 Po pg k 3 k_ 3 k4 � c l c 2 k7

K 1 0 . 37 5 0 . 07 5 - - 0 . 0 3 0 100 . 0 2 0 . 0 6 . 0 0 . 26 12 . 0 2 . 5 0 . 02 0

K 2 0 . 1 5 0 0 . 300 0 . 02 2 5 0 . 0 2 5 0 . 04 6 100 . 0 2 0 . 0 6 . 0 0 . 26 1 2 . 0 2 . 5 0 . 02 0

L 1 0 . 1 50 0 . 0 30 0 . 02 2 5 0 . 02 5 0 . 04 6 100 . 0 2 0 . 0 6 . 0 0 . 26 1 2 . 0 2 . 6 , 0 . 02 0 2 2 o 7 1 3 2 . 8 , 4 3 . 0 , 5 3 . 2 5 , 6 3 . 5

M l 0 . 1 50 0 . 0 30 0 . 0 2 2 5 0 . 0 2 5 0 . 04 6 100 . 0 2 0 . 0 6 . 0 0 . 26 1 2 . 0 3 . 5 0 . 2 50 , 2 0 . 500 , 3 1 . 000 , 4 2 . 500 , 5 5 . 000

N 0 . 1 3 5 0 . 0 30 0 . 017 5 0 . 0 5 5 0 . 074 100 . 0 2 0 . 0 6 . 0 0 . 2 6 2 . 5 0 . 5 0 . 004

0 0 . 4 0 5 0 . 090 0 . 0 5 2 5 0 . 0 5 5 0 . 1 1 2 100 . 0 20 . 0 6 . 0 0 . 2 6 2 . 5 0 . 5 0 . 004

p 0 . 100 0 . 0 28 0 . 1 100 0 . 0 55 0 . 0 18 100 . 0 2 0 . 0 6 . 0 0 . 2 6 2 . 5 0 . 5 0 . 004

Q 0 . 244 0 . 07 1 0 . 06 3 5 0 . 2 3 9 0 . 066 100 . 0 20 . 0 6 . 0 0 . 2 6 2 . 5 0 . 5 0 . 004

a . For exper iments A to Q inc lus ive va lues of kl , k_ 1 , k2 , k_2 , Tc ' , and Tc were not

used . For experiment s A t o 0 inc lus ive , was programmed as 1 . I-' w I-'

Tab l e V I I . - ( c on t . )

I dent ity # Tc 0 k2 k_2 Tc ' O kl

k_ l

Q 2 l 1000 . 00 2 0 0 . 000

Q 3 l , 2 , 3 100 . 00 2 0 . 00 0

Q 4 3 10 . 00 2 . 000

Q 5 3 1 . 00 0 . 2 0 0

Q 6 2 0 0 . 10 0 . 02 0

Q 7 20 , 80 , 240 0 . 0 1 0 . 00 2

Q 8 240 0 . 0 2 , 0 . 00 5 0 . 00 2

Q 9 240 0 . 0 1 0 . 00 1 , 0 . 004 , 0 . 0 10 , 0 . 020

Q 10 24 0 0 . 0 1 , 0 . 10

Q l l - 0 . 10 0 . 020 50 , 100 , l 0 . 1 150 , 200

b . For exper iment s Q 2 t o Q l l , inc l u s ive , the values of so , Ro , R · S0 , Po , 0 Pt , k 3 , k_ 3 , k4 , kffi , C 1 , C 2 , and k7 wh ich were u s ed were the s ame as those g iven above under Ident ity # Q .

f--' w N

1 3 3

thi s would not be true .

Conditions which allowed an exponential increase o f

total prote in and where ( S ) and ( R ) increased exponential ly

at the same rate were found when ( S ) was greater than K and m

Tc ' and Tc were equal to zero . However , when Tc ' or Tc were

di f ferent from zero , it was not poss ible to f ind conditions

o f Eq . ( 14 ) and/or Eq . ( 1 5 ) such that prote in synthes i s was

maintained at a reduced exponential rate . Rather the rate

of prote in synthe s i s a lways decreased initial ly and then

incre ased to the rate occurring in the ab sence of antibiot ic .

I t was not poss ible to f ind conditions which maintained the

i n i t i a l reduced rate of prote in synthe s i s for time s as long

as those seen experimental ly . I n addition , the rate constants

evaluated from the initial inhib ited protein synthe s i s , Tab le

VI I , did not have a l inear dependence upon the total antibiotic

concentration .

Several variations o f the mode l have been sugges ted

by the present work . When ( S ) is less than �m , value s of

rate cons tants were rej ected because the rate of prote in

synthe s i s was not f irst-order . The se non-exponential re sults

might have been caused by the value of oC uti l i zed . These value s

o f rate constants might lead to exponential protein synthes i s

and ( S ) and ( F ) might be produced in proportionate amounts i f

a larger number were uti l i zed for oC

When ( S ) i s greater than Km , the mode l as presently

s tated appe ars to be inherently balanced , i . e . after e s tab l ish­

ment o f equi l ibria between antibiotic and ribosome s it re turns

1 3 4

to the rate o f prote in synthes i s preva i l ing be fore addition

o f antibiotic . Perha lt t 1 ps a erna e va ue s of rate cons tants

can be found which would prolong the time of establ i shment of

thi s equi l ibria . The s tatement o f the equi l ibria between

Tc ' and Tc is not exact as expre ssed in Eq . ( 1 4 ) . It has

only been used in a very l imited manner . Expre s s ion of thi s

equi l ibria in terms o f the concentration of tetracycl ine in

the broth ( Tc ' ) and the concentration within an individual

c e l l (Tci ), might be a de s i rable way of a l tering the present

model such that attainment o f equi l ibria between antib iotic

and ribosomes is not achieved .

I f the s e changes are not sufficient , then the e f fect

o f variation of the pseudo-rate cons tants , c1 and C 2 ,

\vi th time and w i th tetracycline concentration should be

cons i dered . The concentrations of e i ther tffiJA or rRNA in

the cul ture whi ch are contained in these " cons tants "

undoubtedly change during culture growth . I t seems l ikely

that tetracy c l ines would indirectly af fect these " constants "

by v irtue of the i r secondary inhibi t ion of nucleic acid

synthe s i s . The present work has uti l i zed the se parameters

as cons tants solely because this was the s imp l e s t and easiest

procedure . The introduction of such a mod i f i cation would

nece s s i tate a very large number o f integrations and is pro-

bab l y not practical . I t would require a great deal more

knowledge about metabol i c events within the inhibited bacterial

c e l l s than i s presently known in a quantitative manner . I t

should b e rea l i zed that the present work as we l l a s others

( 1 8 , 3 0 , 3 3 and 3 4 ) has measured e f fects on synthe s i s of

und i f ferenti ated nuc leic acids and not on the individual

species of ribonuc leic acids .

Another a l ternative to the present mode l would be to

formulate a s imi lar mode l on the bas i s of concentrations per

ce l l and then sum the se concentrations over the number o f

1 3 5

ce l l s which exi s t a t any given time . I n this type of a mode l

i t should be pos s ible to make certain s impl i fying assumptions :

such a s s teady- s tate concentrations o f various intermediates ,

perhaps R · S , tRNA and rRNA . The summation o f values over

indices which change with time and inhibitor concentration

doe s , however , present unusual problems in analog s imulation

and has not been uti l i zed in thi s s tudy .

1 3 6

V . Summary

S ixteen of the 1 8 tetracycl ine s under inve stigation

have been shown to inhibi t c e l l divis ion , prote in synthesis

and nuc l e i c ac id synthe s i s in cultures of exponenti a l ly

dividing E . c o l i W . The number o f cell s i n inhibited cultures

has been obtained as a function of time of growth by viable

and by total ce l l count methods . Sma l l diffe rences found between

the total number of c e l l s and the number of viable ce l l s are

not b e l i eved to be related to the e ffect of the tetracyclines

in these culture s . Rather the se d i fferences are felt to be

c aused by aggregation o f viable ce l l s during the assay for

v i ab l e c e l l s . A f i l te r technique us ing detergent-extracted

c e l lulose e s ter membranes was devel oped to isolate TCA-ethanol

precipitates of the E . col i ce l l s as a function of the time

o f growth . The hot TCA soluble extract o f these precipitate s

was used to measure nucleic ac ids by the orcinol reaction .

The hot TCA insolub l e material remaining on the fi lter was

tryps in-dige s ted and the protein content e s t imated by the

Fol in-Lowry a s s ay .

At l ow concentrations o f these tetracycl ines , inhibition

is produced after an initial time l ag during which no e ffect

on the te s ted culture parameters was seen . After thi s l ag

time , a l l tes ted culture parameters could be de scribed by

f i r s t order k inetic expre s s ions , such as ; Eq . ( 1 ) , ( 4 ) and

( 6 ) . Generation rate cons tants for te tracy c l ine- inhibi ted

cultures , kv , kt , kp , kna 1 have been obtained from the

1 3 7

l e a s t s quares f i t o f the exper imental data t o these equations .

I t s eems mos t l ike ly that generation rate constants obtained

for a l l culture parame ters are identical for any given

culture ; that i s , balanced growth is mainta ined . Thi s result ,

a lthough not s tr i ctly in agreement with previous reports i s

exp lained b y the f a c t that previous exper iments were e i ther

only s ingle-point measurements or were not determined over

a suffi c ient number of generations such that equi l ibrium

inhibitionwwas not observed by these workers .

Generation rate cons tants for a l l cul ture parameters

were found to be l inearly related to the concentration of

the tetracyc l ine ana log in the culture , Equations ( 2 ) , ( 5 )

and ( 7 ) . The proportional i ty constant s , k� , kf , k� , k�a ' o f

the s e re l ationships have been determined . When the data for

a l l culture parameters were treated together a s ingle

proportional ity cons tant , k�vg ' was obtained . Thi s value

i s a measure o f the antibacte r i a l potency o f the tetracycl ine

analog . These potencies are thought to be sui tab le for use

in s tructure-activi ty relationships s ince they are kinetic

cons tants determined by several d i f ferent a s s ay me thods .

The s imple mode l o f Garrett , Mi l ler and Brown ( 2 4 3 )

which attempts to expl a in the l inear re lationship o f

generation rate constants t o antibiotic concentration has

been pre sented . The i nadequacy o f the kinetic exp l anation

proposed by thi s mode l has been noted and attempts have been

made to derive a more satis fac tory mode l of te tracycl ine

inhib i tion . A mode l based on the b iochemical mode o f action

1 3 8

o f the tetracy c l ines was proposed . T h i s model was investigated

to see i f i t might l e ad directly to the l inear re lationship

o f generation rate constants to antibiotic concentration .

I t was fe l t that thi s might result from the inclusion of

reactions leading to the growth o f " subs trate " and " enzyme "

concentrations in add i tion to the usual enzyme-kinetic

reactions . Cond i t i on s such that the mode l generated output

cons i s tent with the experimenta l l y observed type of inhibition

were not found . O f course , a l l condi tions were not tes ted .

Several mod if i cations o f the mode l have been sugges ted such

that the experimental results might be exp la ined by thi s

type o f mode l but because o f the l arge number o f differential

equations which need to be s olved the se have not been te sted .

The time o f onset o f inhibi tion was approximately 3 0

minutes ( l l/2 generation s ) in these culture s . Thi s time

and the t ime o f recovery from inhibition upon di lution into

antibiotic- free med i a was observed to be shorter for the

inhibi tion of prote i n s ynthe s i s than for the inhibition of

cell divi s i on or nucl e i c acid synthe s i s . This difference in

time o f onset o f inhibi tion was espe c i a l ly pronounced when

the broth was changed such that the rate of growth was

reduced . Under the se condi t ions the time of onset o f

inhibi tion o f prote in synthe s i s d i d not vary whi le the time

o f onset o f inhibition o f c e l l divis ion and nuc leic acid

synthes i s were increased . An increase in broth pH vailiue ,

achieved by changing total phosphate as we l l as phosphate

ion ration , decreased the time of onset of inhibition of

c e l l mas s and c e l l div i s ion . These exper iments can be

mos t ea s i ly expla ined if one assumes that inhibition of

nuc leic ac id synthes i s and inhibi tion of cell divis ion are

the result of a pr ior inhibition of prote�n synthes i s . In

addi tion there i s a f inite lag time , which is broth pH

sensitive , before prote in synthe s i s is inhibited .

1 3 9

Thi s interpretation i s cons i s tent with , and these

resu l t s are further evidence tha t , the pr imary mode of ac tion

of tetracyc lines is to cause an inhibition of protein synthes i s .

S inc e a l l tetracyc l ine s behaved qua l i tatively the same it

seems mo st l ikely that they are a l l acting by the same

mechani sm . However , this cannot be said with certa inty

s ince the d i fferences between time of onset of inhibi tion

of protein synthes i s and the times of onset of inhibition of

c e l l d iv i s ion and nucleic acid synthe s i s were only measured

for culture s inhibited by oxytetracyc line . Indeed it should

be emphas i zed that these conc lus ions with regard to the mode

of act ion of oxytetracycl i ne are based on a s ing le exper iment ,

F ig . XXI .

1 4 0

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