JOURNAL OF BACTERIOLOGY, July 1984, p. 265-270 Vol. 159, No. 1 0021-9193/84/070265-06$02.00/0 Copyright ©3 1984, American Society for Microbiology in Acholeplasma laidlawii B: Novel PPi- Dependent Nucleoside Kinase Activity VICTOR V. TRYON AND DENNIS POLLACK* Department of Medical Microbiology and Immunology, The Ohio State University, Columbuts, Ohio 43210 Received 21 February 1984/Accepted 12 April 1984

Acholeplasma Iaidlawii B-PG9 was examined for 16 cytoplasmic enzymes with activity for purine salvage and interconversion. Phosphoribosyltransferase activities for adenine, guanine, , and were shown. Adenine, guanine, xanthine, and hypoxanthine were ribosylated to their nucleoside. Adenosine, inosine, xanthosine, and guanosine were converted to their base. No ATP-dependent phosphorylation of nucleosides to mononucleotides was found. However, PPi-dependent phosphorylation of adenosine, inosine, and guanosine to AMP, inosine monophosphate, and GMP, respectively, was detected. Nucleotidase activity for AMP, inosine monophosphate, , and GMP was also found. Interconversion of GMP to AMP was detected. Enzyme activities for the interconversion of AMP to GMP were not detected. Therefore, A. laidlawii B-PG9 cannot synthesize guanylates from adenylates or inosinates. De novo synthesis of purines was not detected. This study demonstrates that A. Iaidlawii B-PG9 has the enzyme activities for the salvage and limited interconversion of purines and, except for purine nucleoside kinase activity, is similar to Mycoplasma mycoides subsp. mycoides. This is the first report of a PPi-dependent nucleoside kinase activity in any organism. The only members of the class Mollicutes for which the phosphate ([8-14C]XMP), 56 mCi/mmol. [8-14C]inosine 5'- pathways of purine salvage and interconversion have been monophosphate ([8-14C]IMP) (59 mCi/mmol) was purchased comprehensively studied and described is Mycoplasma my- from Amersham Corp. (Arlington Heights, Ill). Lecithin coides subsp. mycoides (27, 28). Activities of some purine (vegetable) was purchased from Mann Research Labora- salvage enzymes in Mollicutes have been reported previous- tories (New York, N.Y.). Other chemicals were purchased ly (12, 13, 16, 22, 23, 27, 28, 34). Hamet et al. (12) have from Sigma Chemical Co. (St. Louis, Mo.), unless otherwise examined purine salvage but not interconversion enzyme specified. activities in five Mycoplasma species and in Acholeplasma Organisms and culture conditions. A. laidlawii B-PG9 was laidlawii A-PG8. Although, Mclvor and Kenny did not test grown without serum in our modification of Edward medium for specific enzyme activities, they found that Mycoplasma containing penicillin G (100 U ml-1), as described previously species and A. laidlawii were able to incorporate radiola- (2). Bacillus subtilis 60015, which lacks purine nucleoside beled purine and pyrimidine bases and nucleosides into RNA kinase activity (9), was obtained from Ernest Freese (Na- (23). tional Institute of Neurological and Communicative Disor- We have reported that A. laidlawii B-PG9 maintains an ders and Stroke, Bethesda, Md.) and grown in a defined adenylate energy charge comparable to that of Escherichia medium as described by Endo et al. (9). E. coli ATCC 25922, coli and other procaryotes and synthesizes more ATP per which has purine nucleoside kinase activity, was grown in milligram (dry weight) than E. coli (2, 3). To determine the Edward medium without penicillin. pathways by which ATP and other purine 5'-monophos- All cultures were incubated at 37°C. Starter cultures were phates are synthesized in A. laidlawii B, we examined 16 inoculated into temperature-equilibrated media to 1 to 5% enzyme activities involved in purine salvage and intercon- (vol/vol) and incubated statically. Cells were harvested in version and the incorporation of [U-_4C]glycine included in their mid-log phase of growth at 6 to 24 h. the growth medium into purine bases. To detect de novo synthesis of purines, A. laidlawii was grown in tryptose broth containing, per liter, tryptose (Difco MATERIALS AND METHODS Laboratories, Detroit, Mich.), 25 g; NaCl, 5 g; and Tris, 5 g Chemicals. The following radiolabeled compounds were (pH 7.5). After autoclaving, we added sterile glucose solu- purchased from Research Products International Corp. (Mt. tion to 1% (vol/vol) and a liposome suspension to 0.5% (vol/ Prospect. Ill.): [8-14C]adenine ([8-14C]ADE), 50 mCi/mmol; vol). The liposome suspension was composed of phosphati- [8-14C]adenosine ([8-14C]ADO), 47 mCi/mmol; [8-14C]guano- dylcholine-cholesterol (1:1) and prepared as described by sine ([8-14C]GUO), 42.8 mCi/mmol; and [U-14C]GMP, 450 Cluss et al. (8). The liposome suspension was added because mCi/mmol. The following were purchased from ICN Phar- it stimulated growth and increased the cell yield. The con- maceuticals, Inc. (Irvine, Calif.): [8- 4C]AMP, 58 mCi/ centration of adenylates in the tryptose medium was 0.3 ,ug mmol; [$-14C]ATP, 51 mCi/mmol; [8-14C]guanine ([8- ml-1 and about half that in modified Edward medium 14C]GUA), 51 mCi/mmol; and [U-14C]glycine, 92 mCi/mmol. without serum. The following were purchased from Moravek Biochemicals Preparation of cell extracts. Cell-free preparations were (Brea, Calif.): [8-14C]hypoxanthine ([8-14C]HX), 56 mCi/ made essentially as described previously (30). A. laidlawii mmol; [8-14C]inosine ([8-14C]INO), 56 mCi/mmol; [8- cells were harvested by centrifugation at 9,000 x g at 4°C for 14C]xanthosine ([8-14C]Xo), 56 mCi/mmol; [8-'4C]xanthine 30 min. The cells were washed by centrifugation three times ([8-14C]X), 57 mCi/mmol; and [8-14C]xanthosine 5'-mono- in 200 to 300 volumes of cold kappa-buffer. Washed cells were lysed by hypotonic shock in aqueous diluted (1:20) * Corresponding author. 37°C kappa-buffer by incubation at 37°C for 3 to 10 min. The 265 266 TRYON AND POLLACK J. BACTERIOL.

crude lysate was centrifuged at 48,000 x g for 1 h at 4°C. The min. Product mononucleotide was chromatographically sep- supernatant was centrifuged at 250,000 x g for 1 h at 4°C. arated from substrate purine base in solvent A. The supernatant was dialyzed in the cold overnight against (ii) ADO kinase (ATP-adenosine 5'-phosphotransferase; four changes of 100 volumes each of 10 mM N-2-hydroxyeth- EC 2.7.1.20) and nucleoside kinase (ATP-inosine 5'-mono- ylpiperazine-N'-2-ethanesulfonic acid (HEPES; Research phosphotransferase; EC 2.7.1.73) were assayed by the meth- Organics, Cleveland, Ohio) (pH 7.5)-2 mM 2-mercaptoeth- od of Yamada et al. (41). Reaction mixtures contained 50 anol-1 mM MgCl2100 puM phenylmethylsulfonyl fluoride. mM IJEPES (pH 7.4), 1 mM MgCl2, 1 mM ATP, an ATP- This dialyzed cell extract was used immediately for all regenerating system consisting of 2 mM phosphoenolpy- enzyme assays. E. coli and B. subtilis cells were harvested ruvate-0.5 U of pyruvate kinase-[8-14C]ADO for the ADO as described above for A. Iaidlawii. Cell extract of E. coli or kinase and [8-'4C]GUO, [8-'4C]INO, or [8-14C]XO for the B. subtilis was prepared by incubation at 37°C in 1:20 kappa- nucleoside kinase. Incubation time was 6 min. Product buffer with lysozyme (100 ,ug ml-1) for 30 min as described was chromatographically separated from sub- by Endo et al. (9). Lysozyme-treated cells were sonicated strate nucleoside in solvent A. ADO kinase and nucleoside (Sonifier Cell Disruptor; Heat Systems, Melville, N.Y.) by kinase utilizing PP, were assayed as for the ATP-dependent three 10-s exposures while on wet ice. Whole and broken kinases, except that 2 to 4 mM sodium PPi (Fischer Certified cells were centrifuged at 15,000 x g for 30 min at 4°C. The A.C.S.; Fischer Scientific Co., Fairborn, N.J.) was substi- cell extract was dialyzed as described above. The dialyzed tuted for ATP, and no ATP-regenerating system was used. cell extract was used immediately for all enzyme assays. In some experimants, we tested for ADO kinase activity Protein was determined by the method of Bradford (4) with with ATP or PP, over the range of 0.1 to 4 mM. In the G-250 dye reagent formulated by Bio-Rad Laboratories preliminary experiments to test for the effect of contaminat- (Richmond, Calif.). ing membrane ATPase, we used these same reaction condi- Enzyme assays. For all assays, reaction mixtures were tions by substituting [8-14C]ATP for the radioactive ADO for incubated at 37°C in a total volume of 0.1 ml. Each reaction up to 30 min of incubation. In these experiments, we used 2 mixture contained 15 to 25 ,umol of radiolabeled substrate. N formic acid-0.5 M LiCl (1:1) to resolve ATP, ADP, and Concentrations of radioactive substrate were adjusted so AMP on polyethyleneimine plates. that greater than 50% of the label remained at the end of the (iii) Purine-nucleoside phosphorylase (purine nucleoside- incubation period. Reactions were started by the addition of Pi ribosyltransferase; EC 2.4.2.1) was assayed. Reaction temperature-equilibrated dialyzed cell extracts containing 10 mixtures for the base to nucleoside conversion contained 50 to 40 ,ug of protein and incubated with shaking. Reactions mM sodium phosphate or HEPES buffer (pH 7.4), 2 mM were terminated by heating at 100°C for 2 min. After MgC92, 4 mM ribose-1-phosphate, and [8-14C]ADE, [8- preliminary study of each assay, we chose an incubation 14C]GUA, [8-14C]HX, or [8-14C]X. For the nucleoside to time which gave the fastest rate of product formation. 14C- base conversion, the reaction mixtures were the same, labeled substrate and product were separated by thin-layer except that [8-14C]ADO, [8-14C]GUO, [8-14C]INO or [8- chromatography on commercial polyethyleneimine plates 14C]XO replaced their respective base. Incubation time was containing 0.55 mEq of polyethyleneimine g-1 of cellulose 4 min. Nucleosides were chromatographically separated (Analtech, Inc., Newark, Del.). Plates were developed in from bases in solvent A. either 1 M LiCl (solvent A) or 4 N formic acid (solvent B). (iv) 5'-Nucleotidase (5'-ribonucleotide phosphohydrolase; Solvent A was used to separate purine 5'-mononucleotides EC 3.1.3.5) was assayed. Reaction mixtures contained 50 from nucleosides and bases. Solvent B was used to separate mM HEPES (pH 7.4), 2 mM MgCI2, and [8-14C]AMP, individual purine 5'-mononucleotides from each other. Ten [8-14C]IMP, [8-14C]GMP, or [8-14C]XMP. Incubation time microliters of the reaction mixture were spotted in each lane was 8 min. Product nucleosides and their bases were chro- with appropriate nonradioactive markers. Resolved purines matographically separated from substrate in were visualized by UV light, scraped into 7 ml of Budget- solvent A. The purine bases were secondarily formed by the Solve (Research Products International Corp.), and counted action of the phosphorylase. in a LSC 7000 liquid scintillation counter (Beckman Instru- (v) synthetase (IMP-1 aspartate ligase; ments, Inc., Fullerton, Calif.). GDP-forming; EC 6.3.4.4) and adenylosuccinate lyase The Rf for compounds separated in solvent A were ADE, (adenylosuccinate AMP-lyase; EC 4.3.2.2) were assayed by 0.41; ADO, 0.63; AMP, 0.96; GUA, 0.46; GUO, 0.68; GMP, measuring the rate of production of AMP from IMP via 0.96; HX, 0.61; INO, 0.80; IMP, 0.96; X, 0.43; XO, 0.65; and adenylosuccinate in a two-step reaction sequence, as modi- XMP, 0.96. Rf for compounds separated in solvent B were fied from the procedure of Lieberman (17) and Fischer et al. XMP, 0.17; IMP, 0.24; GMP, 0.43; and AMP, 0.90. No (10). Reaction mixtures contained 50 mM HEPES (pH 7.4), 2 significant chromatographic trailing occurred in any in- mM MgCl2, 4 mM GTP, 4 mM aspartate, and [8-14C]IMP. stance. Samples were counted to 2% counting error and Incubation time was 30 min. Product AMP was chromato- corrected for quenching. All radioisotope data were calculat- graphically separated from substrate IMP in solvent B. ed as disintegrations per minute and converted to moles of (vi) GMP reductase (NADPH-GMP oxidoreductase; de- product synthesized per minute milligram of protein by aminating; EC 1.6.6.8) was assayed by a modification of the calculation with the specific activity of the radioactive techniques of Mager and Magasanik (20). Reaction mixtures substrate. The following enzyme assays were carried out. contained 50 mM HEPES (pH 7.4), 2 mM cysteine hydro- (i) ADE phosphoribosyltransferase (AMP-PP, phosphori- chloride, 0.4 mM NADPH, and [8-14C]GMP. Incubation bosyltransferase; EC 2.4.2.7) and HX-GUA phosphoribosyl- time was 4 min. Product IMP was chromatographically transferase (IMP-PPi phosphoribosyltransferase, EC 2.4.2.8) separated from substrate GMP in solvent B. were assayed. Reaction mixtures contained 50 mM HEPES (vii) GMP synthetase (XMP-ammonia ligase; AMP-form- (pH 7.4), 5mM MgCl2, 4 mM phosphoribosylpyrophosphate ing; EC 6.3.4.1) was assayed by a modification of the (PRPP), and [8-14C]ADE for the ADE phosphoribosyltrans- procedure of Sakamoto (33). Reaction mixtures contained 50 ferase, [8-14C]GUA, [8-14C]HX, or [8-14C]X for the HX- mM HEPES (pH 7.4), 4 mM ATP, 2 mM glutamine or GUA phosphoribosyltransferase. Incubation time was 8 ammonium sulfate, 2 mM MgC92, and [8-14C]XMP. Incuba- VOL. 159, 1984 PURINE METABOLISM IN A. LAIDLAWII 267 tion time was 5 min. Product XMP was chromatographically TABLE 1. Purine salvage enzymes in A. laidlawii B-PG9 separated from substrate GMP in solvent B. Enzyme activity (viii) IMP dehydrogenase (IMP-NAD+ oxidoreductase; (nmol of product EC 1.2.1.14) was assayed by a modification of the technique Enzyme min-' mg-' of of Magasanik et al. (19). Reaction mixtures contained 50 mM protein + SD) HEPES (pH 7.4), 2 mM cysteine hydrochloride or 2 mM Phosphoribosyltransferases glutathione (Calbiochem, San Diego, Calif.), 2 mM NAD, ADE 5.08 ± 1.45 7 0.1 mM KCI, and [8-14C]IMP. Incubation time was 50 min. GUA 2.76 ± 1.66 7 We also assayed the hypotonic lysate of washed human HX 3.69 ± 2.11 4 erythrocytes by the procedure of Henderson et al. (14). X 0.08 ± 0.02 3 Product GMP was chromatographically separated from sub- strate IMP in solvent Nucleoside phosphorylases B. (base to nucleoside) (ix) Adenylate deaminases were assayed. AMP deaminase ADE 7.12 ± 3.54 4 (AMP aminohydrolase; EC 3.5.4.6) and ADO deaminase GUA 13.15 ± 4.13 3 (ADO aminohydrolase; EC 3.5.4.4) were assayed by a HX 7.56 ± 1.89 4 modification of the technique of Bagnara and Hershfield (1). X 0.034 ± 0.007 3 ADE deaminase (ADE aminohydrolase; EC 3.5.4.2) was assayed by a modification of the method of Canale-Parola Nucleoside phosphorylases and Kidder (5). All reactions were as described for the 5'- (nucleoside to base) nucleotidase, except that [8-14C]ADO and [8-14C]ADE were ADO 1.97 ± 0.62 8 substituted for the purine 5'-nucleotide. Product IMP GUO 1.30 ± 0.27 5 was INO 1.30 ± 0.49 5 chromatographically separated from substrate AMP in sol- XO 0.009 ± 0.003 3 vent B. Products INO and HX were similarly separated from ADO and ADE in solvent A. 5'-Nucleotidases (x) De novo purine synthesis in growing cells was assayed AMP 12.86 ± 2.76 5 by measuring the incorporation of [U-14C]glycine radioactiv- GMP 0.15 ± 0.02 5 ity added to the tryptose growth medium (0.5 ,uCi ml- ) into IMP 1.82 ± 0.12 5 cellular acid-precipitable purine bases as described by Mar- XMP 0.003 ± 0.001 3 tin and Owen (21). Adenylosuccinate synthetase 0.524 ± 0.184 7 RESULTS and adenylosuccinate lyase Utilization of purine bases and nucleosides. The activities of the purine enzymes are reported as the rate of product GMP reductase 0.643 ± 0.301 3 formation (in nanomoles per minute milligram of protein) (Table 1). A. Iaidlawii B-PG9 is capable of converting all the GMP synthetase NAb (<0.001)' 4 purine bases to their corresponding ribonucleosides via the phosphorylase and to the corresponding ribonucleotides via IMP dehydrogenase NA (<0.001)d 5 the phosphoribosyltransferases. Purine ribonucleosides are converted to the corresponding bases via nucleoside Adenylate deaminases phos- AMP NA (<0.005)" 3 phorylase activity. ADO NA (<0.001) 3 In A. laidlawii B-PG9, no ATP-dependent ADO kinase or ADE NA (<0.001) 3 nucleoside kinase activity was detected. To determine whether the lack of ATP-dependent ADO kinase and nucleo- an, Number of different batches of cells. bNA, No activity detected (minimum amount detectable). side kinase activities was due to contaminating membrane ' GMP synthetase activity in E. coli, 7.42 ± 0.04. ATPase activity, dialyzed cell extract was examined for d IMP dehydrogenase activity in E. ccoli, 1.63 + 0.60. ATPase activity. Under conditions as described for the eAMP deaminase activity in B. subtilis 0.48 + 0.12. assay for ATP-dependent ADO kinase activity, 90 to 91% of [8-14C]ATP was still present in the reaction mixture after 6 in the coupled assay of adenylosuccinate synthetase and min of incubation, and about 85% of the radiolabeled ATP adenylosuccinate lyase activities (Table 1). No AMP was remained after 30 min. This indicated that our inability to detected when ATP or inosine triphosphate were substituted detect ATP-dependent ADO kinase or nucleoside kinase for guanosine triphosphate or when glutamine, arginine, activities was not due to the absence of ATP in the reaction glutamate, or ammonium sulfate was substituted for aspar- mixtures. ate. GMP reductase activity was detected. when NADPH, However, in A. laidlawii B-PG9, ADO, INO, and GUO, but not NADH, was used. but not XO, were converted to the ribonucleotides via a PPi- No GMP synthetase activity was detected in A. Iaidlawii dependent ADO kinase or nucleoside kinase activity (Table B-PG9. No activity was detected when sodium PP, was 2). We do not know whether one or more enzymes are substituted for ATP or when aspartate or ammonium sulfate responsible for these activities. The rate of formation of was substituted for glutamine. GMP synthetase activity was AMP as a result of ADO kinase activity in crude prepara- detected in E. coli (Table 1) and in B. subtilis and human tions ofA. laidlawii B-PG9 was found to be dependent on the erythrocytes (data not shown). concentration of sodium PP1 (Fig. 1). In E. coli, as expected, No AMP deaminase, ADO deaminase, or ADE deaminase ATP but not PP1 was required for these purine nucleoside activity was detected in A. laidlawii B-PG9 AMP deaminase activities (Table 2). In B. subtilis, as expected, no ATP- or activity was detected in B. subtilis (Table 1). PP,-dependent purine nucleoside kinase activity was detect- No IMP dehydrogenase activity was detected in A. ed (data not shown) (9). laidlawii B-PG9. Activity was detected in E. coli (Table 1) Interconversion of nucleotides. IMP was converted to AMP and in B. subtilis and human erythrocytes (data not shown). 268 TRYON AND POLLACK J. BACTERIOL.

TABLE 2. Nucleoside kinase activities in A. laidlawii B-PG9 and E. coli ATCC 25922 Enzyme activity (,umol of product min-' mg-' of Organism Substrate protein ± SD) of the following phosphate donors PP, ATP A. laidlawii B-PG9 ADO 58.9 ± 21.3 NAb 7 GUO 50.8 ± 21.3 NA 4 INO 32.3 ± 8.68 NA 3 XO NA NA 3 E. coli ADO NA 0.401 ± 0.101 5 GUO NA 0.110 ± 0.011 3 INO NA 0.064 ± 0.030 3 XO ND" ND an, Number of different batches of cells. b NA, No activity detected (<0.001). ' ND, Not done.

In some experiments with AMP deaminase, IMP dehy- have shown in this work. This suggests that perhaps the r4te drogenase, and GMP synthetase, we added washed A. of interconversion is insufficient to fulfill the requirements laidlawii B-PG9 membranes (30) to the reaction mixtures (90 for adenylates in A. laidlawii B. However, any comparisons to 110 ,ug of protein) without effect. or opinions drawn from the rates of the enzyme activities De novo synthesis of purines. During growth, A. Iaidlawii reported here must be made provisionally and with great B-PG9 did not incorporate exogenous [U-t4C]glycine radio- caution since our assays were conducted with essentially activity into purine bases. crude cell extracts, and there may be competing reactions. We minimized competing reactions by extensive dialysis of DISCUSSION our cell-free preparations. Control reactions lacking added Except for M. mycoides (32), the lack of a defined medium substrates or cofactors had no activity. Perhaps our inability that supports adequate growth impairs the study of purine to detect some enzyme activities, for example XO kinase, metabolism of members of the class Mollicutes. In prelimi- was due to inadequate sensitivity, the use of reaction condi- nary experiments, we used a defined medium for A. Iaidlawii tions inappropriate for A. Iaidlawii B-PG9, or inhibition by B-PG9 similar to that of Rodwell's C-2 medium (32), as used some undetermined mechanism. by Mitchell and co-workers (27, 28) and formulations sug- The committed step of de novo purine synthesis is the gested by Razin and Cohen (31) and Greenaway and Wase formation of phosphoribosylamine (PRA). PRA is synthe- (11). However, we did not obtain sufficient growth for our sized from ribose-5-phosphate and PRPP, a reaction involv- enzyme assays in these media. ing PPi release and hydrolysis. Both ribose phosphate and Except for the PP1-dependent ADO kinase and nucleoside PRPP have been demonstrated in growing Mollicutes (6, 24). kinase, XMP nucleotidase, and XO phosphorylase activities PRA first reacts with glycine in the 10-step synthesis of the reported here, we found that A. laidlawii B-PG9 has similar enzymatic activities of purine salvage and interconversion as M. mycoides (27, 28). That is, both M. mycoides and A. 100o laidlawii B-PG9 are able to convert purine bases to their respective nucleoside and mononucleotide and guanylates to adenylates via GMP reductase, adenylosuccinate synthe- O 80- tase, and adenylosuccinate lyase (Fig. 2). Both organisms w SODIUM PYROPHOSPHATE are incapable of converting adenylates to guanylates. We did N not detect AMP, ADO, or ADE deaminases, IMP dehydro- wen- I genase, GMP synthetase, or XO kinase activities in A. 60- Iaidlawii B-PG9 (Fig. 2). The activities of these enzymes in z 4 M. mycoides have not been reported but are presumed to be absent based on labeling experiments performed by Mitchell and co-workers (27, 28). we conversion In some experiments, detected of the n nucleosides to their bases in the absence of added phos- a. - phate. This suggests that there may also be some phosphate- independent purine nucleosidase (N-ribosyl-purine ribohy- drolase; EC 3.2.2.1) activity as reported in Trichomonas 20 vaginalis and other protozoa (25). However, we cannot be certain of this because phosphate may be supplied from crude lysate, despite dialysis, and may be adequate to ATP support phosphorylase activity which appears as phosphate- 4-P pq independent hydrolase activity. 0.2 O.5 1.0 2.0 3.0 4.0 M. mycoides is capable of using GUA but not ADE as the mM sole source of purine nucleotides for growth (27, 28, 32). FIG. 1. Effect of various concentrations of sodium PP, or ATP However, A. Iaidlawii B is reported to require both ADE and on the amount of AMP synthesized in the standard reaction mixture GUA for growth (18, 35, 37), despite the presence of the (see text) for adenosine kinase activity by dialyzed lysates of A. enzyme activities necessary to convert GUA to ADE, as we laidlawii B-PG9. VOL. 159, 1984 PURINE METABOLISM IN A. LAIDLA WI! 269

PRA

GLY (

12 15

pp8V \.Ppi v P t N ppj, p t N ppj ADENOSINE 8 * I NOSINE XANTHOSINE GUANOSINE

PRPP F KR-I-P PRPP K-I-R P PRPP X KR-I-P PRPP t KR-I-P

ADENINE 9 * HYPOXANTHINE XANTHINE GUANINE FIG. 2. Proposed salvage pathways for the of purine nucleotides in A. Iaidlaiwii B-PG9. Enzyme activities were found for the following reactions: 1, adenine phosphoribosyltransferase (EC 2.4.2.7); 2, hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8); 3, 5'-nucleotidase (EC 3.1.3.5); 4, PPi-dependent adenosine kinase; 5, PPi-dependent nucleoside kinase; 6 and 7. purine nucleoside phosphorylase (EC 24.2.1); 10, adenylosuccinate synthetase (EC 6.3.4.4); 11, adenylosuccinate lyase (EC 4.3.2.2.); 15, GMP reductase (EC 1.6.6.3). 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, phosphoribosylgly- cinamide synthetase (EC 6.3.3.3).

purine IMP. Our inability to detect incorporation of members of the class Mollicutes (7, 26, 36, 38). O'Brien et al. [U-14C]glycine into purine bases of growing A. laidlawii B- have detected inorganic pyrophosphatase activity in a num- PG9 suggests that the block is in the synthesis of PRA or its ber of Mycoplasma and A(holeplasma species, except A. conversion to IMP. These findings strengthen the view that laidlaivii B, A. laidlawt,ii A, and Acholeplasma axanthilm A. laidlawii and other Molliclites cannot synthesize purines (29). In the absence of inorganic pyrophosphatase, we de novo, an opinion which is also supported by their speculate that more PP, is available for other PPi-dependent requirement for nucleic acid precursors when growing in enzyme activities. defined media (18, 32, 37). Purification and biochemical characterization of the PPi- This is the first report of a PPi-dependent ADO kinase or dependent nucleoside kinase is being conducted. The dis- nucleoside kinase activity in any procaryote or eucaryote, covery of the PPi-dependent nucleoside kinase may have and the first report of any ADO kinase or nucleoside kinase some utility. The property may be useful in studying the activity in the Mollicutes. Five enzymes in procaryotes and phylogenetic relatedness of the Mollicuites within their class eucaryotes are known to utilize PP, as a phosphate donor; and to other procaryotes. If the need for PP, is common to they are carboxytransphosphorylase, pyruvate-phosphate other Molliclutes, this requirement and its associated metab- dikinase, PPi acetylkinase, PP, serine kinase, and PP, phos- olism may act as a marker to detect or serve as the locus to phofructokinase (39). We have recently reported the utiliza- inhibit mycoplasmal infection or pathology. tion of PP1 as a phosphate donor for phosphofructokinase in A. laidlawii B-PG9 (J. D. Pollack and M. V. Williams, Abstr. ACKNOWLEDGMENTS Annu. Meet. Am. Soc. Microbiol. 1984, G20, p. 172). PPi- This work was supported in part by a grant from The Upjohn Co., dependent carboxytransphosphorylase activity in A. Kalamazoo, Mich. laidlawii B-PG9 has been observed (K. D. Beaman and J. D. We thank C. Hieronymous, L. Lemmer, and A. Ottolenghi for Pollack, unpublished data). their help. It will be interesting to know whether these PPi-dependent enzymes are functional in the intact cell. A number of LITERATURE CITED biosynthetic reactions involve the formation of PPi, and it is generally believed that PP, is a metabolic end product that by 1. Bagnara, A. S., and M. S. Hershfield. 1982. Mechanism of hydrolysis thermodynamically drives coupled in deoxyadenosine-induced catabolism of adenine ribonucleotides reactions in adenosine deaminase-inhibited human T-lymphoblastoid the anabolic direction (15). Wood et al. have suggested that cells. Proc. Natl. Acad. Sci. U.S.A. 79:2673-2677. the ability of Entamoeba histolytica, Propionibacteriurm 2. Beaman, K. D., and J. D. Pollack. 1981. Adenylate energy shermanii, and Bacillus symbiosus to utilize PPi may confer charge in Acholeplasina laidlavvii. J. Bacteriol. 146:1055-1058. a degree of selective advantage and may partly account for 3. Beaman, K. D., and J. D. Pollack. 1983. Synthesis of adenylate the high efficiency of growth of these organisms (40). The nucleotides by Mollicutes (Mycoplasmas). J. Gen. Microbiol. presence of deoxyuridine triphosphatase activity in A. 129:3103-3110. laidlawii B-PG9 has been reported (M. V. Williams and J. D. 4. Bradford, M. M. 1976. A rapid and sensitive method for the Pollack, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, G quantitation of microgram quantities of protein utilizing the 19, p. 172), and in this we the of principle of protein-dye binding. Anal. Biochem. 72:248-254. study reported presence 5. Canale-Parola, E., and G. W. Kidder. 1982. Enzymatic activities ADE, HX, X, and guanine phosphoribosyltransferase activi- for interconversion of purines in spirochetes. J. Bacteriol. ties. In addition to the mononucleotide, the product of each 152:1105-1110. of these latter five enzyme activities is PP1. Other enzyme 6. Castrejon-Diez, J., T. N. Fisher, and E. Fisher, Jr. 1963. Glucose activities known to produce PPi, e.g., during the formation metabolism of two strains of Mycoplasina laidlawtiii. J. Bacte- of nucleic acids by polymerases, have been reported in riol. 86:627-636. 270 TRYON AND POLLACK J. BACTERIOL.

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