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LIPIDS AND PHOSPHOLIPASE ACTIVITY OF VIBRIO CHOLERAE
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
BY
Buford L^Brian, M. A. \ \\
Denton, Texas
August, 1972
« < '
Brian, Buford L., Lipids and Phospholipase Activity
of Vibrio choleras. Doctor of Philosophy (Biological
Sciences}, August, 1972, 137 pp., 27 tables, 14 figures,
bibliography, 170 titles.
One purpose of this investigation is to determine the
fatty acid and lipid content of typical Vibrio cholerae cells.
The comparison of cholera lipid constituents with those of
closely-related bacteria might be of taxonomic value.
Furthermore, chemical characterization of the cholera vibrio
could provide useful criteria for identification of these
disease-producing microorganisms.
In an early V. cholerae lipid study, large amounts of
free fatty acid and phospholipid were observed when cholera
lipids were separated by thin-layer chromatography, and the
existence of a powerful cholera lipase was thus suggested.
Therefore, another purpose of this study is to determine
whether cholera cells contain phospholipase activity capable
of rapid hydrolysis of fatty acids from phospholipids.
Companion studies were conducted for the purpose of
development and extension of gas chromatographic techniques.
The esterification reagents boron trichloride and boron
trifluoride in methanol were tested for quantitative con-
version of cyclopropane fatty acids to their respective
methylesters. Hydrogenation and bromination procedures were
applied to the detection of cyclopropane fatty acids in
Pseudomonas aeruginosa. Fatty acids of lymphosarcoma and
liver of tumor-bearing DBA/1J mice were studied during tumor
development. Fatty acids of carcass, liver, and fat bodies
of Cnemidophorus ticrris prior to hibernation were investi-
gated. A new phenylthiohydantoin amino acid derivative, the
. acetate, was introduced and compared to the trifluoroacetate
with respect to gas-chromatographic applicability.
V. cholerae strain 569 3 (Inaba) contains 5.4-6.6%
lipid. Cholera lipids consist of ca 75% phospholipid and
ca 25% free fatty acid. Major phospholipids separated by
thin-layer chromatography are tentatively identified as
phosphatidyl ethanoiamine, phosphatidyl glycerol, and
cardiolipin.
Gas-liquid chromatography of fatty acids from thirty
cholera strains was conducted. Major acids found are myristate
(2-9%), palmitate (21-39%), hexadecenoate (34-46%), stearate
(2-5%), and octadecenoate (12-26%). Cyclopropane-ring-
containing compounds are not observed. The feasibility of
utilizing such chemical analyses for identification of
cholera vibrios is suggested.
Pnospho1ipase activity of the acyl hydrolase type
is j.ound in V. cno^srae strain 569 B {, Inaba) cell sonicates
32 1A
using P and C radioactive phospholipid substrates.
Results indicate that both fatty acids are removed from
phosphatidyl ethanoiamine, and that no detectable amounts
of lys©phosphatidyl ethanolamine are present in reaction
products. The phospholipase appears to be membrane-bound.
Reaction in the presence of EDTA 910 mM) and 3-hydroxyqu ino-
line (1 mM) shows that no divalent cation is required. One
and 10 mM concentrations of the chloride salts of calcium,
barium, magnesium, manganese(ous), zinc, iron (ferrous), and
mercury cause inhibition of the cholera phospholipase. No
inhibition is observed with potassium or sodium chloride
(1 and 10 mM). Triton X 100 optimum concentration is 1 mg/ml.
The of the cholera phospholipase for Escherichia coli -^P
4 -
phospholipid is 63 (~6) uM using sonicate (1.6 mg/ml protein)
in 25 mM borate buffer, pH 7.5 with 1 mg/ml Triton X 100.
An enzyme capable of selectively hydrolyzing membrane
phospholipids could contribute to the outpouring of fluids
and ions into the intestinal lumen, causing the extreme
diarrhea observed in cholera victims. A major contribution
to the understanding of the processes by which intestinal
pathogens cause diarrheal symptoms in disease victims might
be made by elucidation of the effect of bacterial phospho-
lipase activity on intestinal membrane constituents.
LIPIDS AND PHOSPHOLIPASE ACTIVITY OF VIBRIO CHOLERAE
DISSERTATION
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
BY
Buford L^Brian, M. A. \ \\
Denton, Texas
August, 1972
TABLE OF CONTENTS
Page
LIST OF TABLES v
LIST OF ILLUSTRATIONS
Chapter
I. LIPIDS OF VIBRIO CHOLERAE 1
Introduction Materials and Methods Results and Discussion
II. PHOSPHOLIPASE ACTIVITY OF VIBRIO CHOLERAE . . . 42
Introduction Materials and Methods Results and Discussion
III. GAS CHROMATOGRAPHY OF CYCLOPROPANE FATTY ACID METHYLESTERS PREPARED WITH METHANOLIC BORON TRICHLORIDE AND BORON TRIFLUORIDE . . . . 84
Introduction Materials and Methods Results and Discussion
IV. CYCLOPROPANE FATTY ACIDS OF PSEUDOMONAS AERUGINOSA 92
Introduction Materials and Methods Results and Discussion
V. TUMOR AND LIVER FATTY ACIDS OF DBA/1J MICE DURING LYMPHOSARCOMA DEVELOPMENT 100
Introduct i on Materials and Methods Results and Discussion
xxi
Chapter Page
VI. FATTY ACID DISTRIBUTION OF LIPIDS FROM CARCASS, LIVER AND FAT BODIES OF THE LIZARD, CNEMIDOPHORUS TIGRIS, PRIOR TO HIBERNATION 112
Introduction Materials and Methods Results and Discussion
VII. ANALYSIS OF ACETYLATED AND TRIFLUORO-ACETYLATED PHENYLTHIOHYDANTOIN AMINO ACIDS BY GAS CHROMATOGRAPHY 118
Introduction Materials and Methods Results and Discussion
LITERATURE CITED 123
IV
LIST OF TABLES
Table Page
Vibrio Cholerae Strains 6
II. Non-Cholera Vibrios 7
III. Phosphorus Analysis of Thin-layer Chromatography-Separated Phospholipids of Vibrio Cholerae Strain 569 B (Inaba) and Comparison with Escherichia Coli . . . . 17
IV. Percent Yield of the Simmons-Smith Reaction as Determined by Gas Chromatography . . . . 20
V. Gas Chromatographic Reproducibility Using One Sample of Fatty Acid Methylesters (NIH 41) Analyzed Ten Times 22
VI. Effects of Hydrogenation on Percent
Fatty Acid Methylesters 24
VII. Fatty Acid Distribution of Vibrio Cholerae . . 25
VIII. Fatty Acid Distribution of Non-Cholera Vibrios 27
IX. Effects of Incubation Temperature on Fatty Acid Distribution (Percentages) of NIH 41 29
X. Percent Fatty Acids in Lipid Fractions . . . . 30
XI. Phospholipase D Assay 60
XII. Assay of Vibrio Cholerae Sonicate for Phospholipase Activity Using 32P Phosphatidyl Glycerol as Substrate 63
XIII. Effects of Tris-HCl (pH 8.0), EDTA, 8-Hydroxyquinoline and Dialysis on Reaction of Sonicate 68
XIV. Percent Inhibition by Chloride Salts (One and Ten mM) on Phospholipase Activity of V. Cholerae Sonicate 69
v
Table
XV.
XVI.
XVII.
XVIII.
XIX.
XX.
XXI.
XXII.
XXIII.
XXIV.
XXV.
XXVI.
Effects of Triton X 100 Concentration on Phospholipase Activity of Sonicate . .
Phospholipase Activity of XM-100A, PM-30, and UM-10 Membrane Filter Retentates of V. Cholerae Sonicate . . .
Page
71
75
Ratio of the Peak Areas of Cis-9,10-Methylene Octadecanoate (Cyc C-̂ g) to Heptadecanoate (^7) Following Esterification (1 mg of Each Acid) . . . .
Effects of Incubation Temperature on Pseudomonas Aeruginosa Fatty Acids . . . .
Fatty Acids (%) of Pseudomonas Aeruginosa Strains Incubated 40 C
Results of Hydrogenation and Bromination on Pseudomonas Aeruginosa Fatty Acids . .
Mean Net Weight of Tumors and Livers from Tumor-Bearing Mice at Various Stages of Tumor Development
Percent of Lipid in Tumors and Livers from Tumor-bearing Mice at Various Stages of Tumor Development ,
Percentage of Fatty Acids Occurring in Liver Lipids of Tumor-bearing Mice at Various Stages after Implanation . . . . ,
Percentage of Fatty Acids Occurring in Tumor Lipids at Various Stages after Implanation ,
Male and Female C. Tigris Body Measurements (mm), Tissue Dry Weights (mg), and Lipid Content Expressed as Percentages of the Dry Weight of Each Tissue
88
94
95
96
104
106
107
108
115
Male and Female C. Tigris Fatty Acids from Carcasses, Livers, and Fat Bodies Expressed as Percentages (Mean Values) of the Total Fatty Acid Content 116
VI
Table Page
XXVII. Relative Retention Times of Amino Acids Phenylthiohydantoin Acetates and Trifluoroacetates 120
LIST OF ILLUSTRATIONS
Figure Page
1. Growth Curve of Vibrio Cholerae Strain 569 B (Inaba) by Optical Density (O.D.) Readings (540 nm) 9
2. Thin-layer Chromatogram of V. Cholerae (569 B) Lipids with a Standard Mixture of Phospholipids 15
3. Thin-layer Chromatogram of E. Coli (ATCC 11775) Lipids and V. Cholerae (560 B) Lipids 16
4. Gas-liquid Chromatogram of Fatty Acid Methylesters of V. Cholerae Strain NIH 41 21
5. Thin-layer Chromatogram of Major Egg Yolk Phospholipids and Purified Phosphatidyl Choline . 53
6. Assay of Crotalus atrox Venum (2.5 ug) Phospholipase A? Activity on Phospha-tidyl Choline (10 umoles) with Time (22 C) 5 5
7. Assay of Crotalux atrox venom (2.5 ug) Phospholipase A? Activity on 5-50 umoles of Phosphatidyl Choline (10 min, 27 C.) 5 5
8. Thin-layer Chromatogram of Bacterial Phosphatidyl Ethanolamine (PE) and Lysophosphatidyl Ethanolamine (LPE) Prepared by Reaction of PE with Crotalux atrox venom 57
9. Optical Density (o. D.) Readings (526 nm) Following Reaction of 4-20 umoles of Choline Hydrochloride with Ammonium Reineckate (Average of Two Determinations) . . 59
Vlll
Figure Page
10. Thin-layer Chromatogram of E. coli (ATCC 11775) 32p phospholipids 62
11. Thin-layer Chromatograms of Cholera Phospholipase Products Following Reaction with Phosphophatidyl Ethanolamine (21 nmoles) for 1 hr, 37 C 65
12. Assay of Vibrio Cholerae Strain 569 B (Inaba) "Roux Flaslc Supernatant" (6 mg/ml Protein) for Phospholipase Activity Using 32p phosphatidyl Ethanolamine (11 nmoles) as Substrate 66
13. Effect of Enzyme (Sonicate) Concentration on Reaction Rates of V. Cholerae Phospholipase Activity Using -^P (16.6 nmoles) as Substrate 72
14. Initial Velocities (nmoles/hr) of "Roux Flask Supernatant," 6 mg/ml Protein, and Sonicate, 1.6 mg/ml Protein, with Lineweaver-Burk plots 73
xx
CHAPTER I
LIPIDS OF VIBRIO CHOLERAE
Introduction
The introduction of gas chromatography to microbial
lipid chemists (55, 56) began a surge of investigations on
the fatty acid content of bacteria. Reports of fatty acid
and lipid content of many bacterial species have been exten-
sively reviewed (3, 29, 49, 53, 61, 62, 68, 72, 85, 87).
Bacterial fatty acids consist chiefly of straight-chain or
branched-chain saturated compounds, monoenes, hydroxyl- or
cyclopropane-ring-containing acids (85, 87).
Hydroxy fatty acids were found as major constituents
of 1ipopolysaccharides in Escherichia coli (26) and Proteus
(82). Branched-chain (iso, anteiso) compounds were reported
to be in large amounts in Sarcina lutea (52, 100, 102),
Bacillus species (57, 58, 66), and Staphylococcus aureus
(107). Fatty acids containing cyclopropane rings were
reported in Serratia marcescens (6, 7, 64, 65), E. coli
(28, 33, 36, 59, 60, 70, 78, 94, 106), Pseudomonas
fluorescens (16, 34), Pseudomonas aeruginosa (23, 47, 103),
Lactobacillus species (49, 50), Aqrobacterium tumefaciens
( 6 0 )' Streptococcus species (75, 76), Rhodomicrobium
vannielii (88), Klebsiella pneumoniae (37), rugose variants
1
2
of Vibrio cholerae (17, 19), and a strain of Vibrio fetus
(101). The most commonly reported cyclopropane fatty acids
are cis-9,10-methylene hexadecanoate (59) and cis-11,12-
methylene octadecanoate (lactobacillic acid, 50).
The mechanisms involved in the synthesis of cyclopro-
panes were the subject of several papers (30, 31, 35, 48, 71,
84, 86, 90, 109) and were shown to involve construction of a
methylene bridge across the double bond of palmitoleic or
cis-vaccenic acids with S-adenosylmethionine as the carbon
donor to produce the two cyclic compounds described. Bacteria
do not synthesize polyunsaturated acids (85). The chief
monounsaturates reported were cis-9,10-hexadecenoate
(palmitoleate) and cis-11,12-octadecenoate (cis-vaccenate).
Oleic acid (cis-9,10-octadecenoate) was found in the tubercle
baccilus (27). Hexadecenoate and octadecenoate appear to be
major fatty acids of V. cholerae (18).
Differences found in fatty acid content of bacterial
species have prompted some investigators to suggest that
such gas chromatographic data might prove valuable for pur-
poses of identification of bacteria (2, 11, 98). Patty acid
profile patterns could only be useful for characterization
if cultural and analytical techniques were standardized,
since age of culture, incubation temperature, and media
constituents are known to have drastic effects on fatty acid
distribution (65, 66, 70, 78, 94). Furthermore, unreliability
3
of analytical techniques or strain differences introduce
further variables and might have accounted for failure by some
-workers to report cyclopropane acids in P. aeruginosa (9, 38,
91), since others (23, 47, 103) have indicated their presence
in this species. However, use of gas chromatographic data
for characterization of bacteria deserves attention by those
equipped for such analyses.
Thin-layer, gas-liquid, and silicic acid column
chromatography have replaced earlier separation methods such
as paper chromatography, distillation, and electrophoresis
for lipid purification. However, many cases of technical
incompetency cloud the literature in this area, and it
should be emphasized that complete utilization of the
methodology requires a highly-trained technologist.
The most widely-studied species for lipid content is
c o l i (60, 83), which was reported to contain phosphatidyl
ethanolamine, phosphatidyl glycerol, and cardiolipin as major
phospholipids. It seemed feasible to use E. coli for com-
parative purposes in lipid studies of V. cholerae.
Phospholipid chemistry and metabolism of bacteria.
Plants, and animals have been reviewed by many workers (1,
3, 28, 45, 46, 61, 62, 68, 72, 87). Lipids are used for
energy storage as well as for structural elements in cell
membrane construction (92). Attempts have been made to dis-
cern correlations between lipid composition and pathogenecity
4
(18, 81) of microorganisms, but no clear-cut relationship has
been found.
Lipid is known to comprise an integral part of bac-
terial endotoxins (26). Investigation of purified Type 2
toxin (choleragen) of V. cholerae strain 569 B (25, 32) by
Kaur, et al. (69) showed that the exo-entero-toxin contained
30% lipid. Fatty acids of the toxin (90) were similar to
those previously reported in whole-cell vibrios (18).
Further purification of the choleragen (40-43, 73) resulted
in an active toxin with a molecular weight of 84,000 and
with little lipid remaining. Pierce and Greenough (89)
reported that the Type 2 toxin stimulated glycerol produc-
tion in isolated fat cells. The means by which such lipolytic
activity occurs is not known.
A paucity of knowledge is available on the lipids of
¥• cholerae. One investigation (4) of cholera lipids
utilized cholera vaccine. Fatty acids were analyzed by paper
chromatography (4) and bear little resemblance to those
determined by gas—liquid chromatography by Brian and Gardner
(13, 14—19). Blass (8) investigated the nitrogen—containing
phospholipids of the cholera vibrio. Electrophoretic
techniques were used to separate nitrogenous lipid components.
Ethanolamine, ornithine, and several amino acids were found.
In an early V. cholerae lipid study (18), large
amounts of free fatty acid and phospholipid were observed
5
when cholera lipids were separated by thin-layer chromatography,
and the existence of a powerful cholera lipase was thus sug-
gested. Discovery of the presence of lipolytic activity
in the vibrio might provide ample incentive for rapid
extraction of cells with lipid solvents. Cells which are
allowed to stand at room temperature for some appreciable
time would surely be a poor source for lipids (a possibility
given little consideration by some workers).
The purpose of this investigation was to determine
the fatty acid and lipid content of typical V. cholerae
cells. The comparison of cholera lipid constituents with
those of closely-related bacteria might be of taxonomic
value. Furthermore, chemical characterization of the
cholera vibrio could provide useful criteria for identifi-
cation of these disease-producing microorganisms.
Materials and Methods
Bacterial Species. Vibrio cholerae strains investi-
gated are given in Table I. The three major antigenic types
(AB - Ogawa, AC - Inaba, and R - Rough) were determined by
slide agglutination with monospecific rabbit antisera (22).
V. cholerae rugose (108) strains studied have been previously
described (10, 11, 17, 19, 22). Non-cholera vibrios investi-
gated are shown in Table II. Strains which demonstrated
typical vibrio morphology and monotrichous polar flagella-
tion but which failed to agglutinate in anti-cholera sera
6
TABLE I
VIBRIO CHOLERAE STRAINS
Strain Antigenic Components Source
NIH 41 AB (Ogawa) National Institutes of Health
NIH 35 A3 AC (Inaba) II
ATCC 14035 AB American Type Culture Collection
569 B AC R. A. Finkelstein Ca 72 AB, Rough (R) W. Burrows, and C. E.
Lankford, isolated in Calcutta, India, 1953
Ca 113 AB II
Ca 323 R II
Ca 324 R II
Ca 325 R II
Ca 385 R II
Ca 412 AB II
Ca 424 AB II
IRAN 46 AB J. C. Feeley, National Institutes of Health .
Iraq 230 AB 11 B 29112 AB 11 B 1307 AB H "7 "
J 8001 AB II
ROK 350 AB II
M 3735 AB 28/62 AB II
VC 12 Rxl AB II
El 36 AB II
CH-1 AB II
Ubon 13 AB II
0-4 AB II
VN-1 AB II
CRC 31/64 • AB II
1222 AB HK-1 AB II
V 86 AC II
TABLE II
NON-CHOLERA VIBRIOS3
Strain Source Geographic Location
8032 Diarrhea Philippines
485 II Thailand
942 11 India
8288 Freshwater Philippines
8305 II II
6471 II East Pakistan
9682 Sewage United States
aObtained from H. L. College.
Smith and K. Goodner, Jefferson Medical
8
were designated as non-cholera vibrios (96). Morphological
characteristics of vibrios were determined by T. 0. McDonald
(Alcon Laboratories, Port Worth, Texas) using an electron
microscope. Escherichia coli (ATCC 11775) was also studied
for purposes of comparison with cholera lipids. Bacteria
were maintained in the lyophilized form or on slants (2 C)
of Trypticase Soy Broth (BBL) with 2 percent agar (Difco).
Cultural Conditions. Bacterial species were grown
in Trypticase Soy Broth (TSB), previously shown to be essen-
tially lipid-free (18, 75, 76), or in Rome flasks containing
100 ml of TSB with one-two percent agar (Difco). One ml of
a sixteen hour (37 C) broth culture was used to inoculate
each liter of broth or each Roux flask. Broth cultures
(one to ten liters) were incubated (37 C)until the late
stationary growth phase as determined by optical density
(Fig. 1) using a Spectronic 20 colorimeter (540 nm).
Bacteria were harvested in 250 ml bottles by centrifugation
(5000 x g, 15 min, 4 C) using a Sorvall RC-2B refrigerated
centrifuge equipped with a GSA rotor. Roux flasks were
incubated 37 C, 24 hours. Cells were harvested by washing
the agar surface with distilled water followed by centrifu-
gation. Wet cell paste was extracted for lipid, but in
some cases bacteria were lyophilized to determine dry
weight. Bacteria were streaked for isolation onto Petri
plates containing agar media both before and after initial
.6
.5'
£ A c.4 O
2.3 o
.2
.1
"T" 8
—r-11
—i 13 10
Hours
12
Fig. 1—Growth curve of Vibrio cholerae strain 569 B (Inaba) by optical density (0. D.) readings (540 nm).
10
inoculation as well as after "harvest to determine presence of
contaminating microorganisms. If no contamination was appar-
ent, lipid extraction was begun.
Lipid Extraction. Lipids were extracted from known
weight of tissues or bacteria by the method of Folch, et al.
(44) as previously described (11-21, 23). A minimum of
twenty volumes of chloroform:methanol (2:1, volume/volume)
was used per gram of tissue or cells. Both chloroform and
methanol were distilled before use. Lipid was concentrated
either by a stream of nitrogen or by rotary evaporation
under partial vacuum and then diluted to known volumes with
chloroform:methanol (2:1, volume/volume). Portions of lipid
materials were dried (70 C) on pre-weighed aluminum foil for
dry-weight determination. Phosphorus was quantitated by
the Bartlet procedure (5, 74) using a Cary 14 spectrophoto-
meter at 830 nm.
Thin-layer Chromatography. Thin-layer chromatography
(TLC) was performed using 20 x 20 cm, 5 x 20 cm, and 1 x 3
inch glass plates. Silica gel G (Curtin Chemical Co.)
aqueous slurries were spread onto plates with a thickness
of 0.25-0.5 mm and dried overnight at 26 C. Ammonium sul-
fate (1%, weight/volume) was added to the aqueous slurry
when sample charring, following TLC separation, was desired
(79, 104).
11
TLC-separated lipids were visualized by iodine vapors
or by heating TLC plates (220 C) which contained ammonium
sulfate. Sulfuric acid which results from this treatment
effectively decomposed lipids to visible carbon spots.
Phosphatidyl ethanolamine (PE) was detected by spraying TLC
plates with 0.3% ninhydrin in acetone. Phosphatidyl glycerol
(PG) was detected with periodate-schiff reagent (63), and
Dragendorff reagent (97) was used to detect phosphatidyl
choline (PC). Standards of PC and PE were purchased from
Applied Science Laboratories and cardiolipin (CL) from
Supelco, Inc.; PG was produced by reaction of PC with cabbage
transphosphatidylase in the presence of 20% glycerol (70).
Oleic acid, triolein, methyl oleate, and cholesterol oleate
were from Applied Science Laboratories.
TLC solvent systems used were:
Solvent A (105)—Petroleum ether: diethyl ether:
glacial acetic acid (90:10:1, volume/volume).
Solvent B (77)—Chloroform: methanol: water
(60:30:5, volume/volume).
Solvent C (74)—Chloroform: methanol: glacial
acetic acid: water (80:13:8:0.3, volume/volume).
Gas-liquid Chromatography (GLC). GLC (15, 20, 24,
54, 56, 99) was performed using methylesters of fatty acids
hydrolyzed from lipid constituents. Lipids were hydrolyzed—
esterified by boiling one to ten mg lipid with 0.5 N
12
methanolic sodium "hydroxide (2 ml) followed by 3 ml of 10%
boron trichloride in methanol (Applied Science Laboratories)
as detailed earlier (24, 80). Standard fatty acids and
methyl esters were purchased from Applied science Laboratories
unless otherwise noted.
Fatty acid ester mixtures were hydrogenated (18, 39)
using a platinum catalyst followed by bromination (16) to
detect unsaturated and cyclopropane fatty acids. Cyclopro-
pane methylesters (cis-9,10-methylene hexadecanoate, cis-9,
10-methylene octadecanoate, and cis-11,12-methylene octade-
canoate) were synthesized from palmitoleate, oleate, and
cis-vaccenate, respectively, using the simplified zinc-
copper couple of Shank and Schecter (93) and the Simmon-
Smith reaction (95). Synthesized compounds were purified
using a Varian Aerograph (Varian Associates, Palo Alto,
California) gas chromatograph equipped with a hydrogen-
flame detector, column-effluent splitter, and a column
(6 feet x 0.25 inch, O.D.) packed with 15% diethylene
glycol succinate polyester on Chromosorb W (60/80 mesh)
operated at 180 C.
Quantitative GLC of fatty acid methylesters of
lipid extracts was accomplished using a Varian Aerograph
gas chromatograph Model 204 series equipped with dual flame
hydrogen detectors and a i m volt (full scale) recorder.
One-ten ug of fatty acid methylesters was injected with a
13
10 ul syringe with a range setting of lO--^ and attenuation
of 4-16. Columns (10 feet and 5 feet x 0.125 inch, O.D.)
containing 15-20% diethylene glycol succinate polyester
(DEGS) on Chromosorb W, 60/80 mesh (Applied Sciences Lab-
oratories), were operated isothermally at 180 C. Carrier
flow rate (helium or nitrogen) was 20-25 ml/min, hydrogen
was 30 ml/min, and air was 200-300 ml/min. Peaks obtained
were quantitated by a disc integrator (Disc Instruments,
Inc.) or by multiplication of peak height x width at 1/2 peak
height. Quantitative standards (51) were used to test
linearity of detector response over a wide range of
methylesters.
Results and Discussion
Lipids of Vibrio cholerae. Percent lipid on a dry
cell basis of typical cholera vibrios was reported by Brian
and Gardner (18). Average amount of weighed lipid ranged
from 5.8-7.6% (18). Strain 569 B (Inaba) was found to con-
tain 5.4-6.6% lipid. Phosphorus analysis of the lipid
extracts indicated phosphorus levels of ca 3% of weighed
lipid and ca 0.17% of dry cell weight. Since phosphorus
comprises 4-4.4% of a phospholipid, it was calculated that
cholera lipid investigated consisted of 70-75% phospholipid.
The remaining lipid (25-30%) appeared to be free fatty acids.
The observation of large amounts of free fatty acids in
cholera strains by TLC using solvent A suggested that
14
phospholipase activity had occurred.
A standard mixture containing PE, PG, and cardiolipin
(bacterial) was spotted on TLC plates beside cholera (569 B)
lipids and separated by solvent C (Pig. 2). Migration of
569 B lipid components suggested presence of PE, PG, CL, \
neutral lipid (fatty acids), and ninhydrin-positive material
at the origin. PE was ninhydrin-positive, and the PG spot
reacted positively with periodate-schiff reagent. Tentative
identification of cardiolipin was by Rf only (solvents B and
C). Solvent B did not separate PE and PG. Therefore, solvent
C was chosen for subsequent phospholipid separation by TLC.
The separation of E. coli and V. cholerae lipids
indicated no qualitative differences in phospholipid content
(Fig. 3). This finding was not surprising since the cultural
conditions, media, and extraction techniques used with these
two Gram-negative species were similar. Differences in lipid
content of bacteria which appear in the literature often
have been due to inconsistencies in growth and lipid tech-
niques (85). It seemed feasible to use the most vigorously
studied bacterium (E. coli) for comparative investigation
of cholera lipids.
Strain 569 B lipids containing 0.082 umoles of
phosphorus were spotted on TLC plates and separated by sol-
vent C. Spots were visualized with iodine, scraped into
tubes, and analyzed for phosphorus content (5). Table III
15
- S o l v e n t
o N e u t r a l l i p id
O o CL
0 0 PG
0 0 PE
0 • O r i g i n
Fig. 2—Thin-layer chromatogram of V. cholerae (569 B) lipids (left) with a standard mixture of phospholipids (right) Neutral lipid = fatty acid. Phosphatidyl glycerol (PG) was periodate-schiff-positive, phosphatidyl ethanolamine (PE) was ninhydrin-positive. Origin contained ninhydrin-positive material. Solvent was chloroform: methanol: acetic acid: water (80:13:8:0.3, volume/volume). Cardiolipin = CL.
16
Solvent Neutral lipid CL
00 Origin
Fig. 3—Thin-layer chromatogram of E. coli (ATCC 11775) lipids (left) and V. cholerae (569 B) lipids (right). Solvent chloroform: methanol: acetic acid: water (80:13* 8:0.3, volume/volume).
17
TABLE III
PHOSPHORUS ANALYSIS OF THIN-LAYER CHROMATOGRAPHY-SEPARATED PHOSPHOLIPIDS OF VIBRIO CHOLERAE STRAIN 569 B (INABA)
AND COMPARISON WITH ESCHERICHIA COLI
Tentative Identification Per cent Phosphorus V. cholerae E. colia
Origin 8
Phosphatidyl ethanolamine 71 78
Phosphatidyl glycerol 17 16
Cardiolipin 4 6
Neutral lipid (fatty acids) 0
a Calculated from published data (83).
18
shows relative percent of phosphorus found in each spot.
Values obtained from an area of silica gel containing no
visable lipid were subtracted from visable spots. Results
suggest that PE is by far the major component (71%) with
PG second (17%). Cardiolipin (4%) and a spot at the origin
(ninhydrin-positive) appear as minor constituents. The
origin may contain small amounts of phosphatidyl glycerol-
O-aminoacyl ester as reported in Serratia marcescens (64).
However, it is not uncommon in lipid extractions to trap free
amino acids as well as other water-soluble compounds by their
mutual attraction to phospholipid. Table III also shows the
phospholipid composition of E. coli which was calculated from
published data (83). A close relationship between phospho-
lipids of V. cholerae and E. coli was demonstrated. Ikawa
(53) has shown that closely-related bacterial species con-
tain similar lipids. Chemical characterization, in order to
be valuable for taxonomic purposes, must be conducted with
a large number of bacterial species. This report is an attempt
to provide such information of V. cholerae lipids.
Gas-liquid Chromatography (GLC). GLC techniques have
been discussed previously (11-12, 23). National Institutes
of Health fatty acid standards (51) were used to determine
linearity of detector (hydrogen flame) response over a wide
range of carbon numbers. An attempt was made to set up GLC
conditions that would provide less than 10% error on minor
19
components (less than 10% of total fatty acids) and less than
5% error on major acids (more than 10% of total).
Cyclopropane fatty acids were synthesized using a
simple zinc-copper couple (93) and the Simmons-Smith reaction
(95). The reaction mixture was analyzed toy GLC for percent
yield (Tatole IV) and then purified toy preparatory GLC (24).
Purified cyclopropane fatty acid methylesters (99% pure toy
GLC) were used as reference standards. Cyclopropanes were
resistant to mild hydrogenation tout reacted with bromine
(16, 17).
Esterification of fatty acids to their corresponding
methylesters using bacterial lipids containing cyclopropanes
has been reported (20). Boron-trichloride (BCI3) in methanol
(10%) was found to be a satisfactory esterification reagent.
Fatty Acids of Vibrio cholerae. The fatty acids of
typical V. cholerae (strain NIH 41) as revealed by GLC is
shown in Fig. 4. Major fatty acids were myristic (^4)/,
palmitic (C16), hexadecenoic (c16_)/ stearic (C1Q), and
octadecenoic (C2g=) acids. No cyclopropanes were found.
NIH 41 fatty acids were injected into the gas chromatograph
ten consecutive times and analyses made in order to deter-
mine GLC reproducibility (Table V). Average percent seemed
less significant than range obtained. Fatty acids comprising
over 30% of the total deviated less than 5% from the average
while low values deviated considerably. Subsequent reports
20
TABLE IV
PERCENT YIELD OP THE SIMMONS-SMITH REACTION AS DETERMINED BY GAS CHROMATOGRAPHY
Reactant Product Percent Yield
methyl palmitoleate methyl cis-9,10-methylene hexade-canoate 69
methyl oleate methyl cis-9,10-methylene octa-decanoate 53
methyl cis-vaccenate
methyl cis-11,12-methylene octa-decanoate 51
21
So I v e n t
Fig, 4—Gas-liquid chromatogram of fatty acid methyl-esters of V. cholerae strain NIH 41. Column (10 ft. x 0.125 in.) was operated at 180 C (isothermally).
22
TABLE V
GAS CHROMATOGRAPHIC REPRODUCIBILITY USING ONE SAMPLE OF FATTY ACID METHYLESTERS (NIH 41)
ANALYZED TEN TIMES
Fatty acida Per cent found (average) Range
14:0 3.5 3.4 - 3.8
iso 16:0 1.0 0.5 - 1.1
16:0 32.6 31.1 - 33.6
16:1 39.8 38.2 - 40.8
17:0 1.1 0.7 - 1.4
iso 18:0 i. 4 0.4 - 2.5
18:0 4.0 3.5 - 4.7
18:1 15.8 14.8 - 16.8
aNumber preceding colon indicates number of carbons, and number following designates degree of unsaturation.
23
of cholera lipids did not include fatty acids found to be
less than 1% of the total.
Brian and Gardner reported on the use of hydrogenation
followed by bromination for the detection of cyclopropane-
ring-containing fatty acids in bacterial lipids (16). Table
VI shows the effects of hydrogenation of methylesters of
fatty acids of E. coli and V. cholerae. Unsaturated com-
pounds were converted to the corresponding saturates.
Cyclopropane fatty acids (eye 17:0 and eye 19:0) were not
affected by hydrogenation but were eliminated from gas
chromatograms following bromination (16). Brominated fatty
acid methylesters were retained by the gas chromatographic
column, and their elution was not observed. The cholera
vibrio did not appear to contain cyclopropanes as major
lipid constituents, thus indicating the absence of the
cyclopropane fatty acid synthetase enzyme (71). Fatty
acids of E. coli (Table VI) were comparable to those found
by other workers (28, 29, 33, 36, 59, 60, 70, 78, 94, 106).
The small amounts of branched-chain compounds (iso 16:0 and
iso 18:0) found in V. cholerae lipids (16) appeared to be
absent in E. coli.
Tables VII and VIII give fatty acids of a wide range
of cholera and non-cholera vibrios. Qualitatively, the
distributions of acids are identical. Quantitatively, little
differences exist. Fatty acids reported here are similar to
24
TABLE VI
EFFECTS OF HYDROGENATION ON PER CENT FATTY ACID METHYLESTERS
Fatty acida Rtb E. coli V. cholerae Rtb Before After Before After
14:0 0.56 3.8 4.2 3.9 4.6
15:0 0.74 1.2 1.3 t t
iso 16:0 0.87 _d 2.0 2.3
16:0 1.00 43.8 46.2 29.5 62.8
16:1 1.18 2.1 - 34.2 -
17:0 1.34 1.4 t 1.2 1.9
eye 17:0C 1.54 20.9 22.0 - -
iso 18:0 1.56 - - 1.8 t
18:0 1.82 te 7.6 3.1 26.8
18:1 2.08 7.2 - 22.9 -
eye 19:0C 2.82 18.8 17.8 - -
3 Number preceding colon indicates number of carbons, and number following designates degree of unsaturation.
•^Retention time relative to 16:0 (palmitic acid).
cCyclopropane fatty acids (eye 17:0 and eye 19:0) were resistant to hydrogenation but reacted with bromine.
^No peak detected.
et = trace (less than
25
TABLE VII
FATTY ACID DISTRIBUTION OF VIBRIO CHOLERAEa
Strain 14: 0 iso 16:C I 16: 0 16: Fatty Acid*3
1 17:0 iso 18:< D 18 i:0 18: 1
NIH 41 3. 9 2. O 29. 5 34. 2 1.2 1. 8 3. 1 22. 9
NIH 35 A3 4. .4 1. 0 37. 7 36. 9 t c 1. 1 3. 1 14. 7
ATCC 14035 3. ,3 2. 7 28. ,3 42. , 3 1.0 1. 5 2. 6 17. 0
569 B 1. ,7 2. 6 24. ,8 40. ,2 t 2. 5 3. ,1 24. 6
Ca 72 2. ,2 t 38. ,0 39. ,5 1.1 1. 1 2. 18 13. 2
Ca 113 4. ,7 1. 6 37. ,2 36. ,2 t t 2. ,5 17. ,5
Ca 323 3. ,3 1. 8 30. .1 42. ,0 1.1 1. 8 3. ,3 15. ,0
Ca 324 6. ,4 t 34. ,5 41. ,0 t t 3. ,3 12. ,7
Ca 325 9. .0 t 38. ,6 37. .5 t t 2. ,6 12. .0
Ca 385 1. .3 t 26. .0 43. .4 t t 3. .7 23. .9
Ca 412 4. .0 1. 5 33. .0 39. .0 1.2 1. 5 3. .7 15. .0
Ca 424 4. .1 1. 7 34. A 38. .2 1.2 1. 5 2. .9 14. .8
Iran 46 1. .8 2. 0 24, .3 41. .0 t 2. 0 2. .8 25. .0
Iraq 230 2. .0 2. 2 23, .3 40, .0 t 2. 1 3. .5 25, .6
B 29112 2. .0 2. 0 25, .0 41, .0 t 1. 0 3, .0 25, .0
B 1307 3, .0 3. 0 24, .0 44, .0 1.0 2. 0 2, .0 20, .0
J 8001 3, .7 1. 0 28, .0 40, .9 t 1. 0 2, .4 21, .6
ROK 350 2, .1 3. 4 22, .0 39, .7 1.0 2. 6 3, .6 24, .6
M 3735 2, .6 2. 0 22, .6 42, .0 t 2. 1 2, .9 24, .0
28/62 1, .4 2. 0 21, .4 39, .2 1.0 2. 5 5, .0 26. .4
VC 12 Rxl 2, .7 2. 5 23, .6 46, .0 1.1 2. 5 2, .5 18, .0
26
TABLE VII—Continued
Strain 14:0 iso 16:0 16:0 16:1 17:0 iso 18:0 18:0 18:1
El 36 1.7 2.5 24.6 40.3 t 1.7 3.5 25.0
CH-1 2.8 1.8 27.5 41.0 1.1 1.7 3.9 18.8
Ubon 13 1.5 2.7 23.2 41.8 t 2.2 2.8 24.6
0-4 2.9 2.2 27.6 44.6 t 1.7 1.7 17.7
VN-1 1.5 2.1 22.9 39.6 t 2.3 4.0 26.1
CRC 31/64 4.3 1.4 29.3 44.8 t 1.0 2.6 15.4
1222 1.8 1.8 24.2 38.1 t 1.8 4.2 26.5
HK-1 1.8 1.8 22.4 44.3 t 1.3 2.8 24.6
V 86 2.1 2.7 28.4 40.5 1.0 2.0 4.2 18.0
<3.
Percent of each fatty acid.
ID Number preceding colon indicates number of carbons, and number following colon designates degree of unsaturation. c t = trace (less than 1% of total).
21
TABLE VIII
FATTY ACID DISTRIBUTION OF NON-CHOLERA VIBRIOS9
Strain 14:0 iso 16:0 16:0 Fatty Acid13
16:1 17:0 iso 18:0 18:0 18:1
8032 2.4 1.2 26.0 40.0 1.0 1.6 4.8 22.8
485 2.0 3.6 25.2 36.8 1.0 2.1 4.0 24.0
942 2.0 1.0 23.3 35.6 1.2 2.3 9.8 24.2
8288 2.5 2.3 28.8 37.0 1.2 2.2 3.9 21.2
8305 1.6 1.4 26.1 38.8 1.0 1.6 4.4 24.4
6471 4.1 1.3 33.2 41.4 t c t 2.8 14.4
9682 1.6 2.8 25.6 35.8 1.0 2.8 4.9 24.3
aPer cent of each fatty acid.
^Number preceding colon indicates number of carbons, and number following designates degree of unsaturation.
°t = trace (less than 1% of total).
28
those reported for Vibrio metchnikovii (28), Vibrio fetus
(101), and the halophile Vibrio costicolus (67).
Although composition of the culture media is known
to affect bacterial lipids (33, 38, 66, 70, 100), no factor
affects lipid and fatty acid more profoundly than incubation
temperature (7, 23, 65, 70, 78, 83, 94). Table IX gives
changes in fatty acid content of V. cholerae strain NIH 41
grown at 25, 32, and 37 C. As temperature of incubation
increases, unsaturation decreases. Hexadecenoic acid (16:1)
is the major fatty acid at incubation temperatures of 37 C
and below. No other bacterial species has been reported to
contain such a large amount of 16:1 under these growth con-
ditions. It has been suggested that fatty acid profiles may
be used for bacterial identification (2, 61, 85, 87, 98).
In order to utilize effectively GLC data for identification
purposes, incubation temperature, as well as other cultural
conditions, must be rigorously controlled.
Of all the cholera vibrios studied to date, only the
rugose morphological mutant (10, 22, 108) has been found to
contain cyclopropane fatty acids (17, 19). It was suggested
that cyclopropanes may play a role in the survival mechanism
of these resistant variants (17).
Distribution of fatty acids in polar (phospholipid)
and neutral (fatty acid) lipids separated by TLC (Table X)
was investigated (18). The major acids of the phospholipid
fraction were palmitate (47-50%), hexadecenoate (22-24%),
29
TABLE IX
EFFECTS OF INCUBATION TEMPERATURE ON FATTY ACID DISTRIBUTION (PERCENTAGES) OF NIH 41
Fatty Acida
25 C Incubation Temperature
32 C 37 C
14:0 1.7 2.1 3.3
iso 16:0 t b 1.0 1.0
16:0 16.4 21.8 31.2
16:1 51.0 41.5 40.3
17:0 t t 1.1
iso 18:0 t 1.9 1.2
18:0 t 3.1 3.9
18:1 29.5 26.7 16.9
3 Number preceding colon indicates number of carbons, and number following designates degree of unsaturation.
= trace (less than 1% of total).
30
TABLE X
PER CENT FATTY ACIDS IN LIPID FRACTIONS
Extractable lipids Polar lipids Neutral lipids Fatty acida (phospholipids)
NIH 41 Ca 324 NIH 41 Ca 324 NIH 41 Ca 324
14:0 3.3 6.4 3.5 7.4 2.9, 4.5
iso 16:0 1.0 t b 1.0 t t t
16:0 31.2 34.5 46.8 50.2 13.9 13.3
16:1 40.3 41.1 22.3 23.6 64.8 66.7
17:0 1.1 t 1.5 t t 1.5
iso 18:0 1.2 t 1.3 1.0 t t
18:0 3.9 3.3 6.0 5.1 1.6 1.7
18:1 16.9 12.7 16.5 11.8 13.8 10.9
aNumber preceding colon indicates number of carbons? and number following designates degree of unsaturation.
b t = trace (less than 1% of total).
31
octadecenoate (12-17%), sterate (5-6%), and myristate (4-7%).
The major free fatty acid was hexadecanoate (65-67%). If a
phospholipase was responsible for the large amount of free
fatty acid found, the enzyme activity appeared to favor
hydrolysis of hexadecenoic acid.
This report has shown that the fatty acid distribu-
tions from a brpad spectrum of cholera vibrios were similar
but were different from those reported in other bacterial
species. The feasibility of utilizing such data in the
chemical-taxonomic characterization and identification of
these pathogenic microorganisms is thus suggested.
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78. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids of E. coli. J. Bacterid. 84:1260-1267.
79. Marsh, J. B., and D. B. Weinstein. 1966. Simple charring method for determination of lipids. J. Lipid Res. 7:574.
80. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.
39
81. Neilsen, H. S., Jr. 1966. Variation in lipid content of strains of Histoplasma capsulatum exhibiting dif-ferent virulence properties for mice. J. Bacterid. 91:273-277.
82. Nesbitt, J. A., Ill, and W. J. Lennarz. 1965. Com-parison of lipids and lipopolysaccharide from the bacillary and L forms of Proteus P18. J. Bacterid. 89:1020-1025.
83. Okuyama, H. 1969. Phospholipid metabolism in Escherichia coli after a shift in temperature. Biochim. Biophys. Acta 176:125-134.
84. O'Leary, W. M. 1959. Involvement of methionine in bacterial lipid synthesis. J.Bacteriol. 78:709-713.
85. O'Leary, W. M. 1962. The fatty acids of bacteria. Bacterid. Rev. 26:421-447.
86. O'Leary, W. M. 1962. S-adenosylmethionine in the biosynthesis of bacterial fatty acids. J. Bacterid. 84:967-972.
87. O'Leary, W. M. 1967. The chemistry and metabolism of microbial lipids. The World Publishing Co., New York.
88. Park, E. E., and L. R. Berger. 1967. Fatty acids of extractable and bound lipids of Rhodomicrobium vannielii. J. Bacterid. 93:230-236.
89. Pierce, N. F., and W. B. Greenough, III. 1970. Stimu-lation of glycerol production in fat cells by cholera toxin. Nature 226:658-659.
90. Pohl, S., J. H. Law, and R. Ryhage. 1963. The path of hydrogen in the formation of cyclopropane fatty acids. Biochim. Biophys. Acta 70:583-585.
91. Romera, E. M., and R. R. Brenner. 1966. Fatty acids synthesized from hexadecane by Pseudomonas aeruginosa. J. Bacteriol. 91:183-188.
92. Selkirk, J. K., J. C. Elwood, and H. P. Morris. 1971. Study on the proposed role of phospholipid in tumor cell membrane. Cancer Res. 31:27-31.
93. Shank, R. S., and H. Schecter. 1959. Simplified zinc-copper couple for use in preparing cyclopropanes from methylene iodide olefins. J. Org. Chem. 24:1825-1826.
40
94. Shaw, M. K., and J. L. Ingraham. 1965. Fatty acid composition of Escherichia coli as a possible controlling factor of the minimal growth temperature. J. Bacterid. 90:141-146.
95. Simmons, H. E., and R. D. Smith. 1959. A new synthesis of cyclopropanes. J. Am. Chem. Soc. 81:4256-5264.
96. Smith, H. L., Jr., and K. Goodner. 1965. On the classification of vibrios. Cholera Research Symposium, Honolulu, Hawaii, U. S. Government Printing Office, Washington, D. C., pp. 4-8.
97. Stahl, E. 1969. Thin-layer chromatography. (A laboratory handbook.) Springer-Verlag, New York.
98. Steinhauer, J. E., R. L. Flentge, and R. V. Lechowich. 1967. Lipid patterns of selected microorganisms as determined by gas-liquid chromatography. Appl. Microbiol. 15:826-829.
99. Supina, W. R. 1964. Analysis of fatty acids and derivatives by gas chromatography, pp. 271-305. In H. A. Szymanski, Biomedical applications of gas chromatography. Plenum Press, New York.
100. Tornabene, T. G., E. 0. Bennett, and J. Oro. 1967. Fatty acid and aliphatic hydrocarbon composition of Sarcina lutea grown in three different media.
101. Tornabene, T. G., and J. E. Ogg. 1971. Chromatographic studies of the lipid components of Vibrio fetus. Biochim. Biophys. Acta 239:133-141.
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104. Walder, B. L. 1971. A novel charring technique for detection of lipids on thin-layer chromatograms. J. Chromatog. 56:320-323.
105. Walsh, D. E., 0. J. Banasid, and K. A. Gilles. 1965. Thin-layer chromatographic separation and colori-metric analysis of barley or malt lipid classes and their fatty acids. J. Chromatog. 17:278-287.
41
106. Weinbaum, G., and C. Panos. 1966. Fatty acid distribu-tion in normal and filamentous Escherichia coli. J. Bacteriol. 92:1576-1577.
107. White, D. C., and F. E. Frerman. 1968. Fatty acid composition of the complex lipids of Staphylococcus aureus during the formation of the membrane-bound electron transport system. J. Bacteriol. 95:2198-2209,
108. White, P. B. 1938. The rugose variant of vibrios. J. Pathol. Bacteriol. 46:1-6.
109. Zalkin, H., J. H. Tav, and H. Goldfine. 1963. Enzymatic synthesis of cyclopropane fatty acids catalyzed by bacterial extracts. J. Biol. Chem. 238:1242-1248.
CHAPTER II
PHOSPHOLIPASE ACTIVITY OP VIBRIO CHOLERAE
Introduction
Lipid extracts of Vibrio cholerae have been shown to
contain large amounts of free fatty.acids and phospholipids
(5). The possibility that phospholipase activity had
occurred prior to lipid extraction was suggested by the high
free fatty acid content (5) since bacterial lipids are known
to consist chiefly of complex lipid molecules.
The purpose of this study was to determine if
phospholipase activity exists in typical V. cholerae which
is capable of cleaving fatty acids from cholera phospholipids.
The existence of such phospholipase activity might be ample
reason for rapid extraction of cells with lipid solvents.
Phospholipids of bacteria would be rapidly hydrolyzed by an
active phospholipase if cells were allowed to remain for
long periods of time in aqueous solution.
Phospholipases have been reviewed by several workers
(1, 20, 23, 27). Enzymes which remove one fatty acid from
a phospholipid are designated phospholipase A (EC 3.1.1.4).
Phospholipase A (phosphatide acyl-hydrolase) is of two types:
phospholipase A.̂ which cleaves fatty acid from the 1-
position of a phosphoglyceride, has been studied in human
42
4 3
and beef pancreas (27) as well as in rat and calf brain (18);
the enzyme specifically designated phospholipase A2, found in
Crotalus atrox (Western Diamondback rattlesnake)' and Crotalus
adamanteus (Eastern Diamondback rattlesnake) venoms, cleaves
the fatty acyl group from the 2-position of a phosphoglyceride
(10, 19, 21, 40, 43, 47-49) to give a lysophosphoglyceride.
This enzyme is also found in bee, wasp, and scorpion venoms
(23) .
Phospholipase B (EC 3.1.1.5) or lysophospholipase (9,
25, 38) cleaves either both fatty acids from a phosphoglycer-
ide (in some cases, in conjunction with phospholipase A) or
the remaining fatty acid from a lysophosphoglyceride.
Lysophosphoglycerides are known to be powerful hemolytic
agents (20).
Phospholipase C (EC 3.1.4.3, phosphatidyl choline t
cholinephosphohydrolase), found in culture supernatants of
Pseudomonas fluorescens (11), Bacillus cereus (50), and
Clostridia (27), hydrolyzes phosphatidyl choline to
digylceride and phosphorylcholine. The substrate specificity
of this enzyme differs with the enzyme source. This enzyme
(Clostridium perfrinqens) was said to be hemolytic and lethal
(27), and its action on artificial membranes (22) resulted
in decreased membranes resistance.
Phospholipase D (EC 3.1.4.4, phosphatidyl choline
phosphatidohydrolase), found in cabbage, spinach, and sugar
44
beet plastids, cleaves choline from phosphatidyl choline to
give phosphatidic acid and free choline (27). Several
phosphoglycerides act as substrate. An unusual phospholipase
D was found (32, 33) in Haemophilus parainfluenzae which
hydrolyzed cardiolipin to phosphatidic acid and phosphatidyl
glycerol.
Phospholipases found in bacteria (other than those
mentioned above) include phospholipase A in Escherichia
coli (3/ 12, 17, 31, 36, 41), Salmonella typhimurium (3),
and Bacillus megaterium (37), and lysophospholipase in E.
coli (12, 35) and Mycoplasma laidlawii (44). These enzymes
were reported to be membrane-bound.
Materials and Methods
Cultural Conditions. Vibrio cholerae strain 569 B
(Inaba), received from R. A. Finkelstein (The University of
Texas Southwestern Medical School, Dallas, Texas), was used
in this study. A large number of cholera workers (8, 13-16,
26, 34) have utilized strain 569 B in studies of V. cholerae
Type 2 (exo—entero) toxin (6). Bacteria were maintained in
the lyophilized form or on slants (2 C) of Trypticase Soy
Broth (BBL) with 2% agar (Difco).
Cells were grown in Trypticase Soy Broth (TSB) or in
Roux flasks containing 100 ml of TSB with 1% agar. One ml
of a sixteen hour (37 C) broth culture was used to inoculate
each liter of broth or each Roux flask. Broth cultures (one
45
to ten liters) were incubated (37 C) until the late station-
ary growth phase as determined by optical density using a
Spectronic 20 colorimeter (540 nm). Bacteria were
harvested in 250 ml bottles by centrifugation (5000 x g,
15 min, 4 C) using a Sorvall RC-2B refrigerated centrifuge
equipped with a GSA rotor. Roux flasks were incubated 37 C,
24 hr. Cells were harvested by washing the agar surface
with distilled water followed by centrifugation. Bacteria
were streaked for isolation onto Petri plates containing
agar media both before and after initial inoculation as well
as after harvest to determine presence of contaminating
microorganisms. If no contamination was apparent, cholera
enzyme solutions were prepared.
Cholera Phospholipase Preparations. V. cholerae cells
harvested by centrifugation were diluted in distilled water
(3.2-6.0 mg/ml protein) and sonicated (6-9 amps, D. C.) for
5 min while in an ice bath. Temperature was not allowed to
exceed 10 C. The sonicate was diluted 1:1 with 0.05 M
borate buffer, pH 7.5. "Roux flash supernatant," pH 6.0,
was prepared by freezing Roux media (-12 C) after most cells
had been removed by washing. Thawed media yielded ca 50 ml
of liquid per Roux flask (6-8 mg/ml protein) and was assayed
for phospholipase activity directly. Protein determinations
were made by the Lowry procedure (28) using bovine serum
albumin as the standard.
46
Thin-layer Chromatography. Thin-layer chromatography
(TLC) was performed using 20 x 20 cm, 5 x 20 cm, and 1 x 3
inch glass plates. Silica gel G (Curtin Chemical Company)
aqueous slurries were spread onto plates with a thickness
of 0.25-0.5 mm and dried overnight at 26 C. Ammonium sul-
fate (1%, weight/volume) was added to the aqueous slurry
when sample charring, following the TLC separation, was
desired (30, 45).
TLC-separated lipids were visualized by iodine vapors
or by heating TLC plates (220 C) which contained ammonium
sulfate. Sulfuric acid which results from this treatment
effectively decomposes lipids to visible carbon spots.
Phosphatidyl ethanolamine (PE) was detected by spraying TLC
plates with 0.3% ninhydrin in acetone. Phosphatidyl glycerol
(PG) was detected with periodate-schiff reagent (24), and
Dragendorff reagent (42) was used to detect phosphatidyl
choline (PC). Standards of PC and PE were purchased from
Applied Science Laboratories and cardiolipin (CL) from
Supelco, Inc.; PG was produced by reaction of PC with
cabbage transphosphatidylase in the presence of 20% glycerol
(27). Oleic acid, triolein, methyl oleate, and cholesterol
oleate were from Applied Science Laboratories.
TLC solvent systems used were:
Solvent A (46)—Petroleum ether: diethyl ether:
glacial acetic acid (90:10:1, volume/volume),
47
Solvent B (29)—Chloroform: methanol: water
(60:30:5, volume/volume), and
Solvent C (27)—Chloroform: methanol: glacial
acetic acid: water (80:13:8:0.3, volume/volume).
Crotalus atrox Phospholipase A^. C. atrox (Western
Diamondback rattlesnake) venom was obtained from J. Smith
(N. T. S. U.) and lyophilized. Ten mg was placed in 100 ml
of buffer (20, 21) containing (per liter) 0.372 g EDTA, 2.22
g calcium chloride, and 12.8 g sodium chloride. The pH was
adjusted to 7.5 with 0.1 N potassium hydroxide.
The enzyme assay (20) consisted of titration of fatty
acids released after incubation (room temperature) of known
quantities of PC or PE (in 2 ml diethyl ether) with 25 ul of
enzyme solution using phenol red as indicator. A 100 ul
syringe was used to deliver known quantities of 0.02 N sodium
hydroxide. Standard lysophosphatidyl ethanolamine (LPE) was
synthesized by this enzyme system from bacterial PE (Calbiochem)
LPE purity was determined by TLC (solvent B).
Cabbage Phospholipase D. Inner cabbage leaves were
washed with distilled water, and plastids were extracted from
200 g (wet leaves) using methods described by Lowenstein (27).
Plastid preparations were lyophilized. Total yield was 450
mg dry weight.
The assay system consisted of 10 mg plastids, 0.25 ml
48
of 1 M calcium chloride in 1.25 ml of 0.2 M acetate buffer,
pH 5.6, and 1 ml of diethyl ether solution with 15 umoles
PC. Following incubation at 25 C, the aqueous phase was
reacted with ammonium reineckate (27) and optical density
determined at 526 nm with a Spectronic 20 colorimeter.
Cabbage plastid transphosphatidylase activity was utilized
to synthesize PG using the above reaction (15 umoles PC)
with 20% glycerol in the aqueous phase (pH 5.6). PG was
purified by elution through heat-activated (110 C, 1 hr)
silicic acid (39) with acetone. Purity was established by
TLC (solvents B and C), and the presence of PG was determined
by periodate-schiff reagent (83).
Preparation of Purified Egg Yolk Phosphatidyl Choline
(PC). Six chicken eggs were used to prepare purified PC.
Yolks (100 ml) were placed in a 500 ml glass—stoppered
graduated cylinder, and lipid was extracted (27) with
chloroform: methanol (2:1, volume/volume). The lipid extract
was dissolved in chloroform, and phospholipids were precipi-
tated with ten volumes of acetone (repeated several times) at
2 C. The acetone-insoluble egg yolk phospholipid was dis-
solved in 10 ml chloroform: methanol (1:1, volume/volume).
A 2.5 x 30 cm glass chromatography column (teflon stopcock)
packed with 10 g aluminum oxide G (Merck) was used to purify
PC. The phospholipid mixture was placed on the top of the
49
alumina bed and elution of PC effected by addition of chloro-
form-methano1 (10 p.s.i. nitrogen pressure) until Dragendorff-
positive material ceased to elute. PE (a major egg yolk
phospholipid) failed to elute from alumina. PC purity was
determined by TLC in solvent B. Purified PC was quantitated
by phosphorus analysis (2). PC was also purified by silicic
acid column chromatography (39), but this method was more
difficult and time-consuming.
Preparation of Radioactive Substrates. Phosphorus-32
(32P)-labeled phospholipids were prepared by growing E. coli
(ATCC 11775) in the presence of 32P disodium hydrogen
32
phosphate (Na2H P04) purchased from International Chemical
and Nuclear Corporation. One liter (distilled water) con-
taining 10 g Bacto peptone (Difco) and 0.5 mM Na2HP0^ was
inoculated with bacteria and incubated at 37 C. After 32
visible turbidity was apparent, 5 mC of Na2H PO^ (0.5 ml)
was added and incubation continued for two hours. Radio-
active cells were harvested as previously described by
centrifugation. 14
Carbon-14 ( C)-labeled phospholipids were prepared
as above using ^ C sodium acetate (500 uC) purchased from
New England Nuclear. 32 14
Phospholipids ( P and C) were extracted from cells
in 16 x 150 mm test tubes with teflon-lined screw-caps by
the Bligh and Dyer method (4). Chloroform extracts were
50
evaporated (vacuum rotary evaporator), taken up in chloro-
form, and analyzed for phosphorus content (2).
32? radioactivity was assayed by drying samples on
planchets and reading directly with a Geiger-Muller tube and
scaler. 14C was assayed with a Beckman liquid scintillation
counter in scintillation vials containing 10 ml of the
following fluid: toluene (1 liter), 5 g PPO, and 130 mg
POPOP (27) .
Purified E. coli "^PE and "^PG as well as "^C PE
were obtained by separating radioactive phospholipids by TLC
in solvent C. Separated components were eluted into 10 ml
chloroform: methanol (1:2, volume/volume); solvent was
evaporated; and the isolated compounds were analyzed for
phosphorus content.
Total "^P phospholipids were separated in solvent C
and visualized by iodine vapors; spots were scraped onto
planchets and read directly with a geiger counter to deter-
mine percentage of label in "^PE, "^PG, and "^P CL.
Cholera Phospholipase Assay. Phospholipid ("^P)
preparations (0.084-4.81 umoles P) were placed into 16 x 150
mm screw-cap test tubes, and solvent was evaporated with a
stream of nitrogen (30 C). One ml of a 2% solution of Triton
X 100 in water was added to each tube and vortexed for 5 min.
Radioactivity in solution was assayed and compared to chloro-
form solutions. Not less than 95% of the radioactive label
51
was rendered water-soluble in the presence of Triton X 100.
The cholera phospholipase assay system consisted of
1 ml of enzyme preparation ("Roux flask supernatant" or 09 14.
sonicate in 0.025 M borate) plus 50 ul of P or C
phospholipid in Triton X 100 solution. After addition of
phospholipid, tubes were vortexed for 1 min and incubated
at 37 C. Following incubation, lipids were partitioned into
a chloroform phase by the Bligh and Dyer method (4) as
modified by Scandella and Kornberg (41). To the 1 ml of
enzyme reaction mixture was added 2.25 ml methanol and 1 ml
chloroform. Vortexing resulted in a single phase. After
standing (26 C) for 15 min, 1 ml chloroform and 1 ml water
were added and mixed. The resulting two-phase system was
centrifuged 5 min (International Clinical Centrifuge) at 32
maximum speed. Water-soluble P was determined by evaporat-
ing 1 ml of aqueous phase on planchets and counting with a
Geiger-Muller tube and scaler. Tubes containing buffer (no
enzyme), but with 50 ul of labeled phospholipid and incubated
under identical conditions as enzyme solutions, served as
controls. Control readings were subtracted from those
obtained with enzymes since controls indicated background
radioactivity plus ubiquitous water-soluble label. Counts
per min (CPM) thus obtained were compared with CPM obtained
by reading 50 ul (100%) of phospholipid (32P) utilized in
each test.
52
The 14C assay of chloroform-soluble products was
more difficult and was used primarily to identify products.
14 After reaction of cholera phospholipase with C phospholipid
*
and Bligh and Dyer lipid extraction (4), the chloroform phase
was evaporated and taken up in 100 ul chloroform. Ten ul of
lipids were spotted on TLC plates and separated in solvent A.
Free fatty acid and phospholipid were assayed for radio-
activity. Solvent B was used to separate (TLC) both -^C and
32
P chloroform-soluble compounds in an effort to determine
presence of LPE as a reaction product. Standards (PE and LPE)
were spotted with samples to be separated and visualized by
ninhydrin spray.
Results and Discussion
Egg Yolk Phosphatidyl Choline (PC). Separation of
egg yolk phospholipids by TLC in solvent B is shown in Fig. 5
(left). Major components were PE and PC. Also shown is the
PC which was purified by aluminum oxide column chromatography
(right). No PE eluted from the alumina column, but a small
amount of neutral lipid was present. PC was used as substrate
f o r atrox phospholipase A2 and cabbage phospholipase D
assays without further purification.
PC as well as other phospholipid substrates were
quantitated by phosphorus (P) analyses (2). Optical density
(830 nm) readings using a Cary 14 spectrophotometer were
made with 0.025-0.15 umoles of KH2P04. Optical density
53
Solvent.
0 PE
0 0 PC
O r i g i n
Fig. 5-7 Thin-layer chromatogram of major egg yolk phospholipids (left) and purified phoaphatidyl choline (right) Solvent was chloroform: methanol: water (60:30:5, volume/ volume).
5 4
(0. D.) was a linear function of P concentration. When the
Cary 14 instrument was not available, readings were made with
a Beckman spectrophotometer at 735 nm where 0. D. values were
1/2 of those at 830 nm. Duplicate samples of phospholipids
were analyzed by the Bartlet procedure (2) and quantitated
by comparison to similar standard curves.
Crotalus atrox Phospholipase A2. Results of incuba-
tion (22 C) of 10 umoles of.PC with C. atrox venom is shown
in Fig. 6. The reaction appears to proceed in a linear
fashion for at least 20 min. The products of the reaction
were lysophosphatidyl choline (LPC) and free fatty acids.
Fatty acids were released at an initial velocity of 50 umoles/
min/mg of venom under these conditions.
Substrate (PC) was increased from 5-50 umoles and the
venom enzyme allowed to react 10 min at 27 C. Hydrolysis
rate increases were linear until at least 20 umoles of PC
were present (Fig. 7) . Enzyme satiation with substrate was
never reached, but initial velocities began to decrease
before 30 umoles PC.
The purpose of investigating the snake venom phospho-
lipase A2 was to develop techniques for production of LPE
using PE as substrate. Bacterial PE (20 umoles) was reacted
with 2.5 ug of venom (1 hr, 25 C), and LPE was purified by
precipitation from cold ether (27). Fig. 3 shows a TLC
separation of PE and LPE (solvent B). Ether-insoluble LPE,
55
40 Min u f e s
Fig. 6—Assay of Crotalus atrox venom (2.5 ;ug) phospho-lipase A2 activity on phosphatidyl choline (10 jumoles) with time (22 C).
56
20 30 40 Phospha t idy l c h o l i n e (Mmoles)
Fig. 7—Assay of Crotalus atrox venom (2.5 jig) phospho-lipase A2 activity on 5-50 jumoles of phosphatidyl choline (10 min, 27 C) .
57
S o l v e n t
Origin
Fig. 8—Thin-layer chromatogram of bacterial phospha-tidyl ethanolamine (PE) and lysophosphatidyl ethanolamine (LPE) prepared by reaction of PE with Crotalus atrox venom. Solvent was chloroform: methanol: water (60:30:5, volume/ volume). Similar separations were used in attempts to detect 14C or 32P LPE following cholera phospholipase reactions.
58
after several ether washings, gave only one ninhydrin-
positive spot by TLC.
Cabbage Phospholipase D. 0. D. readings (526 nm)
following reaction of ammonium reineckate with 4-20 umoles
of choline hydrochloride are shown in Fig. 9. Linear 0. D.
responses were achieved over the determined range of choline.
Similar standard curves were used to quantitate the cabbage
plastid (10 mg) phospholipase D after reaction with 15 umoles
of PC (Table XI). Initial velocity under the conditions
employed appeared to be 2.8 unmoles/min/10 mg plastids.
Products of the reaction were phosphatidic acid (purified
by methanol precipitation, 2 C) and free choline.
The purpose of investigating phospholipase D was to
develop techniques for the production of LPC from PC by
utilizing the transphosphatidylase (27) activity of the
cabbage enzyme. If a large concentration (20%) of an alcohol
is placed in the water phase, choline is liberated from PC,
and the alcohol is substituted. PG was synthesized using
this system by reacting 10 mg of plastid preparation with
15 umoles PC in a 20% solution of glycerol (pH 5.6) for 2
hr, 25 C. Choline analysis indicated 12 umoles of choline
were released. Separation of reaction products by TLC
(solvent B) revealed a new spot which was periodate-schiff
positive (24), thus indicating presence of phosphatidyl
59
.5
.4"
i c .3 <o cs •o
D O*
..2
6 8 10 12 14 C h o l i n e ( M m o l e s )
16 "20
fr,l ,„Fl9' 9 Optical density (0. D.) readings (526 mil) following reaction of 4-20 Mmoles of choline hydrochloride with ammonium reineckate (average of two determinations).
60
TABLE XI
PHOSPHOLIPASE D ASSAY
Time (min) 0. D. (526 rati) jumoles Choline Released
10 .060 2.8
20 .114 5.4
30 .137 ' 6.4
40 .155
CN
I •
A
50 .187 00 t
61
glycerol. Silicic acid column chromatography (39) was used
to purify PG.> The only TLC spot Which eluted from silicic
acid with acetone was PG. No ninhydrin-positive (PE) or
Dragendorff-positive (PC) material was present.
Radioactive Substrates. E. coli 32P- ancj ^c-labeled
lipid was spotted on TLC plates and separated in solvent C
(Fig. 10).. Geiger counter readings of spots obtained by Op
iodine vapors suggested that 95% of P label was in PE, 5%
in PC, and less than 1% in CL. Similar separations were used
32 3 o 14 to prepare purified PG, °^PE, and C PE. Since the vast
majority of label was in 32PE, crude phospholipid extracts
were utilized in some enzyme assays, and results were similar
to those using purified 32PE.
Cholera Phospholipase. Initial cholera phospholipase
studies were performed using 32PG (4.2 nmoles) and sonicate
with 3 mg/ml protein (Table XII). Since products of the
32
reaction were unknown, PG in the assay system would have
detected phospholipase A (lysophosphatidylglycerol is water-
soluble), lysophospholipase, and phospholipase C activities.
All would have given water-soluble 32P but different
chloroform-soluble products. Product was formed at a linear
rate until ca 25% of 32PG was utilized. An overnight reaction
(21 hr) resulted in 43% of 32P in the water phase.
Chi or of orrn- sol ubl e products wore detected using
purified C PE (21 nmoles) as substrate followed by TLC
62
0
SoIvenf Neutral lipid
€1
SPG
P E
• i O r i g i n
32 Pig. 10—Thin-layer chromatogram of E. coli (ATCC 11775) P phospholipids. Solvent was chloroform: methanol: acetic
acid: water (80:13:8:0.3, volume/volume). Similar separations were used to determine per cent 32p in separated phospholipids as well as preparation of purified 32p phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE), and carbon-14-labeled phosphatidyl ethanolamine. Cardiolipin = CL.
63
TABLE XII
ASSAY OF VIBRIO CHOLERAE SONICATE FOR PHOSPHOLIPASE ACTIVITY
USING 32P PHOSPHATIDYL GLYCEROL AS SUBSTRATE
Hours (37 C) Product (nmoles)3
1 0.45
2 0.87
3 1.05
4 1.24
21 1.76 (43%)
^Average of duplicate tests.
r
64
separation (solvent A). Fig. 11 shows that chloroform-
soluble material corresponded to free fatty acid and phospho-
lipid. Scintillation counting of separated spots indicated
that 4.2 nmoles (ca 20% of CPM) was accounted for by free O p
fatty acid (a reaction rate comparable to P assay with
18.5 nmoles 32P). Phospholipids of both 32P and reaction
mixtures using purified PE were separated by TLC in solvent B
with standards to detect LPE. No LPE was found in 1 hr or
21 hr reactions, and the only radioactive phospholipid
detected was unreacted substrate (32PE and 14C PE). Thus it
appeared that both fatty acids of a phospholipid were removed
by the cholera phospholipase with no appreciable amount of
chloroform-soluble intermediate compound (LPE). The 32P
assay system thus became valid for use with 32PE as sub-
strate, since water-soluble 32P was essential for detection
of activity. Activity was achieved with "Roux flask supernatant"
material using purified 32PE (11 nmoles) as substrate. Water-32
soluble P was released from purified 3^pe a t j_nj_tial
rate of 2.3 nmoles/hr until 30% of substrate was utilized
(Fig. 12). Overnight reactions averaged 50-56% water-
soluble 32P.
Dilution of sonicate (3.2 mg/ml protein) with 0.5 M
borate buffer (pH 7.5) gave a 1.6 mg/ml enzyme solution which
was used to test effects of various parameters on enzyme
65
0
Solvent
C h o l e s t e r y 1 o l e a t e
Methyl o l e a t e
T r i o l e i n
O l e a t e
Origin ( P h o s p h o l i p i d )
Fig. 11—Thin-layer chromatograms of cholera phospho-lipase products (left) following reaction with -^c phospho-phatidyl ethanolamine (21 nmoles) for 1 hr, 37 C. Released fatty acid was 4.2 nmoles based on per cent "^C in unreacted substrate versus product. A standard lipid mixture (right) was used to determine products of the reaction. Solvent was petroleum ether: diethyl ether: acetic acid (90:10:1, volume/volume).
66
Hou rs
Fig. 12—Assay of Vibrio cholerae strain 569 B (Inaba) "Roux flask supernatant" (6 mg/ml protein) for phospholipase activity using substrate.
32 P phosphatidyl ethanolamine (11 nmoles) as
67
activity. Table XIII shows the effects of Tris-HCl, EDTA,
8-hydroxyquinoline, and dialysis of enzyme on activity using
8.3 nmoles of 32P substrate. EDTA and 8-hydroxyquinoline
had no effect, thus suggesting no divalent cation require-
ment. Dialysis of 2 ml sonicate (one liter borate buffer)
had no effect, which suggested the absence of loosely-bound
activators or inhibitors. Use of 0.1 M Tris-HCl buffer
(pH 8.0) also did not increase or inhibit phospholipase
activity. These results were surprising, since many phospho-
lipases require the divalent cation calcium (41). Calcium may
act by sequestering hydrolyzed fatty acids. Other divalent
cations were shown to be inadequate substitutes for calcium.
After observing that divalent cations were not neces-
sary for phospholipase activity by the cholera sonicate,
experiments were conducted to determine effects of such
cations on the enzyme. Table XIV shows the level of inhibition
of the cholera phospholipase by chloride salts of barium,
magnesium, manganese, zinc,•calcium, iron, and mercury at 1
and 10 mM concentrations using 25 mM borate buffer, pH 7.5.
No effect was observed, with KC1 or NaCl. Prior to this
investigation, mercuric chloride was shown to inhibit
phospholipase activity (41). Less than 15% inhibition of E.
coli phospholipase A^ was effected by 0.1 or 1 mM mercuric
chloride, while 0.08 M sodium chloride decreased activity
by 50% (41) . Increased ionic strength did not appear to
68
TABLE XIII
EFFECTS OF TRIS-HCl (pH 8.0), EDTA, 8-HYDROXYQUINOLINE
AND DIALYSIS ON REACTION OF SONICATE
Conditions of assay Product (nmoles)/hr
Borate buffer (0.025 M, pH 7.5) 2.1
Variables " 1 r - -- -1-1 *
EDTA (10 mM) 2.1
8-OH quinoline (1 mM) 1.8
Dialysis 1.9
Tris-HCl buffer (0.01 M, pH 8.0) 2.0
a Reaction time = 1 hr, 37 C.
69
TABLE XIV
PER CENT INHIBITION BY CHLORIDE SALTS (ONE AND TEN mM)
ON PHOSPHOLIPASE ACTIVITY OF V. CHOLERAE SONICATE
Cation Per cent inhibition
1 mM 10 mM
Potassium 0 0
Sodium 0 0
Barium 7 37
Magnesium 12 35
Manganese(ous) 44 68
Zinc 56 95
Calcium 80 95
Iron (ferrous) 37 100
Mercury 100 100
70
affect the cholera phospholipase (Table XIV). Further, buffer
concentration (6-50 mM) did not affect cholera phospho-
lipase significantly. Attempts at pH studies were hampered
by the large buffering capacity of the sonicate and must
await enzyme purification.
32 Since Triton X 100 was essential in making P and
14
C phospholipids water-soluble, the concentration of the
detergent for maximum enzyme activity was determined (Table
XV) using 8.3 nmoles substrate. Maximum activity was
achieved at 1 mg/ml detergent. Lower activity was evident
both below and above the 1 mg/ml level. Triton X 100 con-
centrations below the optimum level may not have been suffi-
cient to allow formation of phospholipid micelles necessary
for enzymatic activity. Above the optimum concentration,
Triton X 100 may have interfered with the activity by com-
peting with the substrate for hydrophobic binding sites on
the enzyme (41). Incubation of the cholera phospho1ipase
with 2-4 mg/ml Triton X 100 for 1 hr, 37 C had no inhibiting
effect, thus suggesting that the detergent did not cause
enzyme denaturation.
A linear relationship was shown between enzyme con-
centrations and initial velocities of the cholera phospho-
lipase (Pig. 13). Substrate (^P) concentration was increased
from 18.5—240.5 nmoles/ml in the presence of the sonicate and
"Roux flask supernatant" as shown in Fig. 14. Reaction rates
71
TABLE XV
EFFECTS OF TRITON X 100 CONCENTRATION ON PHOSPHOLIPASE
ACTIVITY OF SONICATE9
Triton X 100 (mg/ml) Product (nmoles)/hr
0.25 1.27
0.50 2.04
1.00 2.18
2.00 1.34
4.00 0.50
'Reaction time = 1 hr, 37 C.
72
.4 0.6 0.8 1.0 1.2 1A
S o n i c a t e ( m g / m l p r o t e i n )
Fig. 13—Effect of enzyme (sonicate) concentration on reaction rates of V. cholerae phospholipase activity using 32 P phospholipid (16.6 nmoles) as substrate.
73
l/V XIO9 P r o d u c t ( n rno les /hour )
Fig. 14—Initial velocities (nmoles/hr) of "Roux flask supernatant" (A), 6 mg/ml protein, and sonicate (B), 1.6 mg/ml protein, with Lineweaver-Burk plots (below).
74
increased in a hyperbolic fashion with substrate concentra-
tion, but the enzyme was not saturated at the highest sub-
strate level. Lineweaver-Burk plots (Fig. 14) of reciprocal
values suggested a of 60 (SE + 7) uM for "Roux flask
supernatant" and 63 (SE + 6) uM for the sonicate. These
data suggest that both enzyme preparations contained the
same phospholipase activity. The "Roux flask supernatant"
solution was centrifuged (5000 x g, 15 min), and the
particulate fraction was assayed for activity. Activity was
higher in the pellet than in the supernatant. A membrane-
bound phospholipase was thus suggested.
The cholera sonicate was fractionated using an
Amicon Ultrafiltration Cell model 52 and Diaflo membranes
as shown in Table XVI. The substrate was 16.6 nmoles 32P
phospholipid. One-half of the activity was retained on the
XM-100A filter, which was probably due to its membrane-bound
characteristic. The PM-30 and UM-10 filters demonstrated no
apparent phospholipase. Combination of retentates of the
three fractions resulted in recovery of most of the phospho— I
lipase. Since the assay system employed was applicable only
to phospholipase activity, which released both fatty acids
from a phospholipase, the possibility that phospholipases A^
and/or A^ were retained on the PM-30 and UM-10 filters was
investigated. However, no 32P LPE could be found in the
chloroform phase by TLC in solvent B. However, optimum
75
TABLE XVI
PHOSPHOLIPASE ACTIVITY OF XM-100A, PM-30, AND UM-10
MEMBRANE FILTER RETENTATES OF V. CHOLERAE SONICATE
Fraction Product (nmoles/hr)a
Sonicate (100% activity) 4.6 - 4.9
Retentates
XM-100Ab 2.3 - 2.7
PM-30c 0.0 - 0.3
UM-10d o • o - 0.3
XM-100A + PM-30 2.5
00 • CN
1
XM-100A + UM-10 2.9 - 3.2
PM-30 + UM-10
o • o - 0.5
XM-100A + PM-30 + UM-10 4.3 - 4.7
Range of three determinations.
^Retains globular proteins with molecular weights of 100,000 and above.
cRetains globular proteins with molecular weights of 30,000 and above.
^Retains globular proteins with molecular weights of 10,000 and above.
76
conditions for phospholipase A activity, should it exist in
cholera cells, have not been determined.
Scandella and Kornberg (41) isolated the phospho-
lipase activity of E. coli B. The Km for PG using Triton
X 100 (0.5 mg/ml) was 15 uM and in the absence of detergent
was 0.34 uM. No effect on V__„ was noted with different lUcLX
detergent concentrations. A Lineweaver-Burk plot might
measure the affinity of the enzyme for phospholipid vesicles
or miscelles (41). If is a measure of enzyme affinity
for phospholipid, the cholera phospholipase (K^ of 60-63 uM)
compares favorably with the E. coli enzyme in that respect.
Such kinetic data published to date on bacterial phospho-
lipases have come only from Kornberg and co-workers (37, 41).
Isolation and purification of the E. coli enzyme required
1 pound (41) of starting material (bacteria), and the
purified enzyme resulting was 2 mg. It is not surprising
that other reports on bacterial phospholipases discussed
only a few parameters of the enzyme reaction (17, 31-33,
36, 36) .
Bacterial phospholipases (acyl hydrolases) were
found to be localized in the cell envelope (3) and were
tightly bound to their structural elements. The phospho-
lipase A^ was located in the outer membrane (3). This fact
presented a difficult problem in the isolation of the enzyme.
Scandella and Kornberg (41) and Raybin, et al. (37) were able
77
to remove phospholipase A-^rom the cell envelope by the
detergent sodium dodecyl sulfate (SDS). Removal of enzyme
from the cell envelope was a prerequisite to subsequent
purification. The cholera phospholipase activity was com-
pletely destroyed by SDS. Attempts to partially purify the
cholera enzyme by ultrafiltration using the Amicon Dyaflo
membrane (SM-100A) have resulted in partial retention of
activity in every case, thus suggesting that the enzyme
was structurally bound or aggregated in a form which exceeded
molecular weight of 100,000. Prior to future purification
attempts, some means of removal of enzyme from the envelope
which does not destroy activity must be determined.
The possible role of cholera lipase in the disease
mechanism can be hypothesized. An enzyme capable of selec-
tively hydrolyzing membrane constituents (phospholipids)
could contribute to the outpouring of fluids and ions into
the intestinal lumen, causing the extreme diarrhea observed
in cholera victims. Pierce and Greenough (34) have recently
found that the Type 2 (choleragen) cholera toxin (6, 8, 13-16,
26), said to be the fluid transport active factor, stimulated
lipolysis in isolated fat cells. Antisera prepared against
purified toxin destroyed lipolytic activity. Chiappe and
Van Den Bosch (7) recently reported on the lipolysis in
isolated fat cells by phospholipase A and lysophospholipase.
Lipase activity was stimulated by cyclic 3', 5'-AMP. Any
78
relationship between lipolysis of fat cells by choleragen
(34) and phospholipase activity of V. cholerae is not known.
Study of cholera lipases with respect to disease mechanisms
seems important and long overdue. A major contribution to
the understanding of the processes by which intestinal
pathogens cause diarrheal symptoms in disease victims might
be made by elucidation of the effect of bacterial phospho-
lipase activity on intestinal membrane constituents.
>- ;!:I Ij >_J : i v.i.v ' .'-J
1. Boudham, A.D. 1963. Physical structure a^d behavior of lipids ai!-l lipid ixizyuies. Adv. Lipid Res. 1: t' 3-104 .
2. Barfclet, G.H. 1^59. phosphorus in coluain ciiromato-gxaphy. J . Biol. Char,. 2 34 ; 4 b6-4 68 .
3. Bellf 'R.M. , R.D. Mavis, M.J. Osborrx, and P.R. Vagelos. ly?!, Enzymes of phospholipid metabolism; Localisat.1 on in the cytoplasmic ancl outer membrane of the cell envelope of Escharichxa coli and Sq.lT'ionelIa typhiirvur iii-nri. Biochim- Biophys• Acta 249; G2 8~63i>V"~
4. Bixyh, K.C4. f a;»U W.J• Dyer.. 3,959. v, rapid method of total lip,;d or';raci.iOi' find purification. Can. J. B1 o c i i c m. p 11 y s i o 1. 3 7: S i 1 - 917 .
5. Brian, a.P., ana S.w. Gardner. J.it.ds,, Fatty acids from Xt£££:"l Ixp-ds. J. Infect. Diseases i18:47-53- *
6. Burrows, w. 1958. Cholera toxins. Ann. Rev, Microbiol. 22:245-268. '
7. Chiappa Do Cxngoxani» G.E., H. Van Der; Bosch, and L.L.M. Ja.ii , i9 72 . j?no s p ft o i xpa.ae A and iysophospho— xipa^e a.cbi.v3 txes in isolated fat cells; Effect of cyclic 3' , 5 ' -I.ilP. Biochim. Biophys. Acta 2 60:3 o 7-332 .
8. Co ie it u*n f Vv'.d./ J. Katix", M.E, Ivert, G.J. nasai,. and V>. Barrows. 1360. Cholera toxins: Purification and pial.Litij.aa/y ch-iinctcii^atioa oC ileal loop reactive 'i'ypo 2 to:;in. J. ;>acrt:.©r.iol. 96 :1137»i:i.4 3 ,
9. T;aW;;oa, R. M. r. _ ̂ 5S. Phe i,V.atiil'ica tion of tv:o li^id component;; in .liver vhich enable .?.>rj.c:U Ixum
^X!:^cr*3 co ̂ ycirolyse lecithin, aiocben>.
80
10. Deenen, L. L. M., and G. H. DeHaas. 1963. The sub-strate specificity of phospholipase A. Biochim. Biophys. Acta 70:538-553.
11. Doi, 0., and S. Nojima. 1971. Phospholipase C from Pseudomonas fluorescens. Biochim, Biophys. Acta 248:234-244."
12. Doi, 0., and S. Nojima. 1972. Two kinds of phospho-lipase A and lysophospholipase in Escherichia coli. Biochim. Biophys. Acta 260:244-258.
13. Finkelstein, R. A., P. Atthasampunna, M. Chulasamaya, and P. Charunmethee. 1966. Pathogenesis of experimental cholerae: Biological activities of purified procholeragen A. J. Immunol. 96:440-449.
14. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Pathogenesis of experimental cholera. Preparation and isolation of choleragen and choleragenoid. J. Exp. Med. 13:185-202.
15. Finkelstein, R. A., and J. J. LoSpalluto. 1970. Production of highly purified choleragen and cholera-genoid. J. Infec. Diseases 121:563-574.
16. Finkelstein, R. A., and J. J. LoSpalluto. 1972. Crystalline cholera toxin and toxoid. Science 75:529-530.
17. Fung, C. K. and P. Proulx. 1969. Metabolism of phosphoglycerides in E. coli. III. The presence of phospholipase A. Can. J. Biochem. 47:371-373.
18. Gatt, S. 1968. Purification and properties of phospholipase A, from rat and calf brain. Biochim. Biophys. Acta*156:304-316.
19. Hachimori, Y., M. A. Wells, and D. J. Hanahan. 1971. Observations on the phospholipase of Crotalus atrox. Molecular weight and other properties. Biochemistry 10:4084-4089.
20. Hanahan, D. J. 1960. Lipid chemistry. John Wiley and Sons, Inc., New York.
21. Hanahan, D. J., M. Rodbell, and L. D. Turner. 1954. Enzymatic formation of monopalmitoleyl- and monopalmitoyllecithin (lysolecithins). J. Biol. Cherrt. 206:431-441.
81
22. Hendrickson, H. S., and E. M. Scattergood. 1972. The action of phospholipase C on black film bilayer membranes. Biochem. Biophys. Res. Commun. 46: 1961-1969.
23. Kates, M. 1960. Lipolytic enzymes, pp. 165-237. In K. Block (ed.), Lipid metabolism. John Wiley and Sons, Inc., New York.
24. Kates, M. 1967. Paper chromatography of phosphatides and glycolipids on silicic-acid-impregnated filter paper, pp. 1-39. In V. Marinetti (ed.), V. 1. Lipid chromatographic analysis. Marcel Dekker, Inc., New York.
25. Kates, M., J. R. Madeley, and J. L. Beare. 1965. Action of phospholipase B on ultrasonically dispersed lecithin. Biophys. Acta 106:630-634.
26. LoSpalluto, J. J., and R. A. Finkelstein. 1972. Chem-ical and physical properties of cholera exo-enterotoxin (choleragen) and its spontaneously formed toxoid (choleragenoid). Biochim. Biophys. Acta 257:158-166.
27. Lowenstein, J. M. (ed.). 1969. Methods in enzymology. V. 14. Lipids. Academic Press, New York.
28. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.
29. Mangold, H. K. 1969. Aliphatic lipids, pp. 363-421. In E. Stahl (ed.), Thin-layer chromatography. (A laboratory handbook.) Springer-Verlag, New York.
30. Marsh, J. B., and D. B. Weinstein. 1966. Simple charring method for determination of lipids. J. Lipid Res. 7:574.
31. Okuyama, H., and N. Shoshichi. 1969. The presence of phospholipase A in Escherichia coli. Biochim. Biophys. Acta 176:120-124.
32. Ono, Y., and D. C. White. 1970. Cardiolipin-specific phospholipase D activity in Haemophilus para-influenzae. J. Bacteriol. 103:111-115.
33. Ono, Y., and D. C. White. 1971. Consequences of the inhibition of cardiolipin metabolism in Haemophilus parainfluenzae. J. Bacteriol. 108:1065-1071.
82
34. Pierce, N. F., and W. B. Greenough, III. 1970. Stimu-lation of glycerol production in fat cells by cholera toxin. Nature 226:658-659.
35. Proulx, P., and L. L. M. Van Deenen. 1966. Acylation of lysophosphoglycerides by Escherichia coli. Biochim. Biophys. Acta 125:591-593.
36. Proulx, P., and L. L. M. Van Deenen. 1967. Phospho-lipase activities of Escherichia coli. Biochim. Biophys. Acta 144:171-174.
37. Raybin, D. M., L. L. Bertsch, and A. Kornberg. 1972. A phospholipase in Bacillus meqaterium unique to spores and sporangia. Biochemistry 11:1754-1760.
38. Roelofsen, B., R. F. A. Zwaal, P. Comfurious, C. B. Woodward, and L. L. M. Van Deenen. 1971. Action of pure phospholipase A2 and phospholipase C on human erythrocytes and ghosts. Biochim. Biophys. Acta 241:925-929.
39. Rouser, G. G. Kritchevsky, and A. Yamomoto. 1967. Column chromatographic and associated procedures for separation and determination of phosphatides and glycolipids, pp. 99-162. In V. Marinetti (ed.), V. 1. Lipid chromatographic analysis. Marcell Dekker, Inc., New York.
40. Saito, K., and D. J. Hanahan. 1962. A study of the purification and properties of the phospholipase A of Crotalus adamanteus venom. Biochemistry 1:521-532.
41. Scandella, C. J., and A. Kornberg. 1971. A membrane-bound phospholipase A-, purified from Escherichia coli. Biochemistry 10:4447-4456.
42. Stahl, E. 1969. Thin-layer chromatography. (A laboratory handbook.) Springer-Verlag, New York.
43. Uthe, J. F., and W. L. Magee. 1971. Phospholipase Ao: Action on purified phospholipids as affected by deoxycholate and divalent cations. Can. J. Biochem. 49:776-784.
44. Van Golde, L. M. G., R. N. McElhaney, and L. L.' M. Van Deenen. 1971. A membrane bound lysoohospho-lipase from Mycoplasma laidlawii strain B". Biochim. Biophys. Acta 231:245-249.
83
45. Walker, B. L. 1971. A novel charring technique for detection of lipids on thin-layer chromatograms. J. Chromatog. 56:320-323.
46. Walsh, D. E., 0. J. Banasid, and K. A. Gilles. 1965. Thin-layer chromatographic separation and color-imetric analysis of barley or malt lipid classes and their fatty acids. J. Chromatog. 17:278-287.
47. Wells, M. A. 1971. Evidence that the phospholipases A2 °f Crotalus adamanteus venom are dimers'. Bio-chemistry 10:4074-4078.
48. Wells, M. A. 1971. Spectral peculiarities of the monomer-dimer transition of the phospholipases A~ °f Crotalus adamanteus venom. Biochemistry 10:4078-4083'.
49. Wells, M. A., and D. J. Hanahan. 1969. Studies on phospholipase A. I. Isolation and characteriza-tion of two enzymes from Crotalus adamanteus venom. Biochemistry 8:414-424.
50. Zwall, R. F. A., B. Roelofsen, P. Comfurius, and L. L. M. Van Deenen. 1971. Complete purification and some properties of phospholipase C from Bacillus cereus. Biochim. Biophys. Acta 233:474-479.
CHAPTER III
GAS CHROMATOGRAPHY" OP CYCLOPROPANE FATTY ACID
METHYLESTERS PREPARED WITH METHANOLIC BORON
TRICHLORIDE AND BORON TRIFLUORIDE
Introduction
Christie (4) has suggested that the use of boron
trichloride in methanol (BC1^-CH^OH) by Brian and Gardner
(1-3) for esterification of bacterial fatty acids may have
resulted in unreliable gas chromatographic (GC) data.
Minnikin and Polgar (10) showed that methanolic boron
trifluoride (BF̂ -CH-̂ OH) reacted with disubstituted cyclo-
propanes to give methoxyesters and the corresponding olefins,
The possibility that a similar and unnoticed side-
reaction may have occurred when BCl^-CH^OH was used to
esterify cyclopropane fatty acids of bacterial origin (2,
3) was implied (4).
BF^ has been shown to be superior to BCl^ as a
catalyst for fatty acid transesterification (11). Detailed
studies using BF^ or "̂ or esterification (8, 9, 11) did
not include lipids containing cyclopropane fatty acids. A
large number of papers have been published in which BF3 or
BCl^ was used to catalyze the reaction of CH^OH with
bacterial fatty acids. Few authors who used BF^ or BCl^
84
85
in CH^OH "have indicated that another procedure was employed
to check recovery of cyclopropane acids from bacterial
lipids (3).
The purpose of this investigation was to determine
the reliability of GC data following use of commercially
available BF3~CH3OH and BC13-CH OH in the esterification
of bacterial fatty acids containing cyclopropane rings.
Materials and Methods
Esterification of Fatty Acid Standards. A standard
solution was prepared containing 1 mg/ml each of cis-9,10-
methylene octadecanoic (eye C^g) acid (Supelco, Inc.,
Bellefonte, Pa.) and heptadecanoic (C17) acid (Applied
Science Laboratories, State College, Pa.) in chloroform
(CHCI3).
One-ml aliquots were transferred to 15 x 150 mm
screw-cap tubes, and CHC13 was evaporated with a stream of
N 2 (30 C). Two ml of 14% BF3 in CH OH (w/v) or 10% BC13 in
CH30H (w/v), both from Applied Science Laboratories, were
added, and the open tube was placed in boiling water for 2
min (8). The tube was cooled, and the contents were trans-
ferred to a 30-ml separatory funnel. The tube was washed
with 4 ml of CHC13, the CHC13 and 1 ml of water were added to
the separatory funnel, and the contents were shaken and allowed
to separate. The CHCl^ phase containing methylesters was
evaporated with N2 in a screw-cap tube.
86
The fatty acid standards were also esterified in an
open tube for 0.5 rain (100 C), and for 5 min (100 C) while
tightly closed by a teflon-lined screw-cap.
Gas Chromatography of Fatty Acid Methylesters. Dried
methylesters were dissolved in 1 ml of CHCl^, and 1 ul was
injected into an Aerograph (Varian Associates, Palo Alto,
Calif.; Model 204-1C) gas chromatograph with flame ioniza-
tion detectors. Columns (5 ft. x 0.125 in.) containing 15%
diethylene glycol succinate polyester on Chromosorb W (60-80
mesh) were operated at 180 C. Detector and injector tempera-
tures were 220 C, and the flow was 25 ml/min. Range was
10-10, an<^ attenuation was 8. Areas of peaks were calculated
by multiplication of peak height by peak width at 1/2 height.
Esterification of Bacterial Fatty Acids. Escherichia
coli (ATCC 11775) was incubated for 16 hr at 40 C in Trypti-
case Soy Broth (BBL). Higher incubation temperature is
known to favor cyclopropane fatty acid production (7). Lipids
were extracted with CHCl^-CH^OIi (2:1) (5). A solution con-
taining 1 mg/ml C17 and 1.2 mg/ml E. coli lipid in CHC13 was
esterified with BC1 -CH_OH and BF -CHo0H by the method of o J 3 J
Metcalfe, Schmitz, and Pelka (9). Fatty acids were iden-
tified by hydrogenation, bromination, and by comparison with
authentic standards. Cyclopropane methylesters (cis-9,10-
methylene hexadecanoate, eye C17, and cis-11,12-methylene
87
octadecanoate, eye Cn g) were synthesized from palmitoleate
and cis-vaccenate using a simplified zinc-copper couple
(12) and the Simmons-Smith reaction (13).
Results and Discussion
Results of BF^-CH^OH and BCl^-CH^OH were compared
with the reaction of 2 -ml of freshly distilled diazomethane
at 0 C for 30 min (Table XVII). Diazomethane gave quantita-
tive recovery of eye C (99-101%). Recovery of eye
(retention time relative to C-̂-j = 2.03) using BCl^-CH^OH
at 100 C for 2 min (open tube) or 5 min (closed tube) was
similar to that obtained with diazomethane (93-100%).
BFo-CH^OH gave poor recovery of eye C (10-50%) depending o 29
on conditions of the reaction (see Table XVII). Similar
results were obtained with a 10% solution of BF^ in CH^OH
prepared from a BF^-ether complex, BF̂ O(C2H,_)2 (Eastman
Chemical Co.).
Five additional peaks (retention times relative to
c17 = 1.40, 1.64, 1.83, 3.12, and 4.23) were obtained when
BF -CH OH was used (Table XVII). J Results of BC1 -CH OH for esterification of E.
3 3 ~ coli fatty acids indicated that eye C comprised 29% and
eye C^g 16% of the total acids. Chromatograms obtained,
following use of BF CH OH, revealed considerable loss of •3 3
cyclopropane esters as well as additional small peaks.
Therefore, BF^-CH OH appears to be an undesirable esterifi-J 3 cation reagent for fatty acid mixtures containing cyclopropanes,
38
TABLE XVII
RATIO OF THE PEAK AREAS OF CI S - 9,10-METHYLENE OCTADECANOATE (CYC C19) TO HEPTADECANOATE (C17) FOLLOWING
ESTERIFI CATION (1 MG OF EACH ACID)9
"K n
Esterification method eye C-̂ g Other peaks
1. Diazomethane 0.99-1.01
2. Open tube, 0.5 min, 100 C BF3-CH3OH 0.45-0.59 0.14-0.15
3. Open tube, 2 min, 100 C BCI3-CH3OH 0.93-1.00 BF3CH3OH 0.12-0.13 0.31-0.32
4. Closed tube, 5 min, 100 C BCI3CH3OH 0.96-0.98 BF3CH3OH 0.10-0.11 0.46-0.50
Range of three determinations is given,
17 ^Retention time relative to C-j-y = 2.03.
cPeaks other than eye C19 with retention times relative to Cj_7 of 1.40, 1.64, 1.83, 3.12, and 4.23.
89
BCl^-CH^OH, on the other hand, is suitable for this purpose
and appears to quantitatively esterify cyclopropane fatty
acids which are prevalent in the lipids of many bacterial
species.
CHAPTER BIBLIOGRAPHY
1. Brian, B. L., and E. W. Gardner. 1967. Preparation of bacterial fatty acid methyl esters for rapid char-acterization by gas-liquid chromatography. Appl. Microbiol. 15:1499-1500.
2. Brian, B. L., and E. W. Gardner. 1968. A simple pro-cedure for detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552.
3. Brian, B. L., and E. W. Gardner. 1968. Cyclopropane fatty acids of rugose Vibrio cholerae. J. Bacteriol. 96:2181-2182.
4. Christie, W. W. 1970. Cyclopropane and cyclopropene fatty acids. Topics Lipid Chem. 1:1-49.
5. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.
6. Goldfine, H., and C. Panos. 1971. Phospholipids of Clostridium butyricum. IV. Analysis of the positional isomers of monounsaturated and cyclopro-pane fatty acids and alk-l'-enyl ethers by capillary column chromatography. J. Lipid Res. 12:214—220.
7,. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids in E. coli. J. Bacteriol. 84:1260-1267.
8. Metcalfe, L. D., and A. A. Schmitz. 1961. The rapid preparation of fatty acid esters for gas chroma-tographic analysis. Anal. Chem. 33:363-364.
9. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.
10. Minnikin, D. E., and N. Polgar. 1967. Structural studies on the mycolic acids. Chem. Communic., pp. 312-314.
90
91
11. Morrison, W. R., and L. M. Smith. 1964. Preparation of fatty acid methyl esters and dimethyl acetals from lipids with BF -CH OH. J. Lipid Res. 5:600-608. 6 6
12. Shank, R. S., and H. Schecter. 1959. Simplified zinc-copper couple for use in preparing cyclopropanes from methylene iodide and olefins. J. Org. Chem. 24:1825-1326.
13. Simmons, H. E., and R. D. Smith. 1959. A new synthesis of cyclopropanes. J. Am. Chem. Soc. 81:4256-4264.
CHAPTER IV
CYCLOPROPANE FATTY ACIDS OF
PSEUDOMONAS AERUGINOSA
Introduction
Pseudomonas species have been shown to contain cyclo-
propane fatty acids as major constituents of cellular lipids
(2, 4, 7, 15). Several investigations of Pseudomonas
aeruginosa fatty acids (1, 5, 8, 12) failed to demonstrate
cyclopropane acids in lipid extracts. Increased time and
temperature of incubation of some bacteria have resulted
in higher amounts of cyclopropane acids (9, 10, 15). This
investigation was conducted to determine the level of
cyclopropane fatty acid synthetase activity in typical P.
aeruginosa strains.
Materials and Methods
Two serologically distinct strains of P. aeruginosa
Verder and Evans (16) strains 2108 (serogroup III) and 1369
(serogroup II), obtained from Joe A. Bass (N.T.S.U.), were
used in this study. The organisms were grown on Trypticase
Soy Broth (BBL) with 2% agar (2). Incubation time and
temperature are indicated in Tables XVIII, XIX, and XX.
Wet cells were extracted with chloroform-methanol (2:1,
volume/volume) (6), fatty acids were esterified with
92
93
BC13-CH30H (10), and the methyl esters were analyzed by gas-
liquid chromatography (2). Cyclopropane fatty acid methyl-
esters (C-j.7 an<^ 9) were synthesized from methyl palmitole-
ate and methyl cis-vaccenate using an easily-prepared zinc-
copper couple (13) and the Simmons-Smith reaction (14).
Fatty acids were identified by comparison of retention times
with authentic standards on a diethylene glycol succinate
polyester column at 180 C (isothermal) and by results of
hydrogenation (3) and bromination (2). Fatty acids which
were detected in amounts of 0.5% or more in any one sample
were considered as major components.
Results and Discussion ' *
Increasing incubation temperature from 37 C to 40 C
resulted in higher amounts of methylene hexadecanoic and
methylene octadecanoic acids (Table XVIII). Strain 2108
grown at 37 C for 24 hr contained less than 1% total cyclo-
propane acids. Both strains, when incubated at 40 C for 16,
24, and 48 hours, demonstrated and cyclopropane
fatty acids as major lipid constituents (Table XIX).
Increased incubation time resulted in slightly larger amounts
of cyclic acids. Table XX presents effects of hydrogenation
and bromination (2) on P. aeruginosa fatty acids.
Strains 2108 and 1369 of P. aeruginosa demonstrate
cyclopropane fatty acid synthetase activity. This activity
94
TABLE XVIII
EFFECTS OF INCUBATION TEMPERATURE AERUGINOSA FATTY ACIDS
ON PSEUDOMONAS (°/o)
Fatty Acida Strain 2108 Fatty Acida
37 C, 24 hr 40 C, 24 hr
14:0 0.9 . tb
16:0 35.6 40.7
16:1 14.4 6.6
eye 17:0 0.8 1.3
18:0 1.4 2.1
18:1 46.8 45.5
eye 19:0 t 3.7
aNumber preceding colon indicates number of carbons; number following colon designates degree of unsaturation (eye = cyclopropane ring).
ht = trace (less than 0.5% of total).
95
TABLE XIX
FATTY ACIDS (%) OF PSEUDOMONAS AERUGINOSA STRAINS INCUBATED 40 C
Fatty acid Strain 2108 Strain 1369 16 hr 24 hr 48 hr 16 hr 24 hr 48 hr
16:0 44.2 40.7 42.4 40.0 40.2 41.8
16:1 5.6 6.6 5.6 5.9 6.4 4.9
eye 17:0 0.8 1.3 1.4 1.5 1.6 1.9
18:0 2.9 2.1 2.3 2.1 2.0 3.0
18:1 43.1 45.5 44.0 44.7 44.2 41.6
eye 19:0 3.3 3.7 4.2 5.8 5.5 6.7
aNumber preceding colon indicates number of carbons; number following colon designates degree of unsaturation (eye = cyclopropane ring).
96
TABLE XX
RESULTS OF HYDROGENATION AND BROMINATION ON PSEUDOMONAS AERUGINOSA FATTY ACIDS (%)
Fatty acida Retention time
Strain 1369, 40 C, 48 hr Fatty acida Retention
time Hydrogenation results Before After
Bromination results0
16:0 1.00 41.8 46.4
16:1 1.18 4.9 -
eye 17:0 1.54 1.9 1.7 +
18:0 1.82 3.0 45.0
18:1 2.08 41.6 -
eye 19:0 2.82 6.7 6.9 +
Number preceding colon indicates number of carbons: number following colon designates degree of unsaturation (eye -cyclopropane ring).
^Retention time relative to 16:0 (Palmitic acid).
:Plus (+) = peak eliminated.
97
appears to be low in relation to some bacterial species.
Incubation of Escherichia coli (ATCC 11775) 24 hr, 40 C
gave the following fatty acids: C14:0 (6.5%), C16:0 (53.3%),
C16:1 (trace), eye C17:0 (27.7%), CIS:1 (1.6%), and eye
C19:0 (10.3%). The low level of production of cyclopropane
fatty acids by P. aeruginosa could explain their absence in
fatty acid data of several investigators (1, 5, 7, 11).
However, the unknown acids presented by Edmonds and Cooney
(5) might have been cyclopropane acids. In addition to gas
chromatographic retention data, other chemical techniques
should be employed to detect presence of cyclopropane fatty
acids. Hydrogenation and bromination (2) are simple, rapid,
and sensitive procedures for detection of cyclopropane acids
which occur at low levels, as in P. aeruginosa.
CHAPTER BIBLIOGRAPHY
1. Bobo, R. A., and R. G. Eagon. 1968. Lipids of cell walls of Pseudomonas aeruginosa and Brucella abortus. Can. J. Microbiol. 14:503-513.
2. Brian, B. L., and E. W. Gardner. 1968. A simple pro-cedure for detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552.
3. Brian, B. L., and E. W. Gardner, 1968. Fatty acids from Vibrio cholerae lipids. J. Infect. Diseases 118:47-53,
4. Crowfoot, P. D., and A. L. Hunt. 1970. The effect of oxygen tension of methylene hexadecanoic acid forma-tion in Pseudomonas fluorescens and Escherichia coli. Biochim. Biophys. Acta 202:550-552.
5. Edmonds, P., and J. J. Cooney. 1969. Lipids of Pseudomonas aeruginosa cells grown on hydrocarbons and on Trypticase Soy Broth. J. Bacterid. 98:16-22.
6. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.
7. Hancock, I. C., and P. M. Meadow. 1969. The extractable lipids of Pseudomonas aeruginosa. Biochim. Biophys. Acta 187:366-379.
8. James, A. T., and A. J. P. Martin. 1956. Gas-liquid chromatography. The separation and identification of the methyl esters of saturated and unsaturated acids from formic acid to n-octadecanoic acid. Biochem. J. 63:144-152.
9. Kates, M., and P. O. Hagen. 1964. Influence of temperature on fatty acid composition of psychro-philic and mesophilic Serratia species. Can. J. Biochem. 42:481-488.
10. Marr, A. G., and J. L. Ingraham. 1962. Effect of temperature on the composition of fatty acids in
coli. J. Bacteriol. 84:1260-1267.
98
99
11. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 33:514-515.
12. Romero, S. M., and R. R. Brenner. 1966. Fatty acids synthesized from hexadecane by Pseudomonas aeruginosa. J. Bacteriol. 91:183-188.
13. Shank, R. S., and H. Schecter. 1959. Simplified zinc-copper couple for use in preparing cyclopropanes from methylene iodide and olefins. J. Org. Chem. 24:1825-1826.
14. Simmons, H. E., and R. D. Smith. 1959. A new synthesis of cyclopropanes. J. Am. Chem. Soc. 81:4256-4264.
15. Vaczi, L., J. K. Makleit, A. Rethy, and I. Redai. 1964. Studies on lipids in Pseudomonas Pyocyanea. Acta Microbiol. 11:383-390.
16. Verder, E., and J. Evans. 1961. A proposed antigenic schema for the identification of strains of Pseudomonas aeruginosa. J. Infect. Diseases 109:183-193.
CHAPTER V
TUMOR AND LIVER FATTY ACIDS OF DBA/lJ MICE
DURING LYMPHOSARCOMA DEVELOPMENT
Introduction
Fatty acid composition of tumor lipids have been
studied in a number of animal systems (1, 3, 4, 7, 9-12).
A major difference between tumor and liver fatty acids was
found to be the ratio between stearic and oleic acids (9,
11). Selkirk, et al. (9) found a greater percentage of
unsaturated fatty acids in phospholipids of a fast-
developing Morris 3924A hepatoma than on those of slower-
growing Reuber H-35 hepatoma. The ratio of stearic acid
(18:0) to oleic (18:1) was higher in normal liver than in
either hepatoma. Carruthers (3) found that the amount of
18:1 decreased, while that of linoleic acid (18:2) increased
following methylcholanthrene treatment of mouse skin.
Newland, et al. (7) have shown that 18:2 was the predominant
fatty acid in phosphatides of mice infected with mammary
tumor virus.
Lipid levels were shown to be lower in organs of
tumor-bearing mice. The fatty acids 18:0 and 18:2 increased,
while 18:1 decreased in organs during tumor growth (10).
Wood and Harlow (12) found a C24 dienoic acid in
100
101
sphingomyelin and ceramide fractions of Ehrlich ascites
carcinoma cells which did not appear in normal tissues.
Fatty acid studies from one investigation to another
have shown little relationship. This may have been because
of varying time periods the tumors were growing in the
animals being studied. Scholes (8) has pointed out the
importance of determining a time-activity relationship in
studying malignancy-associated changes.
The purpose of this study was to determine what
changes, if any, occurred in fatty acids of tumors and
livers of mice bearing tumors for varying periods of time.
Materials and Methods
Tissue Isolation. Male DBA/lJ mice, twelve weeks
old, were obtained from Jackson Memorial Laboratory, Bar
Harbor, Maine. The tumor line (8) has been maintained in
our laboratory for 135 passages. Transplantation of tumor
(lymphosarcoma) was performed by surgically implanting a
small mass (5-10 mg) of freshly removed, minced tissue
subcutaneously in the region of the anterior axial lymph
node.
Tumor-bearing mice were sacrificed by cervical dis-
location on the 4th, 6th, 8th, 10th, and 12th days following
tumor implant. Mice with tumors die, on an average, 12.5
days following tumor implant (8). DBA/lJ mice which did
102
not receive tumor implants were used as liver controls. Two
separate experiments were conducted in which tumors and livers
from 3 mice were pooled on each day of tissue isolation.
Livers and tumors were removed, pooled, immediately chilled
on ice, and total wet weight determined.
Lipid Extraction. All solvents were distilled before
use. Immediately after weighing, tumors and livers were
homogenized at 5 C for 5 min with a Vertis Blender at high
speed in 20 tissue-volumes of chloroform: methanol (2:1,
volume/volume). Homogenates were filtered through solvent-
washed Whatmann #3 paper and were extracted twice with addi-
tional amounts of chloroform: methanol. Lipid extracts were
washed by the method of Folch, et al. (5). NaCl (0.73%) in
distilled water (0.2 volume) was used to wash extracts. The
lower organic phase was reduced to approximately 5 ml under
partial vacuum (rotary evaporator), then taken to dryness
with a stream of nitrogen. Lipid residue was dissolved in
10 ml of chloroform and stored under nitrogen at -10 C.
Two ml aliquots were used to obtain lipid weights. Aliquots
were placed on pre-weighed aluminum foil cups and dried in
vacuo over CaCl2 ^ hrs at which time constant weight
was obtained.
Fatty .Acid .Analysis. Lipid (10 mg) was dried with a
stream of nitrogen and saponified-esterified by the method
103
of Metcalfe, et al. (6) using 0.5 N KOH in methanol followed
by boron trichloride: methanol (.Applied Science Laboratories,
College Station, Pa.). Fatty acid methyl esters were stored
under nitrogen at -10 C until analyzed by gas-liquid
chromatography.
Methyl esters were injected into a Varian Aerograph
Model 204 -1C gas chromatograph equipped with dual hydrogen
flame detectors and temperature programmer. Columns were 5
feet in length, 0.25 inch in diameter, packed with 15%
diethylene glycol succinate on Chromosorb W (60-80 mesh).
Detectors and injectors were 210 C. Columns were programmed
10 C/min between 150 C and 195 C and held at the upper limit,
or were operated isothermally at 180 C. Helium carrier was
20 ml/min. Methyl esters were tentatively identified by
comparison with authentic standards (Applied Science
Laboratories) and confirmed by hydrogenation (2).
Results and Discussion
Table XXI presents average wet weights (g) of livers
and tumors of each duplicate group of mice studied. The
fact that wet weight was determined could account for some
variation. However, an increase in liver weight is apparent
on the 10th and 12th days following tumor implant. Livers
became noticeably enlarged and proceeded from a normal
brown color to a characteristic gray as the terminal stage
of tumor development was approached. Tumor weight also
104
TABLE XXI
MEAN WET WEIGHT3 OP TUMORS AND LIVERS FROM TUMOR-BEARING MICE AT VARIOUS STAGES OF TUMOR DEVELOPMENT
Day post-implant Liver (g) Tumor (g)
Control*3 2.92 ± 0.16 -
4 00 •
CM 9 + 0.05 -
6 2.79 + 0.21 0.88 + 0.35
8 2.19 4- 0.04 2.42 + 0.25
10 4.59 -f* 0.51 4.75 + 0.20
12 5.16 0.56 4.07 + 0.49
aEach weight presented is the average of livers or tumors from two groups consisting of 3 mice per group + range.
^Control = no tumor implanted.
105
increased rapidly between 8 and 10 days post-implant. Prior
to the 3th day, it was difficult to separate tumor growth
from surrounding fatty material.
Table XXII lists the average percent lipid extracted
from wet livers and tumors. Livers increased in lipid con-
tent on the 8th day, while tumor lipid continued to decrease
from day 6 through day 10. Difficulty in separating fatty
tissues from early tumors could account for some increase
observed in lipid percentage.
Table XXIII shows the distribution of various fatty
acids in liver lipids. Only those acids present in amounts
of 0.5% or more in any one sample are presented. Changes
occurred as tumor development progressed. Palmitic acid
(16:0), stearic acid (18:0), arachidonic acid (20:4), and
a polyunsaturated acid decreased considerably, while
18:1 (oleate) and 18:2 (linoleate) increased significantly.
Total unsaturation increased slightly. Hydrogenation (2)
of selected samples resulted in saturated C14, C^, C1Q,
^20' an<^ ^22 acids. The C unsaturated acid has not been
identified but contains more than one unsaturated bond.
Table XXIV shows the distribution of fatty acids
from tumor lipids. Fatty acids 16:1, 18:1, and 18:2
decreased while 18:0, 20:4, and the C22 unsaturated acid
increased as tumors developed. Total unsaturation did not
appear to change appreciably. Fewer changes are evident in
106
TABLE XXII
PER CENT OF LIPID IN TUMORS AND LIVERS FROM TUMOR-BEARING MICE AT VARIOUS STAGES OF TUMOR DEVELOPMENT3
Days post-implant Livers Tumors
Control*3 5.5 + 0.1 -
4 4.8 + 0.1 -
6 4.5 + 0.3 9.3 + 2.6
8 8.2 + 0.2 6.8 + 0.1
10 8.5 ± 1.4 4.2 + 0.6
12 9.3 2.4 5.0 + 0.2
a Each percentage presented is the average per cent lipid of livers or tumors from two groups consisting of 3 mice per group + range.
^Control = no tumor implanted.
107
TABLE XXIII
PERCENTAGE OF FATTY ACIDS OCCURRING IN LIVER LIPIDS OF TUMOR-BEARING MICE AT VARIOUS STAGES
AFTER IMPLANTATION3
Fatty acid b
0 4 Days post-implant
6 8 10 12
14:0 trace trace trace trace trace trace
16:0 30.4 +2:8
28.3 +1.9
26.7 +0.4
26.2 +0.1
23.5 +0.3
24.6 ±0.5
16:1 1.7 +0.2
1.4 +0.2
1.5 +0.2
2.0 +0.1
2.6 +0.7
2.3 ±0.5
18:0 12.7 +0.' 2
13.3 +0.8
12.4 +0.1
8.4 +0.6
7.7 ±1.4
7.7 ±1.1
18:1 10.9 +0.8
11.6 +0.4
13.1 +0.9
18.0 +0.1
24.3 +0.8
23.3 ±1.7
18:2 20.3 +1.6
17.2 +4.0
23.6 +0.2
29.7 +0.5
29.3 ±1.4
31.2 ±0.4
20:4 10.7 +0.2
12.8 ±0.7
10.4 +0.4
6.6 +0.2
6.4 ±0.1
5.1 ±0.5
22:Unc 13.5 +0.7
15.2 ±1.2
12.3 +1.3
9.2 +0.5
6.8 ±1.7
5.9 +0.4
Average per cent fatty acids from 2 groups consisting of livers from 3 mice per group ± range.
^Number preceding colon indicates number of carbons, and number after colon designates degree of unsaturation.
C ^ Fatty acid with 22 carbons and more than one unsaturated bond,
108
TABLE XXIV
PERCENTAGE OF FATTY ACIDS OCCURRING IN TUMOR LIPIDS AT VARIOUS STAGES AFTER IMPLANTATION3
Days post-implant Fatty acid*3 6 8 10 12
1 4 : 0 2 . 0 + 0 . 2 1 . 9 + 0 . 2 1 . 8 + 0 . 1 1 . 7 ± 0 . 3
1 6 : 0 2 5 . 7 + 0 . 4 2 5 . 9 -r 0 . 4 2 2 . 7 + 0 . 8 2 5 . 1 + 1 . 7
1 6 : 1 4 . 7 + 1 . 0 4 . 0 + 0 . 3 3 . 3 + 0 . 2 3 . 1 + 0 . 1
1 8 : 0 6 . 7 + 1 . 1 7 . 6 + 0 . 2 1 0 . 1 + 0 . 4 9 . 8 + 0 . 5
1 8 : 1 3 2 . 5 + 1 . 2 3 2 . 4 -f 0 . 4 2 9 . 8 i 0 . 5 2 8 . 7 + 0 . 6
1 8 : 2 2 8 . 3 + 1 . 0 2 6 . 6 ± °-5 2 4 . 2 +_ 0 . 1 2 4 . 1 + 0 . 9
2 0 : 4 trace 2 . 2 ± o.i 4 . 2 + 0 . 1 3 . 8 + 0 . 5
22:UnC trace trace 3 . 9 + 0 . 2 3 . 0 + 0 . 4
a Average percentage fatty acids from 2 groups consisting of tumors from 3 mice per group _+ range.
ID Number preceding colon indicates number of carbons, and number after colon designates degree of unsaturation.
cFatty acid with 22 carbons and more than one unsaturated bond.
109
tumor lipids than in liver lipids. Both the C20 and C ^
unsaturated acids decreased in liver and increased in tumor.
It would be presumptive to hypothesize mechanisms
of tumor development or relationships between observed fatty
acids and carcinogenesis based upon the data presented here.
However, apparent changes in fatty acid synthesis during
tumor development suggest feasibility of using this model
animal system for future tumor lipid studies. Future
experiments should include investigation of fatty acid dis-
tribution changes in neutral lipids, phospholipids, and
specific lipid classes in developing tumor as well as in
various organ systems during tumor growth.
CHAPTER BIBLIOGRAPHY
1. Bergelson, L. D., E. V.' Daytlovitskaya, T. I. Torkhovskaya, I. B. Sorokina, and N. P. Gorkova. 1970. Phospholipid composition of membranes in the tumor cell. Biochim. Biophys. Acta 210:287-298.
2. Brian, B. L., and E. W. Gardner. 1968. A simple procedure for detecting the presence of cyclopro-pane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552.
3. Carruthers, C. 1962. The fatty acid composition of dermal and epidermal triglycerides and phosphatides in mouse skin during normal and abnormal growth. Cancer Res. 22:294-298.
4. Cartuthers, C. 1967. The fatty acid composition of the phosphatides of normal and malignant epidermis. Cancer Res. 27:1-6.
5. Folch, J., M. R. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497-509.
6. Metcalfe, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38:514-515.
7. Newland, J., R. F. McGregor, and W. E. Carnatzer. 1965. Phosphorus metabolism in viral-induced neoplasia.
• hk vivo and in vitro studies of the milk and mammary gland lipides of strains of mice susceptible to the mammary tumor virus. Can. J. Biochem. 43:297-307.
8. Scholes, V. E. 1969. Skin nucleic acid phosphorus metabolism of DBA/lJ mice during implanted tumor development and methycholanthrene carcinogenesis. Cancer Res. 29:1416-1419.
110
Ill
9. Selkirk, J.K. , J.C. Elwood, and H.P. Morris. 1971. Study on the proposed role of phospholipid in tumor cell membrane. Cancer Res. 31:27-31.
10. Uezumi, N., H. Shinichi, and K. Kasama. 1969. Lipids of neoplastic tissues. II. Lipid contents and fatty acid compositions of the lipids of several organs of tumor (NF-Sarcoma)-bearing mice. Mie Med.J. 19:141-147.
11. Verrkamp, J.H., I. Mulder, and L.L.M. Van Deenen. 1962. Comparison of the fatty acid composition of lipids from different animal tissues including some tumors. Biochim. Biophys. Acta 57:299-309.
12. Wood, R., and R.D. Harlow. 1970. Tumor lipids: structural analyses of the phospholipids. Arch. Biochem. Biophys. 141:183-189.
CHAPTER VI
FATTY ACID DISTRIBUTION OP LIPIDS FROM CARCASS,
LIVER AND FAT BODIES OF THE LIZARD,
CNEMIDOPHORUS TIGRIS, PRIOR
TO HIBERNATION
Introduction
Temperate-zone lizards are known to store lipids in
their carcass, liver, and abdominal fat bodies (1, 3, 6, 9).
Lipids in several lizard species have been associated with
vitellogenesis (6) and energy storage for winter hibernation
(1, 4). However, no information comparable to that on the
salamander, Ambystoma tigrinum (7), is available on the
exact roles of carcass, liver, and fat body lipids in
vitellogenesis and energy metabolism in lizards. Studies
producing such information must await chemical analyses of
these lipid reserves. The objectives of this study were to
analyze the distribution and fatty acid composition of lipids
in the carcass, liver, and fat bodies in male and female
Chemidophorus tigris lizards.
Materials and Methods
Lizards, collected in late August 1970, in El Paso
County, Texas, by F. G. Gaffney (N.T.S.U.), were frozen and
112
113
stored at 0 C until lipid determinations were made. Five
sexually mature males and females (snout-vent length greater
than 80 mm) were studied. Gastrointestinal tracts, post-
coelomic fat bodies, and livers were removed from carcasses
prior to homogenization in a blender. Lipids were extracted
with chloroform-methanol (2:1, volume/volume) and purified
by the method of Folch, et al. (5). Solvent was evaporated
with a stream of N2 (40 C). Total dry weights of livers, fat
bodies, and carcasses were determined as the weight of dried
lipid-extracted residues plus weight of extracted lipids.
Fatty acids were hydrolyzed and esterified by boiling
lipids in 0.5 N KOH in methanol followed by boron trichloride
(10%) in methanol (8). A known amount of internal standard,
heptadecanoic acid, was added to lipid samples prior to the
hydrolysis step. No heptadecanoate was found in control
samples. Fatty acid methylesters were analyzed using a
Varian Aerograph Model 204-1C gas chromatograph equipped
with hydrogen flarae detectors. Columns were 5 ft. x 0.125 in.
packed with 15% diethylene glycol succinate polyester on
60/80 Chromosorb W. The columns were programmed from
150-200 C, 6 C/min and held at the upper temperature. The
helium carrier flow rate was 20 ml/min. Detector and
injector temperatures were 220 C. Fatty acid methylesters
were identified by comparison with retention times of
authentic standards and by hydrogenation (2). Areas of
114
the chromatograph peaks were determined as peak height x
width at half peak height.
Resul-ts and Discussion
Table XXV shows body measurements, tissue dry weights,
and lipid content of male and female carcasses, livers, and
fat bodies. Females had a larger mean fat body weight. Fat
bodies in both males and females contained the highest mean
percentage of lipids (77 and 88 percent, respectively).
Data (Table XXV) for range of male and female fat body lipid
percentages (66-97 percent) agree with those of Rose and
Lewis (10), who found 96.6-99.7 percent in A. tiqrinum,
a salamander. Table XXVI gives the fatty acid content of
carcasses, livers, and fat bodies of males and females.
Major components were myristic, palmitic, palmitoleic,
stearic, oleic, linoleic, linolenic, and arachidonic
acids. Arachidonic acid was highest in liver lipids (8.2-
14.4 percent), lower in carcasses (4.2-4.5 percent), but was
barely detectable (trace) in any fat body lipid extract.
Qualitatively, the data in Table XXVI are comparable to
those of Rose and Lewis (10) for fat body fatty acids.
Total fatty acid content (mg of fatty acid per 100
mg lipid) in male carcasses, liver, and fat bodies was
determined to be 58-60 mg, 34-46 mg, and 66-89 mg, respec-
tively. Both males and females showed the highest percent-
age of total fatty acid content in fat body lipids (66-94
mg fatty acid per 100 mg of lipids).
115
TABLE XXV
MALE AND FEMALE C. TIGRIS BODY MEASUREMENTS (mm), TISSUE DRY WEIGHTS (mg), AND LIPID CONTENT EXPRESSED AS PERCENTAGES OF THE DRY
WEIGHT OF EACH TISSUE
Males Mean Range
Females Mean Range
Body lengths (mm)
Snout-vent 84 8 1 - 8 9 85 81-88
Tail 209 1 6 3 - 2 4 5 196 1 7 0 - 2 1 2
Tissue dry weights (mg)
Carcass 4 0 8 1 3 4 2 6 - 5 1 6 4 4385 3 8 9 0 - 4 7 8 1
Liver
Fat bodies
Lipid percentages
41 2 6 - 6 7
57 1 4 - 1 7 3
58
142
4 4 - 7 9
5 6 - 3 1 2
Carcass
Liver
Fat bodies
4
28
77
2 - 1 0
1 0 - 4 6
6 6 - 9 3
8
36
88
3 - 1 4
2 0 - 4 8
7 6 - 9 7
116
TABLE XXVI
MALE AND FEMALE C. TIGRIS FATTY ACIDS FROM CARCASSES, LIVERS, AND FAT BODIES EXPRESSED AS PERCENTAGES
(MEAN VALUES) OF THE TOTAL FATTY ACID CONTENT
Males Females Fatty acid Carcass Liver
Fat bodies Carcass Liver
Fat bodies
iViyristic 1.2 Trace 1.5 1.1 Trace 1.4
Palmitic 18.0 11.5 21.4 18.3 16.3 21.6
PaIraitoleic 2.9 1.0 4.2 2.3 2.7 2.7
Stearic 9.1 13.8 6.7 8.6 8.2 6.5
Oleic ' 47.3 25.2 50.5 43.7 37.7 47.1
Linoleic 14.0 29.7 11.3 16.8 23.3 15.5
Linolenic 3.0 4.4 4.4 5.0 3.6 5.2
Arachidonic 4.5 14.4 Trace 4.2 8.2 Trace
CHAPTER BIBLIOGRAPHY
1. Avery, R. A. 1970. Utilization of caudal fat by hiber-nating common lizards, Lacerta vivipara. Comp. Biochem. Physiol. 37:119-121.
2. Brian, B. L., and E. W. Gardner. 1968. Fatty acids from Vibrio cholerae. J. Infect. Diseases 118:47-53.
3. Dessauer, H. C. 1953. Hibernation of the lizard, Anolis carolinensis. Proc. Soc. Exp. Biol. Med. 82:351-353.
4. Dessauer, H. C. 1955. Seasonal changes in the gross organ composition of the lizard, Anolis carolinensis. J. Exp. Zool. 128:1-12.
5. Polch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.
6. Hahn, W. E., and D. W. Tinkle. 1965. Fat body cycling and experimental evidence for its adaptive significance to ovarian follical development in the lizard Uta stansburiana. J. Exp. Zool. 158:79-86.
7. Lewis, H. E., ana F. L. Rose. 1969. Effects of fat body fatty acids on ovarian and liver metabolism of Ambystoma tiqrinum. Comp. Biochem. Physiol. 30:1055-1060.
8. Metcalf, L. D., A. A. Schmitz, and J. R. Pelka. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analyt. Chem. 38:514-515.
9. Moberly, W. R. 1963. Hibernation in the desert iguana Dipsosaurus dorsalis. Physiol. Zool. 36:152-160.
10. Rose, F. L., and H. L. Lewis. 1968. Changes in weight and free fatty acid concentration of fat bodies of paedogenic Ambystoma tiqrinum during vitello-genesis. Comp. Biochem. Physiol. 26:149-154.
117
CHAPTER VII
ANALYSIS OF ACETYLATED AND TRIFLUORACETYLATED
PHENYLTHIOHYDANTOIN AMINO ACIDS
BY GAS CHROMATOGRAPHY
Introduction
Reaction of phenylisothiocyanate with the N-terminal
amino acid of a peptide or protein followed by cleavage in
acid results in formation of a 3-phenyl-2-thiohydantoin
(PTH) amino acid derivative (2, 3). Protein sequence
studies have been facilitated by analysis of the isolated
PTH using thin-layer or paper chromatography (7, 8).
Several PTH amino acids have been successfully separated
and analyzed by gas chromatography as trimethylsilyl
derivatives (5, 6). Roda and Zamorani (9) separated six
trifluoracetylated PTH's by gas chromatography using a
stainless steel column containing 5% SE-30 stationary
phase. Prior to this investigation, glass columns alone
had been used to analyze PTH's (5-8).
This report (1) compares the gas chromatographic
behavior of trifluoroacetylated and acetylated PTH amino
acids using a stainless steel column.
118
119
Materials and Methods
N-acetylation and N-trifluoroacetylation of PTH Amino
Acids. Alanine, glycine, valine, proline, leucine, iso-
leucine, methionine, and phenylalanine PTH's were purchased
from Mann Research Laboratories. Acetates and trifluoro-
acetates were made by a procedure similar to that of Roda
and Zamorani (9). To 2 mg of each PTH in 2 ml of chloroform
was added 0.2 ml acetic anhydride or trifluoroacetic anhydride
(TFAA). Reaction mixtures were allowed to stand at least 30
min at room temperature before gas chromatography.
Gas Chromatography. A Varian Aerograph Model 204-1C
gas chromatography with dual flame ionization detectors was
used. Injector and detector temperatures were 220 C. A
5 ft. x 0.125 in. (O.D.) stainless steel column containing
1% SE-30 on Chromosorb G (acid washed and silanized) was
operated at 180 C isothermally to obtain the data in Table
XXVII or programmed from 150-200 C, 10 C/min then held at
200 C. Helium carrier gas flow rate was 25 ml/min. Two ul
samples were injected with a Range of 10"11 and Attentuation
of 16.
Results and Discussion
Table XXVII gives retention times of acetates and
trifluoroacetates relative to unreacted proline PTH.
Since proline PTH was not N-acetylated, the retention time
120
TABLE XXVII
RELATIVE RETENTION TIMES OF AMINO ACID PHENYLTHIOHYDANTOIN ACETATES AND TRIFLUOROACETATES a
Acetates Trifluoroacetates
Alanine 0. .51 0. .26
Glycine 0. .60 0. .33
Valine 0. .71 0. .37
Leucine, Isoleucine 0. . 96 0. .49
Methionine 2. .36
Phenylalanine 2. .88 1, .51
aProline PTH, 5.75 min = 1.00.
121
of the derivative was not affected by acetic anhydride or
TFAA treatment. Proline was the only compound which could
be successfully chromatographed as the free PTH on the
stainless stell column, and therefore it was chosen as a
marker compound for derivatives. All PTH acetates separated
except leucine and isoleucine. Roda and Zamorani (9) also
were unable to resolve these two as the PTH trifluoroacetates.
Methionine PTH trifluoroacetate gave no peak, and the
solution turned dark brown upon standing for several hours
at room temperature. Chromatography of methionine or
phenylalanine PTH1s was not attempted by Roda and Zamorani
(9). The PTH acetates of these two amino acids were readily
separated (Table XXVII). The order of appearance of alanine
and glycine derivatives appears to conflict with that
reported previously (9).
Thus, from a variety of considerations, acetic
anhydride appears to b'e superior to TFAA for derivation of
simple amino acid PTH's.
CHAPTER BIBLIOGRAPHY
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2. Edman, P. 1950. Preparation of phenyl thiohydantoins from some natural amino acids. Acta Chem. Scand. 4:277-282.
3. Edman, P. 1950. Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4:283-293.
4. Eriksson, S., and J. Sjoquist. 1960. Quantitative determination of N-terminal amino acids in some serum proteins. Biochim. Biophys. Acta 45:290-296.
5. Guerin, M. R., and W. D. Shults. 1969. Gas chromatography of silylated phenylthiohydantoin amino acids. Utility of a sulfur-specific detection. J. Chromatog. Sci. 7:701-803.
6. Karman, R. E., J. L. Patterson, and W. J. A. Vanden-Heuvel. 1968. Gas chromatographic behavior of trimethylsilated phenylthiohydantoin amino acids. Anal. Biochem. 25:452-458.
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122
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