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T ry o n , V icto r V.

PURINE SALVAGE METABOLISM IN MOLLICUTES

The Ohio State University Ph.D. 1984

University Microfilms Internstionel300 N. Zeeb Road, Ann Arbor. Ml48106

Copyright 1984 by Tryon, Victor V.

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University Microfilms International

PURINE SALVAGE METABOLISM IN MOLLICUTES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio S tate University

By Victor V. Tryon, 8.S.

*****

The Ohio State University 1984

Reading Committee: Approved By: J . Dennis Pollack, Ph.D. Frank A. Kapral, Ph.D. Abramo C. Ottolenghi, Ph.D. Adviser Norman L. Somerson, Ph.D. ïépartment of Medical Microbiology Marshall V. Williams, Ph.D. and Immunology Copyright by Victor V. Tryon

1984 To Nancy

n I wish to thank Dr. J. D. Pollack for his considerable efforts on my behalf. Dr. M. V. Williams was of great assistance with the enzyme work. I should also like to thank Drs. F. A. Kapral, A. 0. Ottolenghi and N. L. Somerson for their advice and encourage­ ment. R. Montione provided special help with the graphic photography.

m VITA

November 14, 1953 ...... Born - Oakland, C alifo rn ia

1981 ...... B.S., University of Washington Seattle, Washington

1981-1982 ...... Graduate Student, Department of Medical M icrobiology and Immunology, The Ohio State University, Columbus, Ohio

1982-1984 ...... Research Associate, Department of Medical M icrobiology and Iiranunology, The Ohio State University, Columbus, Ohio

PUBLICATIONS

P ollack, J .D ., V.V. Tryon, and K.D. Beaman. 1983. The m etabolic pathways of Acholeplasma and Mycoplasma: An overview. Yale 0. Med. 56:709-7T6.

Tryon, V.V., and J.D. Pollack. 1984 Purine metabolism in Achole­ plasma laidlawii B-PG9: A novel pyrophosphate dependent nucleoside kinase activity. J. Bacteriol. 159:265-270.

ABSTRACTS AND PRESENTATIONS

P ollack, J .D ., Beaman, K.D., Tryon, V.V., and Robertson, J . New emerging patterns of NADH oxidase localization and lipid synthesis in Mollicutes. Int. Org. Mycoplasmology, Tokyo, Japan, September, 1982.

Tryon, V.V., and J.D. Pollack. Purine salvage is the sole route for the synthesis of cellular adenylates in Acholeplasma laidlawii B-PG9. American S ociety fo r M icrobiology. New O rleans, LA, March 6, 1983.

Tryon, V.V., and J.D. Pollack. Purine salvage and interconversion in Acholeplasma laidlawii B-PG9. American Society for Micro- biology. St. Louis, MO, March 9, 1984.

Pollack, J.D., V.V. Tryon, and M.V. Williams. Pyrophosphate metabolism in A. la id la w ii B-PG9. F ifth In te rn a tio n a l Congress of the I n te r ­ national Organization of Mycoplasmology. Jerusalem, Israel, June, 1984. iv VITA, co n tin u ed

FIELD OF STUDY Major Field: Medical Microbiology and Immunology Studies in Microbial Physiology and Enzymology: Professor J. Dennis Pollack Studies in Mycoplasmology: Professor J. Dennis Pollack TABLE OF CONTENTS

Page

DEDICATION ...... ü ACKNOWLEDGEMENTS ...... i i i VITA ...... iv LIST OF TABLES...... vii LIST OF FIGURES ...... v iii LIST OF PLATES...... ix INTRODUCTION ...... 1 MATERIALS AND METHODS ...... 8 RESULTS...... 19 DISCUSSION...... 32 CONCLUSIONS...... 45 LITERATURE CITED...... 47

VI LIST OF TABLES

Table Page 1 Organisms used in th is study ...... 9 2 Summary of purine enzymes assayed ...... 16,17 3 Purine salvage enzymes in Acholeplasma laidlawii B-PG9 ...... 22 4 Purine salvage enzymes in Acholeplasma axanthum S-743 ...... ■■...... 23

5 Purine salvage enyzmes in Acholeplasma granularum BTS-39 ...... 24 6 Purine salvage enzymes in Mycoplasme gallisepticum S 6 ...... 225

7 Purine salvage enzymes in Spiroplasma floricola 236. 26 8 Purine salvage enzymes in Mycoplasma arqinini G-230. 27 9 Purine salvage enzymes in Acholeplasma florum LI . . 28

10 Nucleoside kinase activities in A. laidlawii B-PG9, A. axanthum S-743, Acholeplasma granularum BTS-39 and coli (ATCC 25922) ...... 29

11 Summary of purine salvage enzyme activities .... 30

12 Known reactions in which PPi replaces ATP as a phosphate donor ...... 40

vn LIST OF FIGURES

Figure Figure Legends Page 1 Effect of various concentrations of sodium pyrophosphate or ATP on the amount of AMP synthesized in the standard reaction mixture for adenosine kinase activity by dialyzed lysates of Acholeplasma laidlaw ii B-P69 ...... 31 2 Proposed salvage pathways for the biosynthe­ sis of purine nucleotides in Acholeplasma laidlaw ii B-PG9 ...... 33 3 Recovery of labeled AMP, adenosine and adenine during the assay of adenine phosphoribosyl- transferase activity in Acholeplasma laidlawii B-PG9 with respect to time ...... 36 4 Proposed salvage pathways for the biosynthesis of purine nucleotides in Acholeplasma florum LI and Spiroplasma floricola ...... 43 5 Proposed salvage pathways for the biosynthesis of purine nucleotides in Mycoplasma gal1isepticum and Mycoplasma arqinini ...... 44

v m LIST OF PLATES

PI ate Page A Autoradiograph of the separation of purine bases, nucleosides and mononucleotides in IM LiCl on PEI-cellulose p lates ...... 13 B Autoradiograph of the separation of the purine mononucleotides in 4N formic acid on PEI-cellulose plates ...... 14

IX PURINE SALVAGE METABOLISM IN MOLLICUTES

By Victor V. Tryon, Ph.D.

The Ohio State University, 1984

Professor J. Dennis Pollack, Adviser

Fifteen cytoplasmic enzymes with activity for purine salvage and interconversion were examined in Acholeplasma laidlawii B-PG9, Achole- plasma axanthum S-743, Acholeplasma granularum BTS39, Spiroplasma floricola 23-6, and Mycoplasma gallisepticum S6. Phosphoribosyltrans­ ferase activity for adenine, guanine, and hypoxanthine were found in all organisms tested. Phosphoribosyltransferase activity for xanthine was detected only in ^ laidlawii B. Nucleoside phosphorylase activity for xanthine was detected in both A. laidlawii and A. granu­ larum. No ATP-dependent nucleoside kinase activity was detected in any organism. Novel pyrophosphate-dependent nucleoside kinase activity fo r adenosine, 1nosine, and guanosine was found in A. laid law ii B and for adenosine and guanosine in A. axanthum and for adenosine only in A. granularum. Cytoplasmic 5'-nucleotidase activity towards the four purine mononucleotides AMP, GMP, IMP and XMP was detected in A. laidlawii B. Nucleotidase activity for AMP, GMP and IMP was detected in A. axanthum and A. granularum. No cytoplasmic 5 '-nucleotidase a c tiv ity was detected in S. f lo r ic o la , in A. florum , or any Mycoplasma

1 2 spp. Adenylosuccinate synthetase-lyase activity was detected in all species examined except S. floricola and A. florum. GMP reductase activity was detected in all organisms tested except M. gallisepticum and M. arqinini. GMP synthetase a ctiv ity was measured only in S. floricola. IMP dehydrogenase, AMP deaminase, adenosine deaminase, and adenine deaminase a c tiv itie s were not detected in any species. This is the first report of pyrophosphate-dependent nucleoside kinase activity in any organism. All species examined rely on guanine phosphoribosyltransferase or the novel pyrophosphate-dependent nucleoside kinase activity for the production of guanylates. In no organism tested can guanine monophosphate be derived from the other purine mononucleotides. This suggests that the guanine phosphoribo­ syl transferase may be a promising site for the biochemical inhibition of mycoplasma infection and pathology. INTRODUCTION

Organisms in the Class Molli cutes are of biological interest be­ cause of their physiologic uniqueness among procaryotes. These organ­ isms lack a cell wall and cell wall precursors, and some require chole­ sterol for growth. Members of this class are also of interest because of their often vexacious association with animal cells in culture

(2,4,51,73,91), and their occasional pathogenic relationship with their human, lower mammal, bird, insect and plant hosts (21-23,39,98,103,104).

The Class Mollicutes consists of one order, the Mycoplasmatales, which is taxonoraically divided into three families (29), the Achole- plasmataceae, Mycoplasmataceae, and the motile, helical Spiroplasma- taceae. Many Mollicutes are trivially referred to, individually and collectively, as "mycoplasmas." The Acholeplasmataceae consists of one genus, the Acholeplasma, with seven recognized species (29,97). The acholeplasmas were first isolated from sewage by Laidlaw in 1936 (41).

Since then, acholeplasmas have been isolated from soil, birds, mammals, bovine sera, plants (93), and vegetables (90). The Mycoplasmataceae consists of two genera. Mycoplasma and Ureaplasma (29). There are more than 70 recognized species of Mycoplasma, but only 1 species with multi­ ple serovars of the urea-hydrolyzing Ureaplasma. The Spiroplasmataceae has one genus with three recognized species (22). Members of this family are commensals and pathogens of arthropods, plants, and perhaps vertebrates (22-24,98,103,104).

1 2

Some species in the Mollicutes are the smallest known procaryotes

capable of independent replication outside of a host cell (29,89).

Organisms in this class are characterized by their small size (0.25

to 1.0 pm in diameter) and relatively small genome (0.5 x 10^ to 1.0 x

109 dalto n s) with a low guanosine plus cytosine content (23-35 mol%).

Physiologically unique among procaryotes, two of the three families

within the class, Mycoplasmataceae and Spiroplasmataceae, require the

addition of sterols, notably cholesterol, to the culture medium for

growth (29,78,79). These organisms also apparently lack the qui nones

at the necessary concentrations and cytochromes necessary for complete

oxidative respiration (69,71). The discovery that NADH oxidase in A.

. laidlawii is a flavin-associated protein (77) and that M. gallisepticum

m aintains an electrochem ical gradient across i t s membrane (7,8,80)

supports the suggestion by Pollack et (69,71) and Reinards et al.

(77) th a t some members o f th e M ollicutes may have flav in -term in ated

respiration. That is, although still unproven, these species may pro­

duce ATP at a flavin locus similar to the site one locus of mitochondria.

The Mollicutes may often be encountered as troublesome contaminants

of cell cultures. These contaminants may be difficult to detect and

are difficult to eradicate. Such contamination may have profound ef­

fects on the host cell properties and subsequently the conclusions

from research based on those altered cell properties. Contamination

of cu ltu red c e lls by members of th e M ollicutes may r e s u lt in changes

in the metabolic parameters of the host, such as the altered incorpor­

ation of nucleic acid precursors (2,33,51,66,81,91,100); induction of

interferon production (6,9); promotion of natural killer cell activity 3

(20); changes in cellular morphology that may appear as virus-induced cytopathy (2,91); induction of macrophage-mediated cytolysis of neo­ plastic cells (45); the selective stripping of alloantigens from

T-cell subpopulations (106); reduction of the yield of herpes and other v iru ses from cu ltu red mammalian c e ll lin e s (49,86). This subject has been reviewed by Stanbridge (91) and more recently by McGarrity

(51-52).

Approximately half of the more than 60 named Mycoplasma species have been associated with disease states (21,89). Three Mycoplasma spp. have been demonstrated to be pathogens in humans; Mycoplasma pneu­ moniae, Mycoplasma hominis, and Ureaplasma urealyticum (21).

As is tru e o f th e pathogens o f b ird s and lower mammals, th ese organ­ isms are e x tra c e llu la r p a ra s ite s . A number o f spiroplasm as and mycoplasma-like organisms infect plants and arthropods (22,23,103,104).

Changes in purine nucleotide metabolism may be important in the parasitic or pathogenic relationship between members of the Mollicutes and their host cell. Hakala et al., (33) reported the inability of a

HeLa cell line to grow when contaminated with Mycoplasma (Acholeplasma) laidlawii when the de novo synthesis of purines in the human cells was inhibited with the folic acid antagonist amethopterin (methotrexate).

This suggests that the presence of the mycoplasmas prevented or inter­ rupted the normal salvage pathways of purine synthesis of the host.

Van Diqgelen et al. (102) and McGarrity (51) demonstrated that

Mycoplasma spp. contam inants may outconç)ete host manmalian c e lls fo r the hypoxanthine in the restrictive hypoxanthine-aminopterin-thymidine

(HAT) medium used for the selection of hybridomas. 4

Tsuchiya and Sugai (96) found samples of erythrocytes from nine patients with M. pneumoniae infections had 47% less ATP in the supernatant from lysed cells than did five healthy controls. Gabridge and Stahl (30) found that the purine base adenine added to tracheal organ culture media protected the hamster tracheal explant cells from observable cytopathic and ciliostatic effects when infected with the usually cytopathic Mycoplasma pneumoniae. The addition of adenine had no effect on the growth rate or cell yield of the myco­ plasmes. The radiolabel was recovered from mycoplasmes removed from the tracheal cells. Gabridge and Polisky (31) determined that hamster tracheal cells infected with a pathogenic strain of M. pneumoniae had significantly lower ATP levels than uninfected cells, or tracheal cells infected with a non-pathogenic strain. More recently, Upchurch and Gabridge (99,100) suggested that the observed interference with host de novo purine synthesis in human lung fibroblasts infected with M. pneumoniae plays a critical role in the induction of cytopathic effects.

In defined media Mycoplasma, Acholeplasma, and Spiroplasma spp. have an absolute requirement for purine precursors (15,32,44,57,76,78,87,95).

This n u tritio n a l requirem ent suggests th a t members of th e c la ss M olli­ cutes are competent in the uptake and salvage of purine bases, ribo- nucleosides or ribonucleotides, but do not have or utilize a functional pathway for the de novo synthesis of purines. The suggestion that these organisms lack a functional de novo pathway is based on these nutritional reports. There has been no published report on the examination of the Mollicutes for the enzymatic activities required for the de novo synthesis of purines. 5

The only member of the class Mollicutes for which the pathwavs of purine salvage and interconversion have been comprehensively studied and described is Mycoplasma mycoides subsp. mycoides (57,58,85). Mitchell and Finch (57) found th at neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleo­ tides, although guanine could act as precursor for all purine nucleotides, including AMP, incorporated into RNA. Later, Mitchell and coworkers (57,58,85) found in cell-free extracts of M. mycoides subsp. mycoides the phosphoribosyltransferase activities necessary for the phosphoribosylation of the purine bases adenine, guanine and hypoxanthine to their corresponding ribonucleotides, AMP, GMP and IMP. These authors also demonstrated the enzyme activities necessary for the interconversion of GMP to AMP through GMP reductase, adenylo­ succinate synthetase and adenylosuccinate lyase. This provided the enzymatic basis for the earlier finding of Rodwell in 1857 (78) that the entire purine requirement of this organism could be met by the addition of guanine alone to the culture medium. Recently, Neale et al. (62,63) have suggested that M. mycoides takes up and incorporates intact purine and pyrimidine deoxyribonucleoside 5'-monophosphates into ONA. Prior to the work of Mitchell and colleagues, the only purine enzyme activity known in any species of Mollicutes was adenosine phosphorylase (35). Shortly after the publication of the work of Mitchell et ^ . , Mclvor and Kenny (53) demonstrated the uptake of tritium -labeled adenine and guanine by Acholeplasma laidlaw ii A, Mycoplasma putrefaciens. Mycoplasma gallisepticum . Mycoplasma 6 pneumoniae^ Mycoplasma hyorhinis. Mycoplasma arq in in i. Mycoplasma hominis, and a bovine Mycoplasma sp. grown in a conplex medium, harvested by centrifugation, and resuspended in Eagle minimum essen­ tial medium with radiolabeled substrates. All species tested, except M. hominis and M. arqinini, also incorporated the labeled nucleosides adenosine and guanosine into RNA. Hamet et al. (34) examined the purine salvage activities but not interconversion enzyme activities in A. laidlawii A-PG8 and eight Mycoplasma spp. commonly found in cell culture contaminants. These organisms were grown in a complex broth medium containing 20% horse serum. Adenine, guanine and hypoxanthine phosphoribosyltrans- ferase activity was found in all species. Adenosine and inosine phosphorylase was found in all species tested Including M. hominis and M. arginini. No adenosine deaminase or adenosine kinase activity was detected. Beaman and Pollack (3,4) reported that during mid-log phase of growth, Acholeplasma laidlawii B-PG9 maintains an adenylate energy charge (EC/\) comparable to that of Escherichia coli and other procary­ otes, including Spiroplasma citri (82), and synthesizes more ATP per milligram (dry weight) than coli. In contrast, these authors (4) found th at other growing and dividing M ollicutes, representative of the recognized metabolic groups within the Mollicutes, maintained EC/\ levels significantly lower, approximately 20% lower, than that of other rapidly metabolizing procaryotes including A. laidlawii B. They speculated that these lowered EC^ values, which are more 7 reflective of senescent cells rather than actively metabolizing cells

(11,16), may be due to some undetermined metabolic deficiency asso­

ciated with the nucleic acid metabolism of these Mollicutes species.

In order to investigate the role purine metabolism of the Molli­

cutes species may have in the vexacious association with host cells

and the state of relative energy deficiency, the pathways by which the

purine mononucleotides are synthesized in A. laidlawii B-P69, Achole­

plasma axanthum, Acholeplasma granularum. Mycoplasma gallisepticum

and Spiroplasma floricola were determined by examining 15 cytoplasmic

enzyme a c tiv itie s involved in purine salvage and interconversion. MATERIALS AND METHODS

Chemicals. The following radiolabeled compounds were purchased from

Research Products International (Mt. Prospect, IL): [ 8 -^^C] adenine

([8-14c]ADE), 50 mCi/ mmol, [8-T4c]-adenosine ([8-14c]AD0), 47 mCi/mmol,

[8-T4c]guanosine ([8-T4c]GU0), 42.8 mCi/mmol and [U-^4q] guanosine

5 '-monophosphate [U-T4c]GMP, 450 mCi/mmol. The follow ing were purchased from ICN (Irv in e , CA): [8-14c]AMP a t 58 mCi/mmol, [8-14c]ATP a t 51 mCi/ mmol, and [8-T4c]guanine ([8-T4c]GUA) a t 51 raCi/mmol, and [U-T4c]glycine at 92 mCi/mmol. The follow ing were purchased from Moravek Biochemicals

(Brea, CA): [2,8-3h]AD0 at 23 Ci/mmol, [8-14]hypoxanthine (8[14c]HX) a t 56 raCi/mmol, [8-T 4c]inosine ([8-T4]iN0) a t 56 mCi/mmol, [8-T4c]xantho- sin e ([8-14q]xo) a t 56 mCi/mmol, [8-T4c]xanthine ([8-14c]X) a t 57 mCi/mmol, and [8-T4c]xanthosine 5 ' monophosphate ([8-T4c]XMP) a t 56 mCi/mmol.

[8-T4c]inosine 5-monophosphate ([8-T4c]lMP) at 59 mCi/mmol was purchased from Amersham International (Arlington Heights, IL). Lecithin (vegetable) was purchased from Mann Research Labs (New York, NY). Other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.

Organisms and culture conditions. The organisms used in this study are listed in Table 1. Acholeplasma laidlawii B-PG9 was grown with­ out serum in our modification of Edward medium containing penicillin G

(100 units ml-1), as described previously (3). Acholeplasma axanthum

S-743 and Acholeplasma qranularum BTS39 were grown as described for

A. laidlawii but with the addition of 25» (vol/vol) horse serum (K.C.

Biologicals, Lenexa, KA; lot number 200031). Spiroplasma floricola 8 TABLE 1 Organisms used in these studies Organism Origin ______Acholepiasma laidlaw ii B-PG9 Laboratory stock Acholeplasma axanthum S-743 J.G. Tully, National Institute of Allergy and Infectious Diseases Acholeplasma qranularum BTS39 J.G. Tully, National Institute of Allergy and Infectious Diseases Acholeplasma florum LI N.L. Somerson, Department of Medical Microbiology and Immunology, The Ohio State University Spiroplasma flo rico la 23-6 J.G. Tully, National Institute of Allergy and Infectious Diseases

Mycoplasma gallisepticum S 6 N.L. Somerson, Department of Medical Microbiology and Immunology, The Ohio State University Mycoplasma arginini G-230 N.L. Somerson, Department of Medical Microbiology and Immunology, The Ohio State University Escherichia coli ATCC 25922 Departmental culture collection Bacillus subtil is 60015 Ernst Freese, National Institute of Neurological and Communicable Disorders and Stroke, Bethesda, MD 10

23-6 and Mycoplasma gallisep ticu m SB were grown in th e same manner but with the addition of 55» (vol/vol) horse serum. The horse serum used in

all studies was heat re-inactivated at 56*C for 60 min. Bacillus

subtil is strain 60015, which lacks purine nucleoside kinase activity

(26), was obtained from Ernst Freese (National Institute of Neurological

and Communicative D isorders and Stroke, Bethesda, MD) and grown in a

defined medium as described by Endo et al. (26). Escherichia coli ATCC

25922, which has purine nucleoside kinase a c tiv ity , was grown in Edward medium without penicillin or serum.

All cultures were incubated at 37'C. Starter cultures were inocu­

lated into temperature-equilibrated media to 1-55» (vol/vol) and incubated

statically. Cells were harvested in their mid-log phase of growth at

6-48 h.

To d etect de novo sy n th esis of purine, A. la id la w ii was grown in

tryptose broth containing, per liter, tryptose (Difco) 25 g; NaCl 5 g;

and Tris 5 g (pH 7.5). After autoclaving, we added sterile glucose

solution to 15S (vol/vol) and a liposome suspension to 0.5% (vol/vol).

The liposome suspension was composed of phosphatidylcholine-cholesterol

(1:1) and was prepared as described by Cluss e t ^ . (19). The liposome

suspension was added because it stimulated growth and increased the cell

yield. The growth stimulating effect of lipids added to the culture

media of Acholeplasma has been reported previously (54,93,97). The con­

centration of adenylates in the tryptose medium was 0.3 pg m l'l, about

one-half th a t in m odified Edward medium w ithout serum.

Preparation of cell-free extracts. Cell-free preparations were made

essentially as previously described (70). Acholeplasma, mycoplasma and 11 spiroplasma cells were harvested by centrifugation at 9,000 x g at 4“C for 30 min. The cells were washed by centrifugation three times in

200 to 300 volumes of cold kappa-buffer. Washed cells were lysed by hypotonic shock in aqueous diluted (1:20) 37*C kappa-buffer by incuba­ tion at 37*C for 3-10 min. The crude lysate was centrifuged at 48,000

X g for one hour at 4“C. The supernatant was centrifuged at 160,000 to

200,000 X g for one hour at 4"C. The supernatant was dialyzed in the cold overnight against four changes of 1 0 0 volumes each of 1 0 mM jT-2 - hydroxyethyl piperazine-N/-2-ethanesulfonic acid (HEPES; Research

Organics, Cleveland, OH) (pH 7.5), 2 mM 2-mercaptoethanol, 1 mM MgCl 2 , and 100 pM phenylmethylsulfonylfluoride (PMSF). This dialyzed cell-free e x tra c t was used immediately fo r a ll enzyme assays. c o li and

subtil is cells were harvested as for A. laidlawii. Cell extract of coli or subtil is was prepared by incubation at 37*C in 1:20 kappa-buffer with lysozyme (100 pg ml"!) for 30 min as described by

Endo e t (26). Lysozyme tre a te d c e lls were sonicated (S o n ifier

Cell Disruptor, Heat Systems, Melville, NY) by three 30 sec exposures while on wet-ice. Whole and broken cells were centrifuged at 15,000

X g for 30 min at 4*C. The cell-free extract was dialyzed as above.

The dialyzed c e ll e x tra c t was used immediately fo r a ll enzyme assays.

Protein was determined by the method of Bradford (12) using the G-250

dye-reagent form ulated by BioRad L aboratories (Richmond, CA).

Enzyme assays. For all assays, reaction mixtures were incubated

a t 37"C in a to ta l volume of 0.1 ml. Each reactio n m ixture contained

15-25 pmols of radiolabeled substrate. Concentrations of radioactive

substrate were adjusted so that greater than 50% of the label 12 remained at the end of the incubation period. Reactions were started by the addition of temperature equilibrated dialysed cell extracts containing 10-40 pg of protein and incubated with shaking. Reactions were terminated by heating at 100"C for two minutes or by inmiediate spotting onto chromatography matrix. After preliminary study of each assay, we chose an incubation time which gave the fastest rate of product formation. labeled substrate and product were separated by thin-layer chromatography on commercial polyethyleneimine (PEI) plates containing 0.55 meq polyethyleneimine (PEI) g"l cellulose (Analtech, Inc., Newark, DE). Plates were developed in either 1 M LiCl (solvent A) (Plate A) or 4 N formic acid (solvent B) (Plate B). Solvent A was used to separate purine 5 '-mononucleotides from nucleo­ sides and bases. Solvent B was used to separate individual purine 5'-mononucleotides from each other. Ten pi of the reaction mixture were spotted in each lane with appropriate nonradioactive markers. Resolved purines were visualized by UV lig h t, scraped into 7 ml of BudgetSolve (Research Products International, Corp., Mt. Prospect, IL) and counted in a LSC 7000 liquid scintillation counter (Beckman Instruments, Fullerton, CA). The Rf for compounds separated in solvent A were ADE, .41; ADO,

.53; AMP, .96; 6 UA, .46, GUO, . 6 8 ; GMP, .96; HX, .61; INO, .80; IMP, .95; X, .50; XO, .65 and XMP, .96. Rf for compounds separated in solvent B were XMP, .17; IMP, .24; GMP, .43 and AMP, .90. No sig n ifi­ cant chromatographic trailing occurred in any instance. Samples were counted to 2% counting error and corrected for quenching. All radioisotope data were calculated as disintegrations per minute, and 1 3

PLATE A

%8 k 1 4

PLATE B

"f

- tÇ.'T»*- *>

m a . wr-: pz%:yz 15 converted to moles of product synthesized min'l mg"^ protein by cal­ culation using the specific activity of the radioactive substrate. The purine salvage enzyme activities assayed in this study are summarized in Table 9. The following enzyme assays were carried out (Table 2).

(i) Adenine phosphoribosyltransferase (APRT) [AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7] and hypoxanthine-guanine phos­ phor i bosyl transferase (HGPRT) [IMP:pyrophosphate phosphoribosyltrans­ ferase, EC 2.4.2.8 ]. Reaction mixtures contained 50 mM HEPES (pH

7.4), 5 mM MgCl 2 , 4 mM phosphoribosylpyrophosphate (PRPP), and

[8-14c]ADE for the APRT or [S-^^cjGUA or [ 8 -l^C]HX or [8-14c]X for the HGPRT. Incubation time was eight min. Product mononucleotide was chroraatographically separated from substrate purine base in solvent A. ( ii) Adenosine kinase [ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20] and nucleoside kinase [ATP:inosine 5'-monophosphotrans­ ferase, EC 2.7.1.73] were assayed according to Yamada et al. (109).

Reaction mixtures contained 50 mM HEPES (pH 7.4), 1 mM MgCl 2 , 1-3 mM ATP, an ATP regenerating system consisting of 2 mM phosphoenol- pyruvate and 0.5 units of pyruvate kinase, and [8-l^C]AD0 for the adenosine kinase and [8-^^C]GU0 or [8-l^C]IN0 or [8-^^C]X0 for the nucleoside kinase. Incubation time was six to eight min. Product nucleotide was chromatograph!cally separated from substrate nucleoside in solvent A. Adenosine kinase and nucleoside kinase u tilizin g pyrophosphate were assayed as for the ATP-dependent kinases except that 2-4 mM sodium pyrophosphate (Fischer Certified A.C.S., Fischer Scientific Co., Fairborn, NO) was substituted for ATP and no ATP regenerating system was used. In some experiments, we tested TABLE 2 Summary of Purine Enzymes Assayed

Number® Trivial Name ______Activity^______

1 Adenine phosphoribosyltransferase ADE + PRPPC 4. AMP + PP1 transferase (APRT)

2 Hypoxanthine-guan1ne phosphor1bosyl- transferase (HGPRT) HX + PRPP IMP + PP1 3 5'-nucleot1dase 5'-ribonucleotide + H?0 ribonucleoslde + Pi

4 PP1-dependent adenosine kinase PP1 + adenosine AMP + PI 5 PPI-dependent nucleoside kinase PP1 + ribonucleoslde -f- ribonucleotide + Pi

6 Purine nucleoside phosphorylase Purine nucleoside + PI purine + a-O-ribose- 1-phosphate

7 Purine nucleoside phosphorylase Purine + a-D-r1bose-l-phosphate -► purine nucleoside + PI

8 Adenosine deaminase Adenosine + H 2 O Inoslne + NH 3

9 Adenine deaminase Adenine + H 2 O ->• hypoxanthi ne + NH3

10 Adenylosuccinate synthetase GTP + IMP + L-aspartate GDP + PI + adenylosuccinate n Adenylosuccinate lyase Adenylosuccinate fumarate + AMP 0» TABLE 2, continued

12 AMP deaminase AMP + H2 O -► IMP + NH3 13 IMP dehydrogenase IMP + NAO+ + HgO ^ XMP + NAOH

14 GMP synthetase ATP + XMP + NH3 -» AMP + PPi + GMP

15 GMP reductase NADPH + GMP NADP+ + IMP + NH3 16 Phosphori bosyl-glyc i nami de synthetase ATP + 5-phosphor i bo sylami ne + glycine -> ADP + Pi + 5'-phospho- r 1bosyl-g 1yc i n ami de

3 Refers to enzyme number in Figure 2. b Reaction written in direction assayed, c PRPP = 5-phospho-ot-D-ribose-l-diphosphate. 16 for adenosine kinase activity using ATP or pyrophosphate over the range 0.1 to 4 mM. In preliminary experiments to test for the effect of contam inating membrane ATPase a c tiv ity , we used th ese same reactio n conditions substituting [ 8 -^^C]ATP fo r th e ra d io a c tiv e adenosine fo r up to 30 min incubation. In these experiments, we used 2 N formic acidzO.5 M LiCl (1:1) to resolve ATP, ADP and AMP on PEI plates.

(iii) Purine nucleoside phosphorylase [purine nucleosideiortho- phosphate ribosyltransferase, EC 2.4.2.1]. Reaction mixtures for the base to nucleoside conversion contained 50 mM sodium phosphate or HEPES b u ffer (pH 7 .4 ) , 2 mM MgCl 2 , 4 mM ribose-1-phosphate and e ith e r [8 -l^C]ADE or [8-14c]GUA or [8-14c]HX or [8-14c]X. For the nucleoside to base conversion, the reaction mixtures were the same, except [8-14c]AD0or [8-14c]GU0or [8-14c]IN0o r [8-14c]X 0replaced their respective base. Incubation time was four min. Nucleosides were chromatographically separated from bases in solvent A.

(iv) 5'-Nucleotidase [5'-ribonucleotide phosphohydrolase,

EC 3.1.3.5]. Reaction mixtures contained 50 mM HEPES (pH 7.4), 2 mM

MgClg and either [ 8 -l*C]AMP or [ 8 -l*C]IMP or [ 8 -"*^C]GMP or [ 8 -l*C]XMP.

Incubation time was eight min. Product nucleosides and their bases were chromatographically separated from substrate nucleotides in solvent A. The purine bases were secondarily formed by the action of the phosphorylase.

(v) Adenylosuccinate synthetase [IMP:l-aspartate ligase, GDP forming, EC 6 .3.4.4] and adenylosuccinate lyase [adenylosuccinate

AMP-lyase, EC 4.3.2.2] were assayed by measuring the rate of production of AMP from IMP via adenylosuccinate in a two-step reaction sequence 17 as modified from the procedure of Lieberman (43) and Fischer et al.

(27). Reaction mixtures contained 50 mM HEPES (pH 7.4), 2 mM MgCl 2 ,

4 mM GTP, 1 mM a sp a rta te and [ 8 -^^C]IMP. Incubation time was 30-60 min. Product AMP was chromatographically separated from substrate

IMP in solvent B.

(v1) GMP reductase [NADPH:GMP oxidoreductase; deaminating,

EC 1 .6 . 6 . 8 ] was assayed by a modification of the techniques of Mager and Magasanik (48). Reaction mixtures contained 50 mM HEPES (pH

7.4), 2 mM cysteine-HCl, 0.4 mM NADPH and [ 8 -^^C]GMP. Incubation time was four to 60 min. Product IMP was chromatographically separated from substrate GMP in solvent B.

(vii) GMP synthetase [xanthosine 5 '-monophosphate:amonia ligase,

AMP form ing, EC 6 .3.4.1] was assayed by a modification of the proce­ dure of Sakamoto (83). Reaction mixtures contained 50 mM HEPES (pH

7 . 4 ) , 4mM ATP, 2 mM glutamine or ammonium s u lfa te , 2 mM MgCl 2 and

[8-T4c]XMP. Incubation time was five to 60 min. Product GMP was chromatographically separated from substrate XMP in solvent B.

(v iii) IMP dehydrogenase [IMP:NAD"'' oxidoreductase, EC 1.2.1.14] was assayed by a modification of the technique of Magasanik et al.

(47). Reaction mixtures contained 50 mM HEPES (pH 7 .4 ), 2 mM

cysteine-HCl or 2 mM glutathione, (Calbiochem, San Diego, CA), 2 mM

NAD, 0.1 mM KCl and [8-T4c]IMP. Incubation time was 60 min. We

also assayed the hypotonic lysate of washed human erythrocytes

following the procedure of Henderson et (36). Product XMP was

chromatographically separated from substrate IMP in solvent B. 18 (ix) Adenylate deaminases. AMP deaminase [AMP aminohydrolase, EC 3.5.4.5] and adenosine deaminase [adenosine aminohydrolase, EC 3.5.4.4] were assayed by a modification of the technique of Bagnara and Hershfield (1). Adenine deaminase [adenine aminohydrolase, EC 3.5.4.2] was assayed according to a modification of the method of Canale-Parol a and Kidder (13). All reactions were as described for the

5'-nucleotidase, except that [8-^^C]AD0 and [ 8 -^^C]ADE were substituted for the purine 5*-nucleotide. Product IMP was chromatographically

separated from substrate AMP in solvent 8 . Products INO and HX were similarly separated from ADO and ADE in solvent A. (x) De novo purine synthesis in growing cells was assayed by measuring the incorporation of [U-T^C]glycine radioactivity added to the tryptose growth medium (0.5 pCi ml"^) into cellular acid- precipitable purine bases as described by Martin and Owen (50). RESULTS

Utilization of purine bases and nucleosides. The activities of the purine salvage enzymes are reported as the rate of product forma­ tion (in nmols per min"^ mg"^ protein) (Tables 3-10). The minimum detectable amount (shown in the Tables) is three times the background ra te determined from control re a c tio n s. Purine salvage enzyme a c tiv i­ ties are reported for A. laidlawii in Table 3; for A. axanthum in

Table 4; for A. qranularum in Table 5; for M. gallisepticum in Table 6 ; for S. floricola in Table 7; for M. arginini G-230 in Table 8 , and fo r

A. florum in Table 9. The enzyme a c tiv itie s detected and not detected

in all species tested are summarized in Table 11. All organisms tested are capable of converting the purine bases ADE, HX and QUA to their corresponding ribonucleosides via the phosphorylase and to the corres­ ponding ribonucleotides via the phosphoribosyltransferases. The purine ribonucleosides ADO, INO and GUO are converted to the corresponding

bases via nucleoside phosphorylase activity. Only A. laidlawii was

found to have the phosphoribosyltransferase activity necessary to

convert X to XMP (Table 3). A. laidlawii and A. qranularum converted

X to XO in the presence of ribose-1-phosphate (Tables 3,5 and 11).

No ATP dependent adenosine or nucleoside kinase activity was

detected in any organism. To determine whether the lack of ATP-

dependent adenosine and nucleoside kinase activity was due to conta­

m inating membrane ATPase a c tiv ity , dialyzed c e ll e x tra c t was examined

for ATPase activity. Under conditions as described for the assay of

19 20

ATP-dependent adenosine kinase a c tiv ity , 90-91% o f [ 8 -^^C]ATP was still present in the reaction mixture after six minutes of incubation and about 85% of the radiolabeled ATP remained after 30 min. This indicates that our inability to detect ATP-dependent adenosine or nucleoside kinase activities was not due to the absence of ATP in th e reactio n m ixtures as th e re s u lt o f contam inating membrane

ATPase a c tiv ity .

However, in laid la w ii B-PG9 ADO, INO, and GUO, but not XO, were converted to the ribonucleotides via a pyrophosphate (PPi) dependent adenosine or nucleoside kinase activity (Table 10).

PPi-dependent nucleoside kinase activity was detected in A. axanthum for ADO and GUO but not for INO and XO (Table 10). In A. granularum

PPi-dependent nucleoside kinase activity was detected for ADO only

(Table 10). We do not know if one or more enzymes are responsible for these activities. The rate of formation of AMP as a result of adenosine kinase activity in crude preparations of laidlawii B-PG9 was found to be dependent on the concentration of sodium pyrophosphate

(Fig. 1). In coli, as expected, ATP but not PPi was required for these purine nucleoside activities (Table 10). In subtil is, as expected, no ATP or PPi-dependent purine nucleoside kinase activity was detected (data not shown) (26).

Interconversion of nucleotides. IMP was converted to AMP in the coupled assay of adenylosuccinate synthetase and lyase activities in th e Acholeplasma and Mycoplasma sp p ., but not in S. f lo r ic o la (Table 7) or ^ florum (Table 9). In ^ laidlawii no AMP was detected when ATP or ITP were substituted for GTP, or when other amino donors such as 21 glutamine, arginine, glutamate or ammonium sulfate was substituted for aspartate. GMP reductase activity was detected in A. laidlawii when NADPH, but not NADH was used.

GMP synthetase activity was detected only in floricola. No activity was detected in any organism when sodium pyrophosphate was substituted for ATP, or when aspartate or ammonium sulfate was sub­

stituted for glutamine. GMP synthetase activity was detected in

coli (Table 3) and in subtil is and human erythrocytes

(data not shown).

No AMP or adenosine or adenine deaminase activities were detected

in any of the Mollicutes tested (Tables 3-9;ll). AMP deaminase activity was detected in B. subtil is (Table 3). ADO deaminase activity was

detected in E. c o l i .

No IMP dehydrogenase activity was detected in any of the Molli­

cutes tested (Tables 3-9;ll). Activity was detected in coli (Table

3} and in subtil is and human erythrocytes (data not shown).

In some experiments with AMP deaminase, IMP dehydrogenase, and

GMP synthetase, we added washed A. laidlawii B-PG9 membranes (70)

to the reaction mixtures (90-110 pg protein) without effect.

De novo synthesis of purines. During growth, laidlawii

B-PG9 did not incorporate exogenous [U-T4c]glycine radioactivity

into purine bases. 22 TABLE 3

Purine salvage enzvmes in A. laidlawii B-PG9.

Enz)me Activity Enzyme [nmol product rain"^ mg-1 prot] n® Substrate ± SO Phosphoribosyltransferases ADE 5.08 ± 1.45 7 GUA 2.76 ± 1.66 7 HX 3.69 ±2.11 4 X 0.08 ± 0 . 0 2 3 Nucleoside phosphorylases (Base to nucleoside) ADE 7.12 ± 3.54 4 GUA 13.2 ± 4.13 3 HX 7.56 ± 1.89 4 X 0.034 ± 0.007 3 Nucleoside phosphorylases (Nucleoside to base) ADO 1.97 ± 0.62 a GUO 1.30 ±0.27 5 INO 1.30 ± 0.49 5 XO 0.009 ± 0.003 3 5'-Nucleotidases AMP 12.8 ± 2.76 5 GMP 0.15 ± 0.02 5 IMP 1.82 ± 0 . 1 2 5 XMP 0.003 ± 0.001 3 Adenylosuccinate synthetase and lyase 0.524 ±0.184 7 GMP reductase 0.642 ± 0.301 3

GMP synthetase HPP (<0 . 0 0 1 )C 4 IMP dehydrogenase NA (<0.001)d 5 Adenylate deaminases AMP NA (<0.005)6 3 ADO NA (<0.001) 3 ADE NA (<0.001) 3

3 n = number of different batches of cells, b NA = no activity detected (minimum detectable amount), c GMP synthetase activity in E. coli 7.42 (0.04) d IMP dehydrogenase activity in E. coli 1.63 (0.60) e AMP deaminase activity in B. subtil is 0.48 (0.12) 23 TABLE 4

Purine salvage enz^es in Acholeplasma axanthum S-743.

Enzyme A ctivity Enzyme [nmol product min“ ‘ mg“ ‘ prot] Substrate (±SD)a

Phosphori bosyltransferases ADE 6.09 ± 1.31 GUA 9.57 ± 0.770 HX 6 . 6 6 ± 0.998 X NAb(< 0 .0 1 0 ) Nucleoside phosphorylases (Base to nucleoside) ADE 11.5 ± 0.044 GUA 1 0 .1 ± 1.73 HX 6.35± 0.473 X NA (<0.005) Nucleoside phosphorylases (Nucleoside to base) ADO 2.97 ± 0.070 GUA 1.27 ± 0.045 INO 1.11 ± 0.070 XO NA (<0.010) 5'-Nucleotidases AMP 0.810 ± 0.163 GMP 0.068 ± 0 .0 0 1 IMP 0.064 ± 0.015 XMP Adenylosuccinate synthetase and lyase 0 . 2 1 0 ± 0 . 0 1 0 GMP reductase 0.050 ± 0.003 GMP synthetase NA (<0.005) IMP dehydrogenase NA (<0.005) Adenylate deaminases AMP NA (<0.005) ADO NA (<0.001) ADE NA (<0.001)

3 n = three different batches of cells b NA = no activity detected (minimum detectable anount) 24 TABLE 5

Purine salvage enzymes in Acholeplasma granularum BTS-39

Enzyme A ctivity Enzyme [nmol product min -1 mg-' prot] Substrate (±SD)a

Phosphori bosyltransferases ADE 8.34 ± 1 .9 5 GUA 4.65 ± 0.512 HX 2.49 ± 0.438 X NAb(<0 . 0 1 0 )

Nucleoside phosphorylases - (Base to nucleoside) ADE 11.12 ± 1.36 GUA 17.04 ± 1.30 HX 6.07 ± 0.119 X 0 . 0 1 2 ± 0 . 0 0 2 Nucleoside phosphorylases (Nucleoside to base) ADO 0.445 ± 0.028 GUO 0.135 ± 0.009 INO 0.639 ± 0.155 XO NA (<0.010) 5'-Nucleotidases AMP 0.039 ± 0.002 GMP 0 . 0 1 0 ± 0 .0 0 1 IMP 0 . 0 2 2 ± 0 . 0 0 2 XMP NA (<0.005) Adenylosuccinate synthetase and lyase 0.035 ± 0.005

GMP reductase 0.008 ± 0 .0 0 1 GMP synthetase NA (<0.005) IMP dehydrogenase NA (<0.005) Adenylate deaminases AMP NA (<0.005) ADO NA (<0.001) ADE NA (<0.001)

& n = three different batches of cells b NA = no activity detected (minimum detectable amount) 25

TABLE 6

Purine salvage enzymes in Mycoplasma gallisepticum S6.

Enzyme A ctivity Enzyme [nmol product min~' mg~‘ prot] Substrate (±SD)a

Phosphor i bosyltransfer ases ADE 1.18 ± 0.309 GUA 18.3 ± 2.36 HX 7.16 ± 1.06 X NAb(<0.010) Nucleoside phosphorylases (Base to nucleoside) ADE 18.7 ± 2.69 GUA 14.9 ± 4.43 HX 3.49 ± 0.802 X NA (<0.010) Nucleoside phosphorylases (Nucleoside to base) ADO 1.01 ±0.171 GUA 0.653 ± 0.101 INO 0.565 ± 0.257 XO NA (<0.010) 5‘-Nucleotidases AMP 0.064 ± 0.009 GMP NA (<0.005) IMP NA (<0.005) XMP NA (<0.005) Adenylosuccinate synthetase and lyase 0.108 ± 0.027 GMP reductase NA (<0.001) GMP synthetase NA (<0.005) IMP dehydrogenase NA (<0.005) Adenylate deaminases AMP NA (<0.005) ADO NA (<0.001) ADE NA (<0.001)

3 n = three different batches of cells i) NA = no activity detected (minimum detectable amount) 25

TABLE 7

Purine salvage enzymes in Spiroplasma floricola 23-5.

Enzyme A ctivity Enzyme [nmol product rain"' mg"' prot] Substrate (±SD)a

Phosphori bosyltransferases ADE 16.1 ± 1.79 GUA 15.3 ± 0.923 HX 6.03 ± 1.95 X NAb(<0.005) Nucleoside phosphorylases (Base to nucleoside) ADE 26.5 ± 4.58 GUA 37.8 ± 7.85 HX 14.3 ± 0.318 X 0.645 ± 0.057 Nucleoside phosphorylases (Nucleoside to base) ' ADO 3.41 ± 0.430 GUO 3.15 ± 0.455 INO 3.93 ± 0.750 XO NA (<0.010) 5'-Nucleotidases AMP NA (<0.005) GMP NA (<0.005) IMP NA (<0.005) XMP NA (<0.005) Adenylosuccinate synthetase and lyase NA (<0.005) GMP reductase 0.037 ± 0.005 GMP synthetase 0.005 ±0.001 IMP dehydrogenase NA (<0.005) Adenylate deaminases AMP NA (<0.005) ADO NA (<0.001) ADE NA (<0.001)

& n = three different batches of cells b NA = no activity detected (minimum detectable amount) 27

TABLE 8

Purine salvage enz^es in Mycoplasma arginini G-230.

Enzyme Activity Enzyme [nmol product min"l mg"' prot] Substrate (± S D )a

Phosphoribosyltransferases ADE 10.7 ± 0.324 GUA 2.11 ± 0.095 HX 2.97 ± 0.014 X NAb (<0.005) Nucleoside phosphorylases (Base to nucleoside) ADE 15.6 ± 1.03 GUA 9.74 ± 1.20 HX 3.39 ± 0.215 X NA (<0.005) Nucleoside phosphorylases (Nucleoside to base) ADO 2.48 ± 0.186 GUA 5.88 ± 1.06 INO 1.64 ± 0.121 XO NA (<0.010) 5'-Nucleotidases AMP NA (<0.005) GMP NA (<0.005) IMP NA (<0.005) XMP NA (<0.005) Adenylosuccinate synthetase and lyase 0.145 ± 0.013 GMP reductase NA (<0.005)

GMP synthetase NDC IMP dehydrogenase NA (<0.005) Adenylate deaminases AMP NA (<0.005) ADO NA (<0.001) ADE NA (<0.001)

3 n = three different batches of cells b NA = no a ctiv ity detected (minimum detectable amounts) c NO = not done 28

TABLE 9

Purine salvage enzymes in Acholeplasma florum 11

Enzyme A ctivity Enzyme [nmol product rain"' mg"' prot] Substrate (±SD)a

Phosphoribosyltransferases ADE 47.0 ± 3.84 GUA 2.64 ± 0.614 HX 2.55 ± 0.331 X NAb(<0.005) Nucleoside phosphorylases (Base to nucleoside) ADE 93.6 ± 8.68 GUA 17.7 ± 2.70 HX 4.24 ± 1.05 X NA (<0.005) Nucleoside phosphorylases (Nucleoside to base) ADO 1.90 ± 0.271 GUA 4.93 ± 0.289 INO 2.19 ± 0.462 XO NA (<0.010) 5’-Nucleotidases AMP NA (<0.005) GMP NA (<0.005) IMP NA (<0.005) XMP NDC Adenylosuccinate synthetase and lyase NA (<0.005) GMP reductase 0.003 ± 0.001 GMP synthetase ND IMP dehydrogenase NA (<0.005) Adenylate deaminases AMP NA (<0.005) ADO NA (<0.001) ADE NA (<0.001)

n = three different batches of cells NA = no activity detected (minimum detectable amounts) NO = not done 29

TABLE 10

Nucleoside kinase activities in A. laidlawii B-PG9,

A. axanthum S-743, ^ qranularum BTS-39 and ^ coli (ATCC 25922).

Enzyne A ctiv ity Cumol product min"? mg~‘ p ro t] ±SD Phosphate Donor Organism S ubstrate PPi ATP n^

h i. la id la w ii ADO 58.9 ± 21.3 NAb 7 GUO 50.8 ± 21.3 NA 4 INO 32.3 ± 8 . 6 8 NA 3 XO NA NA 3 h i. axanthum ADO 0.529 ± 0.160 NA 3 GUO 0.291 ± 0.080 NA 3 INO NA NA 3 XO NA NA 3 h i. granularum ADO 0.161 ± 0 . 0 1 2 NA 3 GUO NA NA 3 INO NA NA 3 XO NA NA 3

E^ c o li ADO NA 0.401 ± 0.101 5 GUO NA 0 . 1 1 0 ± 0 . 0 1 1 3 INO NA 0.064 ± 0.030 3 XO NDC ND •

® n = number of different batches of cells, b NA = no activity detected (<0.001) c NO = not done 30 TABLE 11 Summary of purine salvage enzyme activities.

§ CO o i to E s. a . * 5 3 to *o Q> *c 10 E u (A 4 -» 3 3 c “D c C u to to O o 0 3 tes X S- to s. to CT 4 - 4 - O) fO Enzyme .•.. Substrate C < Phosphoribosyltransferases ADE, GUA, HX +a X + Nucleoside phosphorylases ADO, INC, GUO + XO + Nucleoside phosphorylases ADE, HX, GUA + + X + + PPi Nucleoside kinases^ ADO + + INO + GUO + + XO 5'-Nucleotidases (cytoplasmic) AMP + + + GMP + + + IMP + + + XMP + Adenylosuccinate synthetase and lyase

GMP reductase + +

GMP synthetase ND + ND IMP dehydrogenase Adenylate deaminases

3 + = enzyme activity detected in all different batches of cells tested (2 3). - = no enzyme activity detected in all different batches of cells tested (> 3). c No ATP-dependent kinase activ ity detected ND = not done 31

1 0 0 1

s 8 0 - SODIUM PYROPHOSPHATE N S ■ ë^ 6 0 - ïn CL S < 4 0 - 10 o £ o.

2 0 -

ATP

0.2 0.5 1.0 2.0 3.0 4.0 mM

Fig. 1. Effect of various concentrations of sodium pyrophosphate or ATP on the amount of AMP synthesized in the standard reaction mixture (MATERIALS AND METHODS) for adenosine kinase activity by dialyzed lysates of Acholeplasma laidlaw ii B-PG9. DISCUSSION

Except for H. roycoides subsp. mycoides (57,58,78,79), the lack of a defined medium that supports adequate growth impairs the study of the purine metabolism of the class Molli cutes. In order to reduce the complications from purines contributed by the growth medium, defined media for A. laidlawii B similar to that of Rodwell's C-2 medium for M. mycoides as used by Mitchell and coworkers (57,58), and formulations for laidlawii similar to those suggested by Razin and Cohen (76) and Greenaway and Wase (32) were used. However, sufficient growth of the organism for the enzyme assays in this study was not obtained using any of these formulations. Horse serum is another complicating factor in the assay of purine enzymes in these organisms. In addition to nucleosides, there also may be contaminating purine-metabolizing enzymes in the serum. Purine nucleoside phosphoryl- ase activity has been reported in horse serum used for the cultivation of mammalian cells (67). Except for the PPi-dependent adenosine and nucleoside kinases, XMP nucleotidase and XO phosphorylase activities reported here, we

found th at k . laidlawii B-PG9 has similar enzymatic activities of

purine salvage and interconversion as f 1. mycoides subsp. mycoides (57,58). That is , both mycoides and A. laidlaw ii B-PG9 are able to convert purine bases to their respective nucleoside and mononucleotide, and guanylates to adenylates via GMP reductase, adenylosuccinate synthetase and adenylosuccinate lyase (Fig. 2). 32 33

PRA 6LY

AMP IMP GMP

•AS XMP

PPi, ▼ [

PRPP PRPP PRPP PRPP R-l-P

HYPOXANTHINE XANTHINE GUANINE'ADENINE

Fig. 2. Proposed salvage pathways for the biosynthesis of purine nucleotides in Acholeplasma laidlawii B-PG9. Enzyme acti­ v itie s were found for the following reactions ( 1 ) adenine phosphoribosyltransferase (EC 2.4.2.7); (2) hvpoxanthine- guanine phosphoribosyltransferase (EC 2.4.2. 8 ); (3) 5'-nucleo­ tidase (EC 3.1.3.5); (4) pyrophosphate dependent adenosine kinase; (5) pyrophosphate dependent nucleoside kinase; ( 6 ) and (7) purine nucleoside phosphorylase (EC 2.4.2.1); (10) adenylosuccinate synthetase (EC 6 .3.4.4); (11) adenylosuccinate lyase (EC 4.3.2.2); (15) GMP reductase (EC 1. 6 . 6 . 8 ). No enzyme activity was detected for the following reactions ( 8 ) adenosine deaminase (EC 3. 5 .4 .4 ); (9) adenine deaminase (EC 3.5.4.2); (12) AMP deaminase (EC 3.5.4. 6 ); (13) IMP dehydrogenase (EC 1.2.1.14); (14) GMP synthetase (EC 6 .3 .4 .1 ); (16) phophoribosylglycinamide synthetase (EC 6.3.4.13). 34

Both organisms are incapable of converting adenylates to guanylates. We did not detect AMP, adenosine or adenine deaminases, IMP dehydro­ genase, GMP synthetase or XO kinase activities in A. laidlawii B-PG9

(Fig. 2). The activities of these enzymes in M. mycoides have not been reported, but are presumed by us to be absent based on labeling experiments performed by Mitchell and coworkers (57). Specific activities for purine salvage enzyme reported for M. mycoides subsp. mycoides range from three times (adenylosuccinate synthetase) to 40 times (APRT) greater than those reported here for A. laidlawii B. Specific activities reported for pyrimidine deoxyribonucleoside meta­ bolic enzymes (62,63) are from 10 to 100 times greater in M. mycoides than for those found in A. laidlawii B (M.V. Williams, personal communication). Soluble (cytoplasmic) S'-nucleotidase activity was detected in A. laidlawii B for the four purine 5'-mononucleotides (Table 3 and 11). Although 5'-nucleotidase activ ity is generally considered a marker for plasma membranes, activ ity associated with cytoplasmic fractions has been reported previously in mammalian tissue (84). No ATPase or NADH oxidase activity was associated with this 200,000 x g supernatant from A. laidlawii B. This indicates that our procedures afforded good separation of a cytosolic fraction without detectable membrane contamination. However, this does not preclude that the S'-nucleotidase activity is only loosely associated with the membrane and that despite gentle disruption of the organism the activity is found in the cytosol. The two other acholeplasmas had activity for AMP, GMP and IMP, but not for XMP. The activ ity detected for 35

A. laidlawii B was at our detectable limit {3-5 pmol min"^ mg*^ prot). The lack of activity for XMP in the other acholeplasmas could be due to a lack of sensitivity. No cytoplasmic nucleotidase activity was detected in S. floricola, A. florum, or the two Myco- plasma spp.

In some experiments, we detected conversion of the nucleosides to their bases in the absence of added phosphate. This suggests there may also be some phosphate independent purine nucleosidase

[N-ribosyl-purine ribohydrolase, EC 3.2.2.1] activity as reported in Trichomonas vaginalis and other protozoa (55). However, we cannot be certain of this because phosphate may be supplied from crude lysate, despite dialysis, and be adequate to support phosphorylase activity which appears as phosphate-independent hydrolase activity.

hi. mycoides is capable of using guanine but not adenine as the sole source of purine nucleotides for growth (57,58,78,79). However,

£. laidlawii B is reported to require both adenine and guanine for growth (44,76,95), d e sp ite th e presence o f th e enzyme a c tiv itie s necessary to convert guanylates to adenylates, as we have shown in this work (Table 3; Fig. 2). This suggests that perhaps the rate of interconversion is insufficient to fulfill the requirements for adenylates in P^. laidlawii B. However, any comparisons or opinions drawn from the rates of the enzyne activities reported here must be made provisionally and with great caution since our assays were conducted using essentially crude cell extracts, and there may be

competing reactions. We minimized competing reactions by extensive

dialysis of our cell-free preparations. Control reactions lacking 36

100

AMP

S A a 50 «

ADO

ADE

2 A 8 16 30 REACTION TIME Cniin>

to time. 37

The committed and most regulated step of de novo purine synthesis is the formation of phosphor1bosylami ne (PRA). PRA is synthesized from glutamine and PRPP, a reaction involving pyrophosphate release and hydrolysis. Both ribose phosphate and PRPP have been demonstrated in growing Mollicutes (14,53,55). PRA f i r s t reacts with glycine in the 10 step synthesis of the purine nucleotide IMP. The two carbons and nitrogen of glycine are incorporated into the growing heterocyclic molecule. The inability to detect incorporation of [U-l^C]glycine into purine bases of growing A. laidlawii B-PS9 suggests that the block is in the synthesis of PRA or its conversion to IMP. These findings strengthen the view that A. laidlawii, and perhaps other Mollicutes as well, cannot synthesize purines de novo. This suggestion is also supported by their requirement for nucleic acid precursors when growing in defined media (15,44,76,78,79,87,95). The in a b ility to detect the incorporation of [U-l^C]glycine into purines in A. laidlawii B cannot be taken as complete evidence against the de novo synthesis of purines by this organism. Other factors such as inhibition or repression of the enzymes involved could explain the lack of incorporation of labeled glycine. Control of the first two enzymes in the biosynthetic pathway by the availability of PRPP and glutamine as well as feedback inhibition by nucleotides is generally accepted. Although not proven, i t has been thought th a t the nucleotides may also act as low molecular weight repressors of gene expression. Houlberg and Jensen (37) provide evidence in Salmonella typhimurium that hypoxanthine and guanine, and not nucleotides, may act as co-repressors. These conclusions are based on 1) the lack of 39 activ ity , can be resolved in a second solvent in one dimension (Plate B). The resolution of purines was unaffected by the levels of proteins and salts found in the cell extracts and reaction mixtures in this study. These methods allowed the detection of specific a ctiv itie s between 1 to 5 pmol at the lower lim it. The compounds of in terest were identified by co-chromatography with standards and identified by absorption of short-wave UV light. In early experiments TLC plates were autoradiographed for up to 72 hours before scraping to insure the detection of all labeled compounds. HPLC procedures exist for the separation of purine bases, nucleosides and nucleotides, but these procedures require long elution times (> 40 min) and complicated, non-linear, multiple-solvent elution gradients (25,28,74). This is the first report of a pyrophosphate dependent adenosine or nucleoside kinase activity in any procaryote or eucaryote, and the first*report of any adenosine kinase or nucleoside kinase activity in the Mollicutes. Previously, five enzymes in procaryotes and eucaryotes were known to u tiliz e pyrophosphate as a phosphate donor. These enzymes are carboxytransphosphorylase, pyruvate-phosphate dikinase, PPi acetylkinase, PPi serine kinase and PPi phosphofructokinase (Table 12)(107). Recently, the utilization of pyrophosphate as a phosphate donor for phosphofructokinase in laidlaw ii B-PG9 has been reported

(J.O. Pollack and M.V. Williams, Abst. Anna. Meet. Am. Soc. Microbiol. 1984, G20, p. 172). K.D. Beaman and J.D. Pollack observed pyrophosphate dependent carboxytransphosphorylase activity in laidlawii B-PG9. This activity was labile in their hands and attempts at purification were unsuccessful (unpublished data). 40

TABLE 12

Known Reactions in Which PPi Replaces AiP as a Phosphate Donor®

CARBOXYTRANSPHOSPHORYLASE

PPi + OXALOACETATE P-ENOLPYRUVATE + Pi + CO2

PYRUVATE, PHOSPHATE DIKINASE

PPi + AMP + P-ENOLPYRUVATE -----^ PYRUVATE + ATP + Pi

PHOSPHOFRUCTOKINASE

PPi + FRÜCT0SE-5-P s- FRUCTOSE-1,6 -P + Pi

ACETYLKINASE

PPi + ACETATE — ^ ACETYLPHOSPHATE + Pi

SERINE KINASE

PPi + SERINE ---- > PHOSPHOSERINE + Pi

® From references 107,108 41

It will be interesting to know whether these pyrophosphate- dependent enzymes detected in A. laidlawii B-PG9, A. axanthum S-743 and A. qranularum are functional in the intact cell. A number of biosyn­ thetic reactions involve the formation of pyrophosphate, and it is generally believed that pyrophosphate is a metabolic end product that by simple hydrolysis thermodynmnically drives coupled reactions in the anabolic direction (40). Wood et al. suggested that the ability of Entamoeba histolytica, Propionibacterium shermanii and Bacillus symbiosus to u tiliz e pyrophosphate may confer a degree of selective advantage and may partly account for the high efficiency of growth of these organisms (108). Williams and Pollack (105) have reported the presence of dUTPase activity in A. laidlawii B-PG9 and in this study we have reported the presence of adenine, hypoxanthine, xanthine and guanine phosphoribosyltransferase activities. In addition to the mononucleotide, the product of each of these latter five enzyme activities is PPi. Other enzyme activities known to produce PPi, e.g., during the formation of nucleic acids by polymerases, have been reported in members of the class Mollicutes (17,18,56,88,101). O'Brien e ^ ^ . detected inorganic pyrophosphatase activity in a number of Mycoplasma and Acholeplasma species except laidlawii B, laidlaw ii A and Acholeplasma axanthum (65). In the absence of inorganic pyrophosphatase, these newly dis­ covered PPi-dependent nucleoside kinase activities, and perhaps other PPi-dependent enzymes as yet unexamined in these organisms, may be essential to the metabolism of A. laidlawii B and A. axanthum. 42 and as such, may be useful in studying the phylogenetic relatedness of these organisms within their class and to other procaryotes.

Although not assayed here, A. granularum is reported to have inorganic pyrophosphatase activity (65). The finding of PPi-dependent

ADO kinase activity even in thr presence of inorganic pyrophosphatase suggests th a t th is novel enzyme a c tiv ity may be common to a ll the acholeplasmas except A. florum and thus another example of their physiological uniqueness from other families in the class Mollicutes.

Most s trik in g in th e examination of th e purine salvage enzyme a c tiv itie s of Acholeplasma, Mycoplasma and Spiroplasma spp. is the inability of any organism to synthesize guanylates from other mono­ nucleotides. In addition, the lack of GMP reductase (GMP — IMP) activity in M. gallisepticum and adenylosuccinate synthetase/lyase

(IMP — AS — AMP) activity in S. floricola and A. florum means that guanylates cannot be converted to other purine mononucleotides in these three organisms. These data suggest that guanine phosphoribosyltrans­ ferase and PPi-dependent nucleoside kinase may be promising loci in which to attempt to biochemically inhibit infection or pathology by

Mollicutes. Inhibitors of GPRT activity have been described (38), but nonspecific effects have made them unsuitable for use in vivo in human cells. Perhaps their potential use in the treatment of infec­ tio n s and contam ination of c e ll c u ltu re s caused by members of the

Mollicutes will bring new reason to attempt modification of such inhibitors to enable their use. 43

FIGURE 4

pp. w rN O siN i

«EMNE

Proposed salvage pathways for the biosynthesis of purine nucleotides in Acholeplasma florum and Spiroplasma floricola. Enzyme activities were found fo r the following reactions: ( 1 ) adenine phosphoribosyl­ transf erase (EC 2.4.2.7); (2) hypoxanthine-gunaine phosphoribosyltrans- ferase (EC 2.4.2.8 ); ( 6 ) and (7) purine nucleoside phosphorylase (EC 2.4.2.1) and (15) GMP reductase (EC 1.6. 6 . 8 ). Numbers refer to the enzymes as marked in Figure 2 . 44

FIGURE 5

ivît GWF

fiDrw3$iN£ INDE'NE BUcNOSiNE •] r e

ADENINE HYFD x XNT hiNE GUANINE

Proposed salvage pathways for the biosynthesis of purine nucleotides in Mycoplasma qallisepticum and Mycoplasma arqinini. Enzyme activ i- ties were found for the following reactions: (l) adenine phosphoribo- syltransferase (EC Z.4.2.7); (2) hypoxanthine-guanine phosphoribosyl- transferase (EC 2.4.2. 8 ); (6 ) and (7) purine nucleoside phosphorylase (EC 2.4.2.1); (10) adenylosuccinate synthetase (EC 6 .3 .4 .4 ); (11) adenylosuccinate lyase (EC 4.3.2.2). Numbers refer to the enzmes as marked in Figure 2. 45

CONCLUSIONS

Fifteen cytoplasmic enzymes with activity for purine salvage and interconversion were examined in Acholeplasma laidlaw ii B-PG9, Achole­ plasma axanthum S-743, Acholeplasma qranularum BTS39, Spiroplasma floricola 23-5, and Mycoplasma gallisepticum S 6 , Phosphoribosyltrans- ferase activity for adenine, guanine, and hypoxanthine were found in all organisms tested. Phosphoribosyltransferase activity for xanthine was detected only in ^ laidlawii B. Nucleoside phosphorylase activity for xanthine was detected in both A. laidlawii and A. granu­ larum. No ATP-dependent nucleoside kinase activ ity was detected in any organism. Novel pyrophosphate-dependent nucleoside kinase activity for adenosine, inosine, and guanosine was found in A. laidlawii B and for adenosine and guanosine in A. axanthum and for adenosine only in A. granularum. Cytoplasmic S'-nucleotidase activity towards the four purine mononucleotides AMP, GMP, IMP and XMP was detected in A. laidlawii B. Nucleotidase activity for AMP, GMP and IMP was detected in A. axanthum and A. qranularum. No cytoplasmic S'-nucleotidase activity was detected in S. floricola, in A. florum, or any Mycoplasma spp. Adenylosuccinate synthetase-lyase activity was detected in all species examined except S. floricola and A. florum. GMP reductase activity was detected in all organisms tested except M. gallisepticum and M. arq in in i. GMP synthetase activ ity was measured only in S. floricola. IMP dehydrogenase, AMP deaminase, adenosine deaminase. 46 and adenine deaminase a ctiv itie s were not detected in any species. This is the first report of pyrophosphate-dependent nucleoside kinase activ ity in any organism. All species examined rely on guanine phosphoribosyltransferase or the novel pyrophosphate-dependent nucleoside kinase activity for the production of guanylates. In no organism tested can guanine monophosphate be derived from the other purine mononucleotides. This suggests that the guanine phosphoribo­ syltransferase may be a promising site for the biochemical inhibition of mycoplasma infection and pathology. Recent work with Mycoplasma hominis obtained from N.L. Somerson, The Ohio State University, shows that this organism has a similar pattern of purine salvage and interconversion as the other Mycoplasma spp. examined in th is study. This is , M. hominis lacks the enzyme activities necessary to convert adenylates to guanylates and guanylates to adenylates. As with the other non-acholeplasma species, no pyro­ phosphate-dependent kinases were found in this organism. Preliminary work indicates that M. hominis may lack some of the enzyme activities necessary for the conversion of purine bases to mononucleotides. LITERATURE CITED

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