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 Purine Metabolism 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, xanthine, and hypoxanthine 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, xanthosine 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 nucleotide 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.