AD-A1BS 457 CHEMOTHERRPY AND BIOCHEMISTRY OF LEISHMAIR(U) 1MSSCHUSETTS UNJV AMHERST DEPT OF MICROBIOLOGYL L NOLAN DEC 94 DARHD1-81-C-U

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CHEMOTHERAPY AND BIOCHEMISTRY OF LEISHMANIA

ANNUAL REPORT

LINDA L. NOLAN, Ph.D.

DECEMBER 1984

Supported by

U. S. ARMY MEDICAL RESEARCH AND DEVELOPMENT CO'MAND -

Fort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMD17-81-C-1198

University of MassachusettsAmherst, Massachusetts 01003

DOD Distribution Statement ..

APPROVED FOR PUBLIC RELEASEDISTRIBUTION UNLIMITED '..~

EECTEOCT 5 1987 u

Dh

The findings in this report are not to be construed as anofficial Department of the Army position unless so designated

by other authorized documents

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SECURITY CLASSIFICATION OF TIS PAGE (ftars Date Entere)REPOT DCUMNTATON AGEREAD INSTRUCTIONS

_____ REPORT___DOCUMENTATION _____PAGE BEFORE COMPLETINGFORM

1. REPORT NUMBER ,3OVT ACCESSION NO. 3. RECIPIENT'S CATALOG NUMBER

4. TITLE (and Subtitle) 5. TYPE OF REPORT & PERIOD COVEREDAnnual Report

Chemotherapy and Biochemistry of Leishmania Jan.l 1984-Dec.31,19846. PERFORMING ORO. REPORT NUMBER

7. AUTHOR(&) S. CONTRACT OR GRANT NUMBER(&)

Linda L. Nolan, Ph.D. DAMDl7-8i-C-1198

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT PROJECT, TASK

Department of Microbiology AREA & WORK UNIT NUMBERJ

University of Massachusetts 61102-A-3MI61102BS10.AF.08oAmherst,_MA__01003 _____________

11. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE

U.S. Army Medical Research and Development December 1984Coimmand; Fort Detrick, Frederick, MD 21701-5012 13. NUMBEROPPAGES

1.MONITORING AGENCY NAME & AODRESSI different from Controlling Office) 1S. SECURITY CLASS. (of this report)

Unclassified

1S. DECL ASSI FI C ATION/ DOWN GRADINGSCHEDULE

16. DISTRIBUTION STATEMENT (a( this Report)

Approved for public release; distribution unlimited

17. DISTRIBUTION STATEMENT (at the abstract entered In Block 20, If different fhem Report)

I8. SUPPLEMENTARY NOTES

1S. KEY WORDS (Continue on, reverse vids It nacoaarv and Identify by block num~ber)

Leishmania/mode of action purine anologs

2% A§4rR ACT (Cmtie ma ro wevu as t~ It roweasay sa Identy by block numnber)

~A comparison of the enzymes of pathogenic protozoa to those of man is offundamental importance to the search for much needed chemotherapeutic agents.The enzymes involved in purine salvage are of particular interest becausemost pathogenic protozoa lack the ability to synthesize purines de novo andconsequently are obligate salvager-s of preformed purines.

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Summary \

On May 17, 1984 workshop on antileishmanial drug development was set up

at WRAIR for the purpose of establishing a major intermural, multidisciplinarydrug development research effort. This antileishmanial research program is beingdirected through the Division of Experimental Therapeutics, and includes threelaboratories at WRAIR and three U.S. university laboratories. The role of thislaboratory in the program is to test compounds in vivo using different Leishmaniaspp. and to determine the molecular mode of action of promising compounds.Compounds provided by WRAIR were tested singly, and for synergy, in combination.

The most promising compounds to date sent by WRAIR appear to be the foliowing:

BK63005 3-B-D-Ribofurano-sylpyrazolo-[4,3-d] pyrimidin-7-thioneBK74731 oxoformycin B;BK63863 Thiopurinol riboside

BK71338 oxoformycin A'BK86124 Allopurinol riboside

9-Deazainos ine

The following compounds sent by Dr. Peter K. Chiang (Department of Biochemistry,WRAIR) were found to be very inhibitory to promastigotes of Leishmania mexicanaamazonensis WR227 : 4'-thioadenosine, deoxyaristeromycin and 5-deoxv-5 (isobutylthio)-3-deazaadenosine (deaza-SIBA).

Sinefungin, a naturally occurring antifungal nucleoside antibiotic containing an

ornithine residue, obtained from Dr. M. Robert-Gero (ICSN-CNRS, Gif Sur Yvette,France) was also found to be very inhibitory.

A DNA-dependent RNA polymerase has been isolated and characterized fromLeishmania mexicana WR #227. The initial stages of purification utilized high ionicstrength extraction and protamine sulfate treatment. The enzyme was further purifiedby differential elution on Heparin-Sepharose, DEAE-Sephadex, and Carboxvmethvl-Sephadex chromatography. Analysis of the chromatographicallv purified RNA polvmeraseon nondenaturing gels revealed two electrophoretic forms. The enzyme isolated hascharacteristics of true DNA-dependent RNA polymerase since it requires DNA and all fournucleoside triphosphates for the synthesis of RNAase-sensitive products. Analysisof ammonium sulfate and metal ion optima, as well as relative activities of theenzyme with Mn2 + versus Mg2 + are similar to those reported for other RNA polymeraseIII in eukaryotes.

Formycin A triphosphate was found to he a competitive - ubstrate Ior this enz%'TrL,and cordycepin triphosphate was found to be inhibitory, although the mode ofinhibition was not determined.

We have partially purified a DNA polvmerase from L. mexicana kVR #227 which IN-ethvlmaleimide sensitive, aphidicolin resistant and showed different sensitivitiesto 2-acrylaminopurine deoxvribonucleoslde-5'-triphosphate than mammalian DNAnolymerase The purification scheme restl ted in removal of over 99 of proteinwi th over ai 2'32 fold increase in specific activi tv. 7 1 I :

-DO4

V i, . ,

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TABLE OF CONTENTS

Summary .............. ............................. 3

Resume of Progress ........... ....................... 5

Distribution List ........... ........................ 51

4

4

- . .

RESUME OF PROGRESS

Culture Methods:

The organisms used in this project have been obtained from the WalterReed Army Institute of Research through the courtesy of Dr. Joan Decker-Jackson

and Dr. Jonathan Berman. The organisms used most have been Leishmania mexicanaamazonensis WR 227 and L. donovani WR 130 (Khartoum strain-drug sensitive visceralleishmaniasis). Other organisms presently being cultivated in this laboratoryare IL. braziliensis WR 424 (Murray isolate from Panama causing cutaneousleishmaniasis), L. braziliensis WR 063 (Terborgh isolate from Peru, causingmucocutaneous leishmaniasis). These organisms are maintained by weekly transfersinto Schneider's medium [Grand Island Biological Co., Grand Island, N.Y. (Gibco)]containing 10% heat inactivated fetal bovine serum (HIFBS: GIBCO).

For growing large batches of leishmaniae promastigotes, Brain Heart InfusionMedium (BHI) containing 37 g Difco Brain Heart Infusion/liter water, 10% heatinactivated serum and 26 og hemin/ml was used. Cells were grown at 26*C in 2000ml wide Fernback flasks containing 250 ml of BHI and harvested during the exponentialgrowth phase (about day 4).

For defined biochemical experiments the medium of Steiger and Black was used.

This medium was used for all transport, uptake and reversal experiments. Thecells were depleted of purines by transferring an inoculum from Brain HeartInfusion into Steiger and Black medium with purine omitted, but with 5% heatinactivated fetal bovine serum. The cells were incubated in this medium for 48 hrat 26'C. The cells were then aseptically centrifuged at 5000x v for 10 min andresuspended to the desired number into fresh Steiger and Black medium Minus purine.By treating the cells in this manner, we avoided as much as possible interferenceof the metabolism of the compound being tested by the purines in the medium.

RE iX (Steiger and Black)

Components per liter:

A) 8.Og NaCL C) 300 mg L-glutamine400 mg KCL 1.0 g NaHCO3200 mg MgSO4 .7120 14.25 g HEPES (=b0 mM)

60 mg Na2HPO 4 .2H20 20 mg adenosine

60 mg K112PO 42.Og glucose D) 1 mg D-biotin

1 mg choline chloride3) 200 mg L-arginine I mg folic acid

100 mg L-histidine 2 mg i-inositol100 mg L-iosleucine I mg niacinamide300 mg L-leucine I mg D-pantothenic acid250 mg L-lysine. HCL (hemi-calcium salt)50 mg L-methionine 1 mg pyridoxal.HCl

100 mg L-phenylalanine 0.1 mg riboflavine300 mg L-proline 1 mg thiamine.HC1400 mg L-threonine30 mg L-tryptophan E) 2.5 mg haemin50 mg L-tvrosine100 mg L-valine

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The experimental design for the testing of promising purine compounds for

antileishmanial activity was as follows. The promastigote form of the followingorganisms were used for testing.

(1) Leishmania mexicana WR #227(2) Leishmania donovani WR #130

(3) Leishmania braziliensis WR #424

Medium used was the defined medium of Steiger and Black lacking purine, butsupplemented with 5% inactivated fetal calf serum and 0.05 mg/ml gentamicin.L. braziliansis does not grow well in this medium at 5% serum, so it was grown.. 10% serum. The leishmania were initially grown in Brain Heart Infusion andwhen in log phase (2xlO 6 cells/ml) these cells were used as an inoculum (0.5ml)and asceptically transferred to 4.5m1 of Steiger and Black medium (as described)in 14.5 cm x 1.5 cm test tubes. The compound to be tested was added (at 0.1 -5004M) and the cells were incubated in a slanted position at 26'C. Optical Density

readings at 660nm were taken every 24 hrs. for a total of 96 hrs. Growthexperiments were done in duplicate.

Compounds found to be inhibitory were retested in medium containing differentpurines in order to determine if the inhibitory action could be reversed withthe addition of a particular purine. Purines tested for reversal of inhibitoryaction included: (1) Adenine (2) adenosine (3) guanine (4) guanosine(5) hypoxanthine (6) inosine.

Information gained from purine reversal experiments provides clues as towhich purine enzyme or uptake process is being affected. Depending on whichpurine reverses we then test the anolog in vitro on one of our enzyme systems.For exampie, if adenine reversed the inhibitory action we would test thoseenzyme systems which used adenine as a substrate.

Our laboratory is set up to do the following in vitro enzyme assays:

1. Adenine deaminase

2. Adenine phosphoribosyltransferase3. Guanine deaminase4. Hxpoxanthine-Cuanine phosphoribosvltransferase5. Nucleosidases6. Xanthine phosphoribosvltransferase

7. Adenylate deaminase

8. Adenylosuccinate synthetase

Indorporation of 14C-uridine into RNA, 14C-phenvlalanine into protein and14 C-thymidine into DNA is determined in order to acertain if macromolecularsynthesis is being inhibited.

If we find that no salvage enzyme is inhibited but macromolecular svnthcsisis, we then test the following enzymes and systems:

i. DNA polymerase2. RNA polymerase3. incorporation of compound into RNA throush usk, of mass spectrowetrv

if no isotope is availabl.4. inhibition of protein synthusis usinf' a cell-free lei.Thmanial lvsate

svstem.

:7

By using the above test systems, we can determine the mode of actionof the compound so that this information can be used to (1) synthesizebetter derivitives (2) explain possible host toxicity and lead to protocolsto avoid this complication (3) help understand and combat resistance to thecompound.

Compounds (sent by WRAIR) Tested

1) BK 63863 Thiopurinol riboside2) BK 74731 Oxotormycin3) BK 86124 Allopurinol riboside4) BK 86133 5-Azaxanthosine5) BK 86142 7-Ribosyl-3-deazoguanine6) BK 63005 3-3-D-Ribofurano sylpyrazolo-[4,3-d] pyrimidine-7-thione

7) BK 48464 6-Aminoallopurinol Riboside8) BK 95141 3-Ethoxy-6-methylthio-l-B-D-ribofurano sylpyrazolo-[3,4-d]

pyrimidine-4(5H)-one

9) BK 95169 3-Ethoxy-l-6-D-ribofuranosylpyrazolo-[3,4-d] pyrimidine-4(5H)-one10) BK 95187 6-Methylthio-f-B-D-ribofuranosyl-4(5H)-oxopyrazolo- [3, 4-d]

pyrimidine-3-ca rboxamide11) BK 95203 7-Amino-5-chloro-3-8-D-ribofuranosyl-pyrazolo- [4, 3-d] pyrimidine

(5-chloro formycn)12) BK 95730 6-Aminoimidazo[4,5-C] pyridin-4(5H)-one (3-Deazaguanine)

Compounds Which Were Significantly Inhibitory at the End of 96 irs

Organism Concentration of BK 63005 (% Inhibition)10 "M 100 1M 500 uM

L. donovani 130 74 82.4 83.8

L. mexicana 227 68.4 83.5 83.0L. braziliensis 424 34.6 52.9 52.9

Organism Concentration of BK 74731 (% Inhibition)

10 WM 100 ujM 500 ijM

L. donovani 130 31.8 78.7 84.9L. mexicana 227 15.5 71.6 83.5L. braziliensis 424 20.7 36.0 42.2

Organism Concentration of BK 63863 (% Inhibition)

10 uM 100 WM 500 -.M

L. donovani 130 60.6 74.3 70.1L. mexicana 227 58.3 71.9 75.4

L. braziliensis 424 3.1 17.6 24.3

Organism Concentration of BK 86124 (% Inhibition)10 WM 100 uM 500 wM

L. donovani 130 46.1 50.1 48.7L. mexicana 227 30.4 41.1 67.9L. braziliensis 424 1.8 5.8 8.7

7

' " % % ' 44 i*-~%~

Organism Concentration of BK 48464 (% Inhibition)10 pM 100 liM 500 PM

L. donovani 130 9.8 35.2 56.2L. mexicana 227 -- 13.8 20.4L. braziliensis 424 -- 27.9 41.3

Compounds Which Showed Up To 20% Stimulation

1. BK 86133 5-Azaxanthosine

2. BK 86142 7-Ribosyl-3-Deazoguanine

These compounds appear to be broken down and metabolized as natural purines.

Figures 1-5, show the response of different Leishmania spp. to the mostinhibitory purine analogs (sent by WRAIR).

Compounds which were inhibitory were tested for reversal of inhibition bynatural purines. The concentration of the analog was 50vM and that of thenatural purine was 200 uM. Table 1 shows the ability of natural purines toreverse the inhibition by the analogs. The purines which were most effective inreversing inhibition by the particular analog are as follows:

Purine Anolog Purine Most Effective in Reversing Inhibition

3-B-D-Ribofurano-sylpyrazolo- Inosine[4,3-d] pyrimidine-7-thione

BK 63005

Oxyformycin AdenosineBK 74731

Thiopurinol riboside Guanosine (not very effective)BK 63863

Allopurinol riboside AdenosineBK 86124

6-Aminoallopurinol riboside Adenosine, Hypoxanthine (equal)BK 48464

6-Aminoimidazo[4,5-C] pyridin Guanosine-4(5H)-one (3-deazaguanine)BK95730

Other purine anologs (not sent by WRAIR) have been shown to be very potentgrowth inhibitors of promastigotes of L. mexicana #227. The following tablecompares the toxicity of these compounds to some of the most promising anologssent by WRAIR.

8*

8

Fi~g. 1

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Sinefungin .005Formycin B 0.1Aphidlcolin 24'Thioadenosine 3Oxoformycin A 4Deoxyaristeromycin 85-Deoxy-5(isobutylthio)-3-Deazaadenosine 20(deaza-SIBA)Cordycepin 259-Deazainosine 40Oxoformycin B 50Allopurinol riboside 200

Tables (2-4) and figs (6-7) show inhibition of these various compoundsalone and in combination with others. As can be seen oxoformycin A and B showno additive effect when used in combination. Oxoformycin A is an adenosine analogand oxoformycin B is a xanthosine analog, so it was believed that the toxicityof these compounds would be additive. The fact that oxoformycin B was found toinhibit growth is an important finding, since this compound has been reportedto be non toxic to both eukaryotes and prokaryotes (1). It is because of thisthat oxoformycin B and 9-deazainosine (also proposed to be non-toxic (2) havebeen combined together and with sinefungin for studies on growth inhibition.Sinefungin is >l0OX more active in Leishmania than mammalian cells, and hits a"hot" target (one which cannot be easily modified or mutated--DNA polymerase).It appears that sinefungin does not have to be metabolized to be toxic, and itis not incorporated into DNA because of its structure. Combining sinefunginat extremely low levels 5-2.5 nM with non toxic compounds which add to the toxicityof sinefungin via another mode of action should provide a safe and rational approach

to chemotherapy of leishmaniasis.

Sinefungin is a naturally occurring antifungal antibiotic nucleoside, containingan ornithine residue (3). This compound has been found to display antiparasiticactivity against malarial parasites (4), T. cruzi (5) and against Leishmaniaspp. (6). We have found that sinefungin inhibits partially purified DNApolymerase activity from L. mexicana #227. The preparations we used had beensubjected to cell homogenation, centrifugation, and DEAE cellulose chromatography.

Using our partially purified DNA polymerase from L. mexicana we have foundthe K for dATP to be 55.5m (Fig. 8), the K. for sinefungin to be l5nM (Fig. 9).mSinefungin was found to be a competitive inhibitor of dATP (Figures 10, 11).Fig. 12 shows the effect of increasing concentrations of sinefungin onL. mexicana DNA polymerase. Figure 13 shows the inhibition of growth ofpromastigotes of L. mexicana by sinefungin.

It is interesting to note that S-adenosyl-homocysteine (SAH) andS-adenosyl-methionine (SAM) (33 tiM) which are similar in structure to sinefungin(Fig. 14) did not inhibit DNA polymerase activity in the above preparation.

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We developed a purification s$leme which resulted in removal of over99% of the protein with over a 282 fold increase in specific activity(Table 5). The DNA polymerase at this stage of purification was N-ethyl-maleimide sensitive and aphidicolin resistant and was not inhibited bysinefungin. These results suggest several possibilities (1) our initialisolation procedures contained an enzyme other than DNA polymerase which wasinhibited by sinefungin and subsequently interferred with our assay and (2)during the isolation procedure the DNA polymerase was modified and sinefunginno longer was inhibitory. We know that sinefungin inhibits nuclear DNAsynthesis by 80% in vivo experiments. Aphidicolin a DNA polymerase t inhibitorin bacteria, plants and eukaryotes (7), is an extremely potent growth inhibitorof leishmania, but we have not been able to detect a DNA polymerase which isinhibited by this compound. Solari et al., (8) reported that Trypanosoma cruziDNA polymerase (predominate form) also failed to be inhibited by aphidicolin,but that aphidicolin inhibited growth. It is unclear at present, whetheraphidicolin inhibits other crucial metabolic processes or inhibits a DNApolymerase which we presently are unable technically to detect. We are currentlydetermining the mode of action of aphidicolin and sinefungin in more detail.

RNA Polymerase

In experiments to elucidate the mechanism of action of formycin B (FoB),we isolated total RNA from [VH]-FoB exposed cells (8uM for 6 hr) and fractionatedthis into tRNA, rRNA and mRNA by a sucrose gradient (15-30%). Figure 15,demonstrates that [3H]-FoB metabolites were incorporated into all RNA species.To determine the effect FoB and its metabolites had on RNA polymerase ofL. mexicana #227 we partially purified this enzyme.

Isolation and characterization of RNA polymerases in parasitic protozoawill not only help elucidate the mechanism of action of some promising anti-leishmanial agents, but will eventually help us understand the mechanism ofpromastigote to amostigote transformation.

RNA Polmerase Solubilization. RNA polymerase was soiubilized from L. mexicanausing- procedures similar to those described for other tissues (9-13). Cells(I liter) were harvested in 250 ml centrifuge bottles and centrifuged at12,000 rpm for 10 7,in. The cell pellet was washed twice in Buffer A containing

0.05 M Tris-HCl (pH 7.9, 23°C), 25" (v/v) glycerol, 0.1 mM EDTA, 0.5 mMdithioerythritol, 5 n:M MgCI 2 and 1 mM phenylmethylsulfonyl fluoride. Thecell pellet (usually 4-6 g wet wt.) was suspended in 10-15 ml of Buffer Asupplemented with 0.3 M (NH,) SO and 1:100 of the following stock solutionof protease inhibitors (i00x). soybean trypsin inhibitor (4.8 mg ml- 1 ),

aprotinin (4.8 mg ml-1 ) and leupeptin (2 mg mil-1 ). The cells were lysed by30 strokes in a Potter-Elvehjem glass-Teflon homogenizer. The suspension wassonicated 6 to 10 times for 10 S with 1 min cooling intervals. This was carriedout with a Braun Sonifier 2000 at 4°C. The suspension was centrifuged at15,000 rpm for 30 min at 40C. This supernatnat fluid was either subjected topoly (ethylene imine) treatment or to protamine sulfate treatment, dialysisand column chromatography. DNA was removed by adding sufficient 2" Protaminesulfate to the crude enzyme to result in a 1:10 dilution. The precipitatefrom the protamine sulfate step was removed by centrifugation at 15.000 rpmfor i3 min at 4C. The supernatant was dialyzed in 2 L of Buffer A containinc0.1 M (NH4) SO,. The precipitate that formed after dialysis was removed bycentrifugation at 13,000 rpm for 10 min at 40 C.

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RAW-

Iteparin-Sepharose CL-6B (Pharmacia Fine Chemical Co.) was equilibrated withsix column volumes of Buffer A contaning 0.1 M (NH4 ) 2 so 4 . From 4-7 ml of thedialyzed crude extract was applied to the column and 1 ml fractions werecollected. After the column was washed with two volumes of the above bufferand no further protein was eluted from the column, NA polymerase was elutedwith Buffer A containing 0.5 M (NH.)2 SO4 . Peak fractions were pooled anddialyzed overnight in 2L of Buffer A containing 0.075 M (NH4 )2 SO4.

DEAE-Sephadex A25. The gel was prepared by suspending 8g of the powder into100 ml of 0.05 M Tris HCI (pH 7.9), 0.1 mM EDTA and 1 M (NH 4 )2SO4 . The gelwas swelled by heating in a 90*C water bath for 1.5 hr. The gel was cooledovernight to room temperature and then heated again to 70*C. When the gel

slurry reached room temperature the buffer was decanted, and the above bufferwith 0.075 M (NH ) 2 So 4 was added. The gel was poured into a 1.5 x 17 cmcolumn and equilibrated with several column volumes of Buffer A containing0.075 M (NH 4) 2SO4

The dialyzed fractions from the Heparin-Sepharose chromatography wereapplied. Buffer A (60 ml) containing 0.075 M (Ni1 ) SO4 was washed throughthe column. The RNA polymerase was then eluted wt9 a linear gradient0.075-0.5 M (NH4 ) S) S in a volume of 100 ml. Active fractions were pooledand dialyzed overnignt in 2 L of Buffer A containing 0.05 M (NH ) SO

4 2 4

Carboxymethyl-Sephadex Chromatography. The gel was prepared as described forDEAE Sephadex chromatography except that the column was equilibrated withBuffer A containing 0.05 M (Ni,) SO 4 . The pooled material from the DEAE-Sephadex column was applied an the enzyme was eluted in a 0.05-0.3 M(I'M4 )2 So4 linear gradient.

Sephacryl S-300 Chromatography. The gel was equilibrated in Buffer A withoutglycerol, and poured into a 1.5 x 100cm column. The packed column measured

1.5 x 75cm. RNA polymerase obtained from DEAE chromatography was subjectedto gel filtration on Sephacryl S-300 for a molecular weight determination.Since RNA polymerase is very unstable in the absence of glycerol, 0.1 ml ofglycerol was placed in the tubes collecting the fractions (I ml). The re-

sulting glycerol concentration in the fractions collected was 10%.

ioly (ethylene imine) Precipitation. A 10 (v/v) solution of poly (ethyleneimine) was prepared and neutralized as described (14). In order to deter-minethe optimal conditions for the use of this method of purification, crudeextract was divided into 1 ml fractions and each of these was mixed with theindicated volumes (Fig. 16) of 10% poly (ethylene imine) and allowed tostand 15 min so that complete precipitation could take place. These fractionswere then centrifuged in a Fisher microfuge for 10 min at 7,500 x g. Thesupernatant fluid from each fraction was collected and assayed for RNApolymerase activity.

RNA Polymerase Assay. Assays for monitoring column fractions were conductedin 96 well microplates. When drug studies were beinez performed, assays were

conducted in 1.5 ml microcentrifuge tubes to prevent protein or drug fromsticking to the surface. The radioactive assav used was a modification of thatused bv Roeder (10). The following components were present in each tube in afinal volume of 51 ,i: 0.05 M Tris HCI (pH 7.0): 2 N MnCI,; 0.6 mM ATP;0.6 mM GTP; 0.6 mM CTP; 0.05 maM UTP; 0.088 mg/ml denatured alf thvmus DNA;

31

Fig. 16

10

* 8'0

a.4

-2

10 30 50 70 90 110 130IJl 10% POLYMIN P

32

-"4,'-''*.*.,' . './*'.. :. . , .... , ,. ,. _ . -. * -.- * - , ,- ., , ,, .. ., ,. . , ,.,... .. • . . , _ . . . . - ; .: -. . ,- - - .... , .. .% . ..%,

ftm ftAW Mnan ILI aTL-W an %A YLPIM Mr Pin lanw PL mrr wna na n. .r i arvi

and 0.2 W Ci [3 H UTP. Unless otherwise stated the (NH4 ) SO. concentration was0.1 M. Each reaction was begun with the addition of 20 ii oi enzyme solution inBuffer A. After 20 min at 30*C, reactions were terminated by pipetting 10 ulof a solution containing 2.5% SDS and 0.15 M sodium pyrophosphate (14). Thereaction mixture was then pipetted onto DEAE-cellulose discs (Whatman DE81)followed by immersion in 0.5 M Na HPO . The filters were washed six timesfor 5 min each in this buffer, twice in distilled H90, twice in 95% EtOH, oncein ether, and air-dried (10). The discs were counted in Fisher Scinti Verse IIin a Beckman scintillation counter.

One unit of activity is defined as the incorporation of 1 pmole of U Pinto RNA in 20 min under standard assay conditions (10).

Polvacrylamide Gel Electrophoresis. RNA polymerase which had been chromatographedon Heparin-Sepharose, DEAE-Sephadex, and Phosphocellulose was subjected to non-denaturing polyacrylamide gel (7%) electrophoresis using the method of Davis (14).One tube was sliced into 4 mm sections immediately after electrophoresis and theslices were assayed in 3 times the standard assay volume. The slices wereincubated 90 min at 30*C, and the reaction mixture was then spotted on DE81cellulose pads and prepared for scintillation counting as described above. Theother gels were stained overnight in 0.2% comassie blue R-250, 50% methanol and10% acetic acid. The stained gel was destained in a Bio-Rad destainer utilizing25% methanol and 10% acetic acid.

Protein Assays. Protein concentrations were either determined by the dye-bindingmethod of Bio-Rad Labs (595 nm) or by a modification of the Bio-Rad Labs method.The modified method was performed in 96 well microplates by adding 80 ul ofBio-Rad dye and 20 il of a column fraction. The plate was then read in a Dynetech580 microplate reader at 575 nm.

Denatured DNA. Calf thymus DNA (Sigma, 0.5 mg/mI) was heated in a water bathat 90*C for 5 min and then quickly chilled on ice.

RESULTS

RNA polymerase activity could not be detected in crude preparations ofL. mexicana. It appears that the removal of DNA and inhibitory proteins arenecessary to detect this enzyme. In our experiments 96-99% of the proteinconcentration was removed from the initial preparation as well as the DNA before RNApolvmerase could be detected. The method utilizing poly (ethylene imine) precipi-tation provides a rapid procedure for removing inhibitory substances. Poly(ethylene imine) is very basic and is expected to precipitate acidic moleculesincluding nucleic acids, nucleoproteins (chromatin and ribosomes), and acidicproteins (including RNA polymerase II) (11). The results of precipitating RNApolymerase with a 10% poly (ethylene imine) solution are shown in Fig. 16. Theenzyme activity in the supernatant initially increases, reaches a maximumand then decreases. The initial increase results from the removal of one or moresubstances which inhibit RNA polymerase. The subsequent decrease in activityreflects the coprecipitation of the enzyme with the poly (ethylene imine), ashas shown to be the case with other systems (11). Fig. 16 shows that all theRNA polymerase activity is effectively precipitated at anv concentration of poly(ethylene imine) above 0.Z.% (v/v) crude extract. Addition of 30 111 of a 10"poly (ethylene imine) solution ml-I of crude extract removed P7-99 of the proteinand nucleic acids.

33

The supernatant fluid from the poly (ethylene imine) precipitation wassubjected to DEAE-Sephadex chromatography and yielded only one active RNApolymerase peak. Also, the method of Smith and Braun (13) utilizing poly(ethylene imine) precipitation of RNA polymerase activity and subsequent releaseof RNA polymerase activity with (NH4 )2 SO failed to detect more than oneRNA polymerase when chromatographed on DEE-Sephadex. Chromatography on

DEAE-Sephadex appears to be the most effective means for resolution ofthe various enzymes in a single chromatographic step. RNA polymerases I, 11and III from most cell types are eluted from this ion-exchange matrix at

concentrations of approximately 0.05-0.15, 0.15-0.25 and 0.20-0.35 M ammoniumsulfate respectively (16). Since we wanted a method which would allow us todetect all three polymerases poly (ethylene imine) precipitation wasabandoned. It appeared that this method was inadequate for the solubilizationof all RNA polymerase types in L. mexicana promastigotes.

To determine the effect of sonication on releasing RNA polymeraseactivity, we sonicated the crude homogenate for different lenghts of time,precipitated inhibitory material with 30 A poly (ethylene imine) m1-1 ofcrude homogenate, centrifuged (7,500 x g for 5 min), and assayed for RNApolymerase activity. Fig. 17 demonstrates that sonication is necessary torelease RNA polymerase activity, but once the enzyme(s) are released, they areinactivated by further sonication.

We found chromatography on Heparin-Sepharose after homogenation, sonicationand protamine sulfate treatment of the crude extract to be a very efficientstep in the purification of RNA polymerase. The protein concentration wasreduced by 95%. RNA polymerase was eluted with a 0,5 M (NH 4 )2 SO step gradientin Buffer A. The inhibitory substances were removed and one peak of RNA poly-merase activity was detected (Fig. 18). Pooling of active fractions from theHeparin-Sepharose and subsequent rhromatography on DEAE-Sephadex increased the

specific activity 66 fold (Fig. 19). Further chromatography on Carboxymethyl-Sephadex (CM) of pooled active fractions resulted in a 2.25 fold increase in

specific activity from the previous step (Fig. 20). Table 6 shows a purificationscheme for RNA polymerase. During Heparin-Sepharose and CM-Sephadex chromato-graphy only one RNA polymerase peak was observed. Several times during DEAE-Sephadex chromatography a small RNA polymerase peak could be detected closelyassociated with the major RNA polymerase peak. Assay of each of the peak fractions,

revealed similar sensitivities to a-amanitin and (NH ) SO, concentrations. Itis not known whether the appearance of this second pIea is the result of proteolvsisof the enzyme or represents a multiple form of an at-amanitin-sensitive RNApolymerase.

The results of gel electrophoresis under nondenaturing conditions of RNApolymerase which had been subjected to protamine sulfate treatment, Heparin-Sepharose, DEAE-Sephadex, and phosphocellulose chromatography is shown inFig. 21. Only two protein bands could be detected when stained with commassieblue. RNA polymerase activity could not be detected when gels were sliced andassayed immediately after electrophoresis. Gel filtration on Sephacrvl S-300of RNA polymerase pooled from a CM-Sephadex column revealed a molecuaar weightof 360,000 (Fig. 22-23). Since this column did not contain glycerol whichstabilizes the polymerase the specific activity after gel filtration was very low.Gel filtration with 25% glycerol in Buffer A takes almost 48 hr to complete.Because of this, the glycerol was omitted, but added to the fractions afterfiltration.

34

Fig. 17

RVA POLYM~ERASE ACTIVITY

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42

Assay of RNA polymerase from the pooled fractions of a DEAE-Sephadexcolumn revealed that the enzyme reaction was linear up to 20 min (Fig. 24).RNA polymerase isolated from DEAE-Sephadex chromatography showed 50% inhibitionat a 4 g/ml c-amanitin concentration (Fig. 25). The ci-amanitin up to 20 wg/mldid not completely inhibit this enzyme. This same enzyme preparation dialyzedagainst Buffer A without metal ions was tested for optimum metal ion concentra-tion in the RNA polymerase assay. Fig. 26 shows that the optimum concentrationfor MnCl2 was 1 mM and that increasing the MnCl concentration beyond 3 mMgreatly inhibited the enzyme. The enzyme showe optimal activity with a MgCl2concentration between 2-4 mM, but this metal was only 14% as effective in enhanc-ing optimal activity when compared to MnCl2.

DEAE-Sephadex pooled fractions dialyzed against Buffer A without (Ni ) SOwere used to determine the optimum salt concentration for the RNA polymeraseassay. As shown in Fig. 27, an (NH4) 2SO4 concentration between 0.02-0.06 Mgave optimal RNA polymerase activity. Increasing concentrations beyond 0.1 M(4114)2So4 inhibited the enzyme.

Since the purpose of our isolation of RNA polymerase was to provide anadequate system to test promising nucleotide analogs, we tested the effect offormycin A-triphosphate (FoA-TP) and cordycepin triphosphate on RNA polymeraseisolated from DEAE-Sephadex chromatography. As shown in Fig. 28, FoA-TP appearsto be a competitive substrate for the RNA polymerase. The addition of increasingconcentrations of ATP (to 0.6 mM) in the presence of 0.4 mM FoA-TP relieves thecompetition, but does not completely reverse it. We have demonstrated previouslythat FoA-TP is incorporated into RNA and that the mRNA containing FoA nucleotidestranslates less efficiently than normal mRNA in L. mexicana promastigotes (17).We are in the process of determining the effect of tRNA containing FoA nucleotideson tR.NA function.

'p Cordycepin triphosphate is known to affect RNA polymerase in a variety oforganisms, but the exact kinetic nature of this inhibition is unknown (1).Cordycepin triphosphate inhibited the RNA polymerase in a linear manner from10-60 .M, hut increasing the concentration up to 100 M did not completely inhibitthis enzyme. This result may be due to more than one RNA polymerase speciespresent in our preparation showing different sensitivities to this nucleotide,or the nucleotide may compete with the natural nucleotides (which are all presentin the assay) and under these conditions was not able to bind to all theavailable sites.

The enzyme isolated has characteristics of true DNA-dependent RNA polymerasein that it requires DNA and all four nucleoside triphosphates for the synthesisof RNAase-sensitive products. The reported ammonium sulfate optima and metalion optima, as well as relative activities of the enzyme with Mn2+ versus lg 2 +are similar to those reported for RNA polymerase III (9). Also, a-amanitinsensitivity and elution characteristics during chromatography indicate that thisenzyme is RNA polymerase III (12).

RNA polymerase III is present in a variety of cells and tissues althoughits concentration is subject to great variability (9). Eukarvotic cells containseveral distinct RNA polymerase proteins, which are localized in different sub-cellular fractions and play different functional roles in the cell. DNA-denendentRNA polymerase III represents one of the three major classes of nuclear RNApolymerases in eukarvotes. The class 111 RNA polvmerases from a variety of celltypes have been shown to have characteristic catalytic and chromatopraphic

43

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RNA POLYNERASE ACTIVITY IN RESPONSE TO TIME

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properties and characteristic subunit structures, all of which distinguishthese enzymes from the homologous class I and II RNA polymerases (9). The classIII RNA polymerases have been implicated in the synthesis of tRNA, 5 sRNA,and other small cellular RNAs and in the synthesis of two small viral RNAspecies in adenovirus-infected cells (9).

We have tested the following compounds sent by WRAIR on isolated RNA andDNA polymerase. Our lack of inhibition probably results from the fact thatmany of these compounds are active when phosphorylated to the triphosphate.

Compounds Tested with No Effect on DNA Polymerase (500,om)

BK86124 Allopurinal ribosideBK74731 OxoformycinBK63005 3-B-D-Ribofurano sylpyrazolo-[4,3-d] pyrinlidine-7-thioneBK48464 6-Aminoallopurinol RibosideBK71338 Oxoformycin A

Compounds Inhibitory to DNA Polymerase at 500um

BK63863 11.52% Thiopurinol riboside

Compounds Inhibitory to RNA Polymerase at 500m

BK74731 9.5% Oxoformycin BBK71338 13.7% Oxoformycin A

44

49

d -a . . . • A J.. ". . .. J. .

- .M. A :R , . . . .... I Ar -7I

Literature Cited

1. Suhadolnik, R.J. 1979. Nucleosides as Biological Probes, John Wiley,New York, 169.

2. Fish, W.R., Marr, J.J., Berens, R.L., Looker, D.L., Nelson, J., LaFon, S.W.and Balber, A.E. 1985. Antimicrob Agents Chemother. 27, 33.

3. Hamill, R.L. and Hoehn, M.M. 1973. J. Antibiotics 26, 463.

4. Trager, W., Tershakovec, M., Chiang, P.K. and Cantoni, G.L. 1980.Expl. Parasitol. 40, 83.

5. Nadler, J., Lederer, E., and Tanowitz, H.B. in press. J. Parasitol.

6. Bachrach, U., Schnur, L.F., El-on, J., Greenblatt, C.L., Pearlman, E.,

Robert-Gero, M., and Lederer, E. 1980. FEBS Leters 121, 287-291.

7. Wist, E. and Prydz, H. 1979. Nucleic Acid Res. 1583.

3. Solari, A., Tharaud, D., Yolanda, R., Aldunate, J., Murello, A., and Litvak, S.

1983. Biochem Internat. 7(2) 147.

9. Roeder, R.G. 1976. RNA Polymerases, Cold Spring Harbor Laboratory,

Cold Spring Harbor, N.Y.

10. Roeder, R.G. 1974. J. Biol. Chem. 249, 241.

ii. Jendrisak, J.J. and Burgess, R.R. 1975. Biochem. 14 (21), 4639.

12. Sklar, V.E.F., Jaehning, J.A., Gage, P., and Roeder, R.G. 1976.J. Biol. Chem. 251(12), 3794.

13. Smith, S.S., Braun, R. 1978. Eur. J. Biochem. 82, 309.

14. Lowe, P.A., Hager, D.A., Burges, R.R. 1979. Biochem. 18, 1344.

15. Davis, B. 1964. Ann. N.Y. Acad. Sci. 121, 404.

16. Roeder, R.G. and Rutter, W.J. 1969. Nature 224, 234.

17. Nolan, L.L., Berman, J.D., Gir, L. 1984. Biochem. Internat. 9, 207.

50

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