Uptake and Utilization of S-Adenosyl-L-Methionine and S-Adenosyl-L-Homocysteine in an Adenine Mutant of Saccharomyces cerevisiae

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1 JOURNAL OF BACTERIOLOGY, May 1969, p Copyright 1969 American Society for Microbiology Vol. 98, No. 2 Printed in U.S.A. Uptake and Utilization of S-Adenosyl-L-Methionine and S-Adenosyl-L-Homocysteine in an Adenine Mutant of Saccharomyces cerevisiae RICHARD C. KNUDSEN, KEITH MOORE, AND IRVING YALL Department of Microbiology and Medical Technology, The University of Arizona, Tucson, Arizona Received for publication 2 January 1968 An adenine mutant of Saccharomyces cerevisiae as able to utilize S-adenosyl- L-methionine (SAM), S-adenosyl-L-homocysteine (SAH), or adenine as sources for groth and ribonucleic acid (RNA) synthesis. Exogenous SAM or SAH as degraded after entering the cell, and the adenine moiety as reutilized in the endogenous synthesis of the thionium compounds. Part of this endogenous synthesis proceeds from an undetermined de novo system of purine synthesis hich contributes significantly to the synthesis of SAM and less so to the RNA purines. Some of the methyl groups of exogenous SAM-methyl-3H ere incorporated into ribosomal and transfer RNA. Methionine partially antagonized this incorporation. The uptake of SAM-adenine-8-'C as not affected by the presence of equal quantities of SAH or adenine in the medium. Exogenous SAM affected the uptake of exogenous SAH-adenine-8-'C or adenine-8-'c. Exogenous SAH inhibited the uptake of exogenous adenine-8-1c. S-adenosyl-L-methionine (SAM) is knon to be accumulated in large amounts in cells hen yeasts are gron in high concentrations of methionine (7, 12). Hoever, comparatively little is knon of the fate of exogenously supplied SAM in yeast. Svihla and Schlenk (1) reported that yeast, hen gron in high concentrations of SAM, could accumulate SAM in the vacuole, although not to the same extent as in the presence of methionine. Yall et al. (13) reported that an adenine. mutant of Saccharomyces cerevisiae could a purine source for groth (13). It as thought that these particular characteristics ould provide unique insight in the determination of the intracellular fate of SAM and SAH. MATERIALS AND METHODS Microorganism. The microorganism used for this study as an adenine mutant of S. cerevisiae (SC ); the groth characteristics and purine requirements of this mutant have been previously described (12, 13). use SAM as a purine source. Experimental groth. The groth medium used as Considerably more is knon about the intracellular fate of exogenously supplied S-adenosyl- perimental conditions and preparation of inocula have the synthetic medium detailed by Roman (6). Ex- L-homocysteine (SAH). Duerre demonstrated been described elsehere (13), except that the inocula that SAH is degraded upon entering the S. ere gron in the presence of 6,moles of adenine per cerevisiae cell (). flask in an L-methionine-free medium. The cells ere There ere to objectives in the present study: harvested by centrifugation of an exponentially groing culture and ere resuspended to a concentration (i) to determine the intracellular fate and relationship of the purine portion of SAM and SAH containing no purine or methionine supplement. A of 7 mg (dry eight) per ml in a synthetic medium in yeast; (ii) to determine the effect exogenously 1-ml amount of cell suspension as transferred to each supplied SAM and SAH ould have on their test flask. Test flasks consisted of 1 ml of synthetic endogenous synthesis. medium (including supplements) in 5-ml Erlenmeyer flasks, and ere usually incubated for either 18, The microorganism selected for this study as an adenine mutant of S. cerevisiae hich had 2, or 8 hr ith shaking at 25 C. Uptake of radioactive compounds as determined by assaying the previously been shon to accumulate SAM in medium. Turbidity as measured at 5 nm on a large quantities hen gron in high concentrations of methionine (12), and hich had also eight as calculated from turbidity measurements Bausch & Lomb Spectronic-2 colorimeter. Dry been found to use exogenously supplied SAM as by means of previously prepared standard curves. 629 Donloaded from on April 26, 218 by guest

2 63 KNUDSEN, MOORE, AND YALL J. BACTERIOL. Isolation of SAM, SAH, and ribonucleic acid (RNA) adenine. After the appropriate period of experimental groth, the cells ere harvested at 25 C by centrifugation at 3, X g and ere ashed tice ith ice-cold medium containing no purine supplements. The cells ere extracted ith 3 volumes of 1.5 N perchloric acid (PCA) at 25 C for 1 hr ith agitation. The cell residue as removed by centrifugation at 5, X g for 1 min and as ashed tice ith 2 volumes of 1.5 N PCA. The PCA extract as neutralized to a ph of 6 to 7 in an ice bath ith solid KHCO3. The KCO precipitate as removed by centrifugation in the cold at 5, X g for 15 min. S-adenosyl-L-methionine as isolated from the neutralized PCA extract on a column of Doex-5 Na+ by the procedure of Shapiro and Ehninger (9). S-adenosyl-L-homocysteine as isolated from the.1 M NaCl eluate from the Doex-S Na+ column by acidifying the eluate to.5 N HCI and adding this to a Doex-S H+ column (9). RNA, nucleosides, and nucleotides ere eluted ith.5 to 1 N HCI. Guanine and some adenine ere eluted ith 3 N HCI, and SAH as eluted ith 6 N HCI. The RNA fractions ere combined and hydrolyzed by placement in an oven for 2 to 3 hr at 8 C, cooled to room temperature, and added to a Doex-5 H+ column. The column as ashed ith 1 N HCI until the absorbancy at 26 nm as less than.2. Guanine as eluted ith 2 N HCI, and adenine as eluted ith 3 N HCI. HCI fractions ere collected on a fraction collector equipped ith an LKB Uvicord absorption meter and recording device. Specific activities ere calculated from the HCI eluates. The specific activities of SAM and SAH ere checked by pooling the eluates from several flasks and precipitating and purifying them by a procedure ith phosphotungstic acid (8). Absorbancy as measured on a Beckman DU spectrophotometer. For SAM and SAH in 6 N HCI eluates, an em = 1,7 at 26 nm as used; for adenine in 3 N HCI eluates, an em = 13,1 at 263 nm as used. Absorbancy readings beteen.6 and.1 per ml ere used for calculation of specific activities. RNA studies. Cells ere disrupted by shaking ith a Mickle disintegrator for 1 min at 3 C, and the RNA as extracted by the method of Moore (Ph.D. Thesis, Univ. of Arizona, Tucson, 1969). Sedimentation analysis of the RNA as accomplished by centrifuging at 13, X g for 5 hr at 3 C in a 5 to 25% linear sucrose gradient, in an SW-39 rotor of the Spinco model L preparative ultracentrifuge. To-drop fractions ere collected from the bottom of the tubes, and 2.5 ml of ater as added to each fraction. After ultraviolet analysis,.5-ml samples ere removed from each fraction for scintillation counting. Chemicals. Adenine-8-1C, adenine-2-8-3h, "IC-sodium formate, and SAM (iodide) ere obtained from Calbiochem, Los Angeles, Calif. S-adenosyl-L-homocysteine-adenine-8-1C (SAH-Ad-8-1C) as prepared by the procedure of Duerre (2), hereas SAM and S-adenosyl-L-methionine-adenine-8-1C (SAM-Ad-8-1C) ere prepared by the procedure of Schlenk and Depalma (8). S-adenosyl-L-methionine-methyl-3H (SAM-Me3H) as obtained from Ne England Nuclear Corp., Boston, Mass. Radioactive assays. The technique used for radioactive measurements has been described (13). In cases of exceptionally lo counts, 1 to 2 ml of the 6 N HCl eluates as placed in scintillation vials, evaporated to dryness over NaOH in vacuo at 37 C, and reconstituted in.2 ml of distilled ater to hich scintillation fluid had been added. RESULTS Figure 1 shos the groth measured turbidimetrically, and the uptake of SAM-Ad-8-_C, SAH-Ad-8_1C, and adenine-8-'c. Also included is a control containing no purine supplement. In the presence of any of the three purine supplements, good groth occurred. SAH gave the poorest groth of the three. In the absence of any purine supplement, there as only a slight amount of groth. This groth may be attributed to the supply of purine in the purine pool, carried over from the flasks in hich the inocula ere gron. Figure 2 shos the competitive effect of a z I) Ui. I~z 'a o Adenine-8-"C No Supplement io.5 5 io o A - SAM-adenin -8'*C,e, } / - /P 7~~~~~ SAH-odenine-8-'C r. a I TIME (HOURS) FIG. 1. Groth and per cent uptake of radioactive ribosides or adenine by the mutant yeast cells. (A) SAM-adenine-8-1C; (B) SAH-adenine-8-1C; (C) adenine-8-1c; (D) no purine supplement. Each purine supplement as at a concentration of jsmoles per 1 ml of medium. Cells ere gron for 3 hr ith shaking at 25 C. Samples ere removed at various time intervals, and the optical density (OD) as observed at 5 nm ith a Bausch & Lomb Spectronic-2 colorimeter before assaying for radioactivity. Symbols: *, radioactive supplement;, turbidity. Ordinate scale is linear in OD In z Donloaded from on April 26, 218 by guest

3 VOL. 98, 1969 ADENINE MUTANT OF S. CEREVISEAE 631 z a cr Co I Co U. a. z. E to I- IU) z -J a.) ~ Donloaded from is TIME (HOURS) FiG. 2. Groth and per cent uptake of radioactive ribosides or adenine in the presence of an unlabeled supplement by the mutant yeast cells. (A) SAM-adenine-8-1C plus SAH; (B) SAM-adenine-8-1C plus adenine; (C) SAH-adenine-8-lC plus adenine; (D) SAH-adenine-8-1C plus SAM; (E) adenine-8-1c plus SAM; (F) adenine- 8-1C plus SAH. For concentrations of the purine supplements and groth conditions, see Fig. 1. Symbols:, radioactive supplement;, turbidity. Ordinate scale is linear in optical density. second purine supplement on the uptake of the radioactive supplements used in the experiments shon in Fig. 1. In the presence of adenine or SAH, SAM gave much better groth than ith SAH plus adenine. SAM appeared to inhibit the initial uptake of labeled adenine or SAH. Unlabeled SAH or adenine did not cause a reciprocal inhibition on the uptake of labeled SAM. The rate of uptake of adenine-8-'c in the presence of SAH shoed the most marked inhibition. Exogenously supplied SAM can serve as a methyl donor to RNA (Fig. 3). The organisms ere exposed to SAM-Me-3H in the presence of various supplements, and the RNA as extracted and analyzed by gradient centrifugation. Proportional amounts of RNA, to correspond ith the yield under each condition, ere placed on the gradients. When the cells ere gron ith SAM-Me-3H as sole supplement, radioactivity as found in the peaks corresponding to ribosomal and transfer RNA (Fig. 3A). When adenine and the radioactive thionium compound ere used, the amount of RNA more than doubled as indicated by absorption at 26 nm. More radioactivity as incorporated into the peaks, but the specific activity as loered (Fig. 3B). When 6,umoles of L-methionine as added to 1 ml of medium containing adenine and SAM-Me-3H, ribosomal and transfer RNA of loer total and specific activity resulted (Fig. 3C). When,umoles of L-methionine as used in addition to the purine supplements, the synthesis and the on April 26, 218 by guest

4 632 KNUDSEN, MOORE, AND YALL J. BACTERIOL. OD26 6 OS I. D26 L -GD26 CPM A CPM : o CPM '12 2 8~~~~~~~~~~~~~~~~~..1 6 *.-.~ S C D FIG. 3. Effect of adenine and L-methionine on the uptake of radioactivity from SAM-Me-3H into RNA extracted from cells of an adenine-requiring yeast strain. (A) SAM-Me-'H; (B) SAM-Me-3H plus adenine; (C) SAM-Me-3H plus adenine plus 6,umoles of L-methionine; (D) SAM-Me-3H plus adenine plus,umoles of L-methionine. Cells ere gron for 8 hr ith shaking at 25 C ith 2 ganoles of radioactive SAM, 2,moles of adenine, and the requisite amounts of L-methionine hen required. RNA as extracted from cells and analyzed by means of sucrose gradient centrifugation. Abscissa numbers represent fractions. Peaks at approximately 18 and 2 represent ribosomal RNA; peak at approximately 3 represents transfer RNA. Radioactivity introduced 1,8, counts/min. incorporation of methyl groups from SAM into RNA ere inhibited (Fig. 3D). This amount of the amino acid stimulates endogenous SAM synthesis (12), but loers the cell yield (13) and total RNA. Tlhe intracellular concentrations of SAM and SAH in the mutant, hen gron in the presence of exogenous purines, are shon in Table 1. The per cent uptake of the purine supplements (as shon in Fig. 1 and 2) indicates that 5 to 6% of the purine supplement as taken up into the cells at the time of harvesting. These results sho that, although large quantities of SAM, SAH, adenine, or a combination of these ere taken up by the cells, only relatively small quantities of SAM and SAH ere reisolated. None of the exogenous purine supplements appreciably altered the intracellular concentrations of SAM, although 8 jumoles of purine supplement combination caused an increase in the concentration of SAH. L-Homocysteine, hoever, caused a marked increase in the concentration of SAM and a slight increase in that of SAH. The specific activities of the reisolated SAM, SAH, and RNA adenine, in the presence of exogenously supplied SAM-Ad-8-'C, SAMmethyl-3H, and SAH-Ad-8-1C, are shon in Table 2. The specific activity of the RNA adenine as about 1% loer than that of the exogenous purine supplement, a dilution that ould be expected if these compounds ere serving as sole purine supplements in this adenine mutant. But the specific activities of the reisolated SAM and SAH shoed a marked reduction, demonstrating that some unknon source of purine is contributing to the synthesis of these compounds in this supposedly adenineless mutant. This reduction in specific activity as most notable in the reisolated SAM in both cases, the specific activity of SAM having been reduced to less than 5% of the specific activity of SAH. The exogenous supply of SAM-adenine-8-1C and SAH-adenine-8-'C (Fig. 1) should be sufficient to repress endogenous synthesis of SAM and SAH, but this as clearly not the case. The reduced specific activities of the endogenous compounds demonstrate that the exogenous compounds do not inhibit endogenous synthesis. TABLE 1. Effect of exogenous purine sources or homocysteine on the formation of SAM and SAH by an adenine-requiring yeast strain Supplementa Cell yield Yield (jumoles) Yieldb (m, eight) SAM SAH SAM SAH SAH () SAM () SAH () + adenine () SAM () + adenine () D, L-Homocysteine (1) + adenine (8) Adenine (8) a Figures in parentheses represent micromoles per 1 ml of medium. Flasks ith umoles of purine supplement ere harvested at 18 hr, and those ith 8 psmoles of total ere harvested at 2 hr. Cells ere gron ith shaking at 25 C; inoculum, 7 mg (dry eight) per flask. b Expressed as micromoles per gram (dry eight) of cells. Donloaded from on April 26, 218 by guest

5 VOL. 98, 1969 ADENINE MUTANT OF S. CEREVISEAE 633 The specific activity of SAM-methyl-3H shoed a greater reduction in specific activity than the adenine-8-'c moiety of SAM. This presents the possibility that the adenine moiety of SAM-Ad- 8-1C (and also of SAH-Ad-8-1C) is derived from a recycling of adenine through the purine pool back into SAM and SAH via either adenosine triphosphate or adenosine. That this is the case is shon in Table 3, in hich SAH-Ad-8-1C and SAM-Ad-8-1C are the purine supplements ith unlabeled adenine and vice versa. The RNA adenine under these circumstances shoed a reduced specific activity of about 5%, indicating that the labeled purine as diluted, as expected, by the unlabeled purine. In each of the experiments, the specific activity of the reisolated SAM and SAH as much loer than that of the RNA adenine, more so ith TABLE 2. SAM than ith SAH. The reduced specific activity of these compounds can be attributed, once again, to the unknon purine source. Increasing the amount of exogenous purine from to 8,umoles by the addition of another purine supplement to the medium did not inhibit this endogenous source. The possibility that part of the endogenous supply of SAM and SAH is derived from a recycling of the adenine moiety of these compounds as confirmed in the experiments that shoed that exogenous adenine-2-8-3h in the presence of either SAM or SAH can contribute to the formation of endogenous SAM or SAH. The adenine moiety of exogenous SAM and SAH mixes ith exogenously supplied adenine in the purine pool, and the mixture is then used for SAM and SAH biosynthesis. Incorporation of radioactivity from exogenous purine sources into reisolated SAM, SAH, and RNA adenine from an adenine-requiring yeast straina Supplementa Specific activityb Per cent specific activityc SAM SAH RNA SAM SAH RNA adenine adenine SAH-adenine-8-1C... 5,3 12,2 6, SAM-adenine-8-1C... 1,9 29, 51, SAM-methyl-3H... 21, 19 a Cells ere gron ith shaking for 18 hr at 25 C; all purine supplements ere added at a level of approximately,moles per 1 ml of synthetic medium. Specific activity of exogenous SAH-adenine-8-1C as 51, counts per min per,amole, of SAM-adenine-8-1C as 56,8 counts per min per /smole, and of SAM-methyl-3H as 11, counts per min per,amole. b Specific activity refers to counts per min per micromole. - Per cent specific activity is the specific activity of the endogenous purine X 1, divided by the specific activity of the exogenous purine. TABLE 3. Incorporation of radioactivity from one or more exogenous purines into reisolated SAM, SAH, and RNA adenine by the yeast mutant cells Specific activityb Per cent specific activityc Supplementa ' SAM SAH adenine SAM SAH adeniane SAH-adenine-8-1C () + adenine ()... 1,9 9,3 2, SAM-adenine-8-1C () + adenine ()... 6,8 25, 28, SAH () + adenine-2-8-3h ()... 66, 285, 11, SAM () + adenine-2-8-3h ()... 12,5 297, 57, Adenine-2-8-3H (8) , 5, 75, a Figures in parentheses represent micromoles per 1 ml of medium. Cells ere gron ith shaking at 25 C for 2 hr. Specific activity of SAH-adenine-8-1C as 51, counts per min per ;mole, of SAMadenine-8-'C as 53, counts per min per jumole, and of adenine-2-8-3h as 873, counts per min per umole. b Same as Table 2. c Same as Table 2. Donloaded from on April 26, 218 by guest

6 63 KNUDSEN, MOORE, AND YALL J. BAcrERIOL. TABLE. Dilution of specific activities of SAM, SAH, and RNA adenine from inocula of an adenine-requiring yeast strain gron in adenine-2-8-3h Supplementa Specific activityb Per cent specific activityc SAM SAH RNA adenine SAM SAH adenine Inoculumd Adenine-2-8-IH (6)... 29,1 719,6 97,2 Test flasks* SAH () + adenine ()... 17,6 76, 53, SAM () + adenine ()..., 3,8 5, Adenine (8)... 6,2 68,1 52, a Figures in parentheses represents micromoles of supplement per 1 ml of Roman's medium. bspecific activity is expressed as counts per min per micromole. c Per cent specific activity is the specific activity of the purine isolated from the cells in the test flasks, divided by the specific activity of the same purine extracted from inocula cells. d Inocula ere gron for 18 hr ith shaking at 25 C. Specific activity of adenine-2-8-3h as 1,66, counts per min per pmole. e Test flasks ere gron for 2 hr ith shaking at 25 C; inoculum, 7 mg (dry eight) per flask. When adenine-2-8-3h as the sole purine supplement, labeled SAM and SAH ere reisolated. The specific activities of these compounds ere greatly reduced hen compared to the specific activity of the RNA adenine. This demonstrates that the synthesis of SAM and SAH via the unknon purine source is not a special effect induced by exogenous SAM or SAH. It is possible that the marked reduction in specific activity of SAM and SAH (Tables 2 and 3) could come from one of to sources. At the time of inoculation, there could be in the cells a large supply of SAM hich can be stored in the vacuoles of yeast in high concentrations (1), and hich does not turn over or turns over at a negligible rate during cell groth. The other possibility is that this dilution could come from a recycling of RNA purine present in the cells at the time of inoculation. To test these hypotheses, the cells ere gron in adenine-2-8-3h. The cells ere harvested, and a sample as taken to measure specific activity and concentration of both SAM and SAH. Equal portions of cells ere used to inoculate test flasks containing unlabeled purine supplements (Table ). The inoculum gron in the presence of adenine-2-8-3h shoed a reduction in specific activity of SAM, and a lesser reduction in specific activity of SAH hen compared to the specific activity of the RNA adenine. When an identical experiment as performed ith adenine-8-1c, similar results ere obtained, demonstrating that the reduction in specific activity as not an artifact of the tritium labeling. The purine-containing compounds isolated from the cells taken from the test flasks after 2 hr of groth shoed considerable reductions in specific activities. The residual compounds from the inocula, as indicated by the determinations shon in Table, played a minor role in the dilutions of SAM and SAH but could be more significant in the case of RNA adenine. The ability of the mutant strain to incorporate radioactivity from 'C-formate into purine compounds as investigated. Table 5 shos the specific activities of SAM and RNA adenine extracted from cells gron in the presence of adenine and radioactive formate for 8 hr. Radioactivity as found in adenine from RNA hydrolysis, guanine (not shon), and SAM. The SAM had more than eight times the specific activity found in adenine. The SAM resulting from this experiment as combined ith 1,umoles of commercial SAM iodide. The mixture as precipitated ith phosphotungstic acid, and the SAM as reisolated by means of paper chromatography. The radioactivity continued to be associated ith the thionium compound, hich TABLE 5. Incorporation of 1C-formate radioactivity into SAM and RNA adenine by an adenine-requiring yeast straina Compound isolated (counts Specific per min activity per pmole) SAM RNA adenine ,67 19,1 a Cells ere gron ith shaking for 8 hr at 25 C in the presence of adenine (,moles/1 ml of synthetic medium) and 1C-formate (3,3, counts per min per flask). Donloaded from on April 26, 218 by guest

7 VOL. 98, 1969 ADENINE MUTANT OF S. CEREVISEAE 635 had the same specific activity as before the precipitation procedure. The SAM as hydrolyzed ith dilute alkali to give adenine and S-ribosylmethionine (5). All of the radioactivity in the molecule as associated ith the adenine moiety. DISCUSSION Exogenous SAM, SAH, or adenine can serve as groth factors for the adenine mutant of S. cerevisiae used in this study. Adenine-8-tC is taken up most readily and is stored before usage (Fig. 1). S-adenosyl-L-methionine is taken up at a steady rate and is used as a source of groth material. A problem of permeability, perhaps requiring the synthesis of adaptive enzyme(s), seems to exist for SAH. A previous report (13) indicated that cells of this strain ere unable to utilize SAH for groth in the absence of adenine hen the inoculum as in the stationary phase (8 hr). The SAH as utilized as a sole source of purine hen an inoculum in the logarithmic phase (18 hr) as used, but only after a 9-hr delay. The rate of uptake of SAM-Ad-8-1C is essentially unaffected by the presence of exogenous SAH or adenine (Fig. 2). Hoever, the presence of exogenous SAM inhibits the uptake of SAH- Ad-8-1C or adenine-8-'c until 12 hr after inoculation, at hich time approximately 28% of the SAM has been taken into the cells (Fig. 1). Exogenous adenine does not alter the uptake of SAH-Ad-8-_C, but the thionium compound does affect the uptake of adenine-8-1c. The effects of SAM or SAH on adenine utilization can be explained by postulating common entry sites on the cell surface for the three purine compounds. The utilization of adenine is controlled by the rates of utilization of SAM or SAH. SAM is readily taken up by the cells and used as a purine source at a steady rate. It does not seem to accumulate and thus does not serve as a pool barrier to adenine. SAH is taken up and used more sloly, as is indicated by the cell groth rate in Fig. 1. This compound may fill an intracellular pool hich ould result in an inhibition of adenine uptake. Cummins and Mitchison (1) reported that prefilling ith a variety of purine bases and ribonucleosides resulted in an inhibition of adenine uptake by Schizosaccharomyces pombe. The ork reported in this paper and elsehere (13) strongly suggests that SAM is taken up by the cells and rapidly demethylated to SAH. Some of the methyl groups are used by the cell for the methylation of ribosomal and transfer RNA (Fig. 3) and the biosynthesis of ne SAM (Table 2). The purine products resulting from the breakdon of SAH are still questionable since, in experiments done in this laboratory, both adenine and adenosine labeled ith 1C have been found to be produced from SAM-Ad-81C. A crude extract from this organism has been found to split SAH to adenosine. Duerre's recent ork on SAH utilization in yeast () indicated that exogenously supplied SAH as degraded to homocysteine and adenosine upon entering the cell. It has been shon previously ith this mutant (12) that adenosine cannot serve as a purine source for groth, although limited amounts of adenosine- 8-1C could be incorporated into cellular SAM. This does not preclude the possibility that the sites of enzyme utilization of adenosine for groth may be impermeable to the exogenous riboside. A hydrolytic nucleosidase that cleaves SAH to ribosylhomocysteine and adenine has been demonstrated in bacteria (3). The organism used in this study requires the addition of a purine-containing compound for groth. The specific activity data given in this paper indicate that exogenous SAM, SAH, or adenine can serve as precursors for RNA purine and for each other. In addition, an endogenous purine synthesizing system exists that seems to favor SAM but hich can also contribute to RNA purine (Table 5). Table 2 shos that a substantial reduction in specific activity occurs in reisolated SAM or SAH in 18 hr hen exogenous SAM-Ad-8-1C or SAH-Ad-8-1C are supplied. Table shos that SAM and SAH carried over in the inocula are negligible factors in this dilution by 2 hr. Takahashi and his co-orkers (11) reported that an adenine-requiring mutant of S. cerevisiae does not gro in adenine-free medium. Hoever, the addition of a relatively small amount of adenine results in the accumulation of purine in the cell of about 1 times as much as that originally supplied. The precise mechanisms that trigger adenine biosynthesis in these organisms remain to be ascertained. ACKNOWLEDGMENT This investigation as supported by grants GB-1838 and GB- 516 from the National Science Foundation. LITERATURE CITED 1. Cummins, J. E., and J. M. Mitchison Adenine uptake and pool formation in the fission yeast Schizzosaccharomyces pombe. Biochem. Biophys. Acta 136: Duerre, J. A Preparation and properties of S-adenosyl- L-homocysteine, S-adenosyl-L-homocysteine sulfoxide and S-ribosyl-L-homocysteine. Arch. Biochem. Biophys. 96: Duerre, J. A A hydrolytic nucleosidase acting on S- adenosylhomocysteine and on 5'-methylthioadenosine. J. Biol. Chem. 237: Duerre, J. A In vivo and in vitro metabolism of S- adenosyl-l-homocysteine by Saccharomyces cereviseae. Arch. Biochem. Biophys. 12: Parks, L. W., and F. Schlenk The stability and hydrol- Donloaded from on April 26, 218 by guest

8 636 KNUDSEN, MOORE, AND YALL J. BACTERIOL. ysis of S-adenosylmethionine, isolation of S-ribosylmethionine. J. Biol. Chem. 23: Roman, H. L A system selective for mutations affecting the synthesis ofadenine in yeast. C. R. Trav. Lab. Carlsberg 26: Schlenk, F., and R. E. Depalma The formation of S-adenosylmethionine in yeast. J. Biol. Chem. 229: Schienk, F., and R. E. Depalma The preparation of S- adenosylmethionine. J. Biol. Chem. 229: F. Shapiro, S. K., and D. J. Ehninger Methods for the preparation and analysis of adenosylmethionine and adenosylhomocysteine. Anal. Biochem. 15: Svihla G., and F. Schlenk S-Adenosylmethionine in the vacuole of Candida utilis. J. Bacteriol. 79: Takahashi, T., F. Yoshitsugu, and H. Takahashi Inhibition of yeast groth by methionine. Part I. Nature of the inhibition. Agr. Biol. Chem. 31: Yall, I Biosynthesis of S-adenosylmethionine by Saccharomyces cerevisiae. L. Adenine and methionine requirements. J. Bacteriol. 83: Yall, I., S. A. Norrell, R. Joseph, and R. C. Knudsen Effect of L-methionine and S-adenosylmethionine on groth of an adenine mutant of Saccharomyces cerevislae. J. Bacteriol. 93: Donloaded from on April 26, 218 by guest