The Role of Biotin-Dependent Pyruvate Carboxylase

Transcription

1 Agric. Biol. Chem., 43 (7), 1513 `1519, The Role of Biotin-Dependent Pyruvate Carboxylase in L-Lysine Production* Osamu TOSAKA, Hajimu MORIOKA and Koichi TAKINAMI Central Research Laboratories of Ajinomoto Co., Kawasaki, Japan Received January 30, 1979 The promotive effect of biotin ( ƒÊg/liter) on L-lysine formation was investigated in Brevibacterium lactofermentum. This effect was observed only when glucose or pyruvate was used as sole carbon source, and accompanied with the specific incorporation of 13CO2 int ƒá-ch2 group of L-lysine. Brev. lactofermentum AJ 3445 (AECr) could grow on pyruvate medium supplemented with biotin at more than 200 ƒêg/liter, while the same growth was observed with the addition of TCA cycle members or glutamate to pyruvate medium. Phosphoenolpyruvate (PEP) carboxylase deficient mutant derived from AJ 3445 could not grow on glucose as sole carbon source, but on glucose plus 200ƒÊg/liter of biotin. AJ 3445 grown on lactate medium containing 500ƒÊg/liter of biotin and KHCO3 contained the biotindependent pyruvate carboxylase. These data suggest that this promotive effect of excess biotin on L-lysine formation may be brought about through the activation of pyruvate carboxylase by biotin. In the previous paper,1) we found that L- lysine formation was stimulated considerably by addition of excess biotin ( ƒÊg/liter) into culture medium. Furthermore, L-lysine formation is considered to be due to the ac tivation of the direct carboxylation of pyruvate to oxaloacetate in the presence of excess biotin. The anaplerotic synthesis of oxaloacetate from three carbon precursors is usually ac complished in bacteria through the action of one of the two CO2-fixing enzymes, the biotindependent pyruvate carboxylase (EC ), which catalyses reaction I: Pyruvate+ATP+ HC _??_ Oxaloacetate+ADP+P (I) or the biotin-independent phosphoenolpyru vate carboxylase (EC ), which catalyses reaction II: Phosphoenolpyruvate+HC _??_ Oxaloacetate+P (II) Since both pyruvate and phosphoenol pyruvate (PEP) carboxylases are supported to fulfil the same function in cell metabolism,2) it was thought that they were not likely to be * Biosynthesis of L-Lysine and L-Threonine in Brevibacterium. Part VIII. simultaneously present in the same cell. However, both carboxylase are present in Pseudomonas citronellolis,3) Ps. fluorescens4) and Azotobacter vinelandii.5) In Brev. flavum, Shiio et al. have demonstrated that PEP carboxylase is present at significant levels in cell free extracts. On the other hand, pyru vate carboxylase has never been detected. However, the effect of excess biotin on L- lysine formation can not be explained by biotinindependent enzyme, PEP carboxylase. In the present paper, we demonstrated that extract of Brev. lactofermentum contained both pyruvate carboxylase and PEP carboxylase. Some of characteristics of the partially purified these enzymes are presented, and the relationship between biotin level and the incorporation of CO, into L-lysine are discussed. MATERIALS AND METHODS Microorganisms and culture conditions. Brevi bacterium lactoferinenturn AJ 3445 (AECr), AJ 3799 (AECr, alanine-) and Pseudomonas citronellolis ATCC were employed. Medium 1, medium 2 and medium 3 and culture conditions were the same as those described previously.6) For solid media, 2 agar was added. Lactate medium had the following composition: sodium lactate, 20g; ammonium sulfate,

2 1514 O. TOSAKA, H. MORIOKA and K. TAKINAMI 10g; KH2PO4, 1g; SO4 E7H2O, 0.4g; FeSO4 E7H2O, 2mg; MnSO4 E4 `6H2O, 2mg; biotin, 500ƒÊg; thiamine E HCl, 200ƒÊg; KHCOg, 1g; L-alanine, 400mg; nico tinamide, 5mg; adenine, 100mg; monosodium gluta mate, 100mg and distilled water to bring the total volume to I liter, adjusted to ph 7.5 with KOH. Preparation of cell free extracts. The cultures were harvested by centrifuging at 8,000g for 10min at 4 Ž. The cells were washed with 0.1M Tris-HC1 buffer, ph 7.5, containing 0.1M KCl, 5mM MnSO4, 0.5mM ATP, 0.1mM dithiothreitol, and 0.1mM EDTA. The cells were suspended in the same buffer and disrupted by ultrasonic oscillator. The homogenate was centri fuged at 20,000g for 30min at 4 Ž. The supernatant was applied to a Sephadex G-25 column which had been equibrated with the same buffer. A part of enzyme eluting with buffer was collected, (NH4)2SO4 was added to 80% saturation, and the solution was immediately centrifuged at 20,000g for 30min. The supernatant solution was discarded, and the precipitated protein was dissolved in the original buffer. Assay methods. Protein was determined by the method of Lowry et al.7) with bovine serum albumin as the standard. (1) Pyruvate carboxylase was assayed spectro photometrically by measurement of oxaloacetate pro duction with malate dehydrogenase (MDH)8) or citrate synthetase. (a) MDH coupled method. The reaction mixture (3ml) contained: Tris-HCl (ph 7.2), 200 ƒêmoles; MgCl2, 30 ƒêmoles; KHCO3, 40ƒÊmol; ATP, 4ƒÊmol; NADH, 0.6ƒÊmol; bovine serum albumin, 1mg; MDH, 7.5 units; sodium pyruvate, 4ƒÊmol and enzyme. After equilibration to 25 Ž the reaction was initiated by addition of pyruvate and the initial rate of NADH oxidation was measured at 366nm. One unit of pyruvate carboxylase activity catalyzes the oxidation of 1ƒÊmol of NADH perminute. (b) Citrate synthase coupled method. The assay mixture (final volume, 3ml) contained: Tris-HC1 buffer (ph 7.5), 200ƒÊmol; ATP, 4ƒÊmol; MgCl2, 54ƒÊmol: bovine serum albumin, 1.8mg; acetyl-coa, 0.25ƒÊmol; KHCO3, 40ƒÊmol; biotin, 10ƒÊg; 5,5 L-dithiobis (2- nitrobenzoic acid), 0.4mg; L-leucine, 10ƒÊmol: citrate synthase, 10ƒÊmol; sodium pyruvate, 4ƒÊmol and enzyme. A control mixture without enzyme was used as a blank. The reactions were started by addition of pyruvate and the increase of the optical density at 412nm was measured. duction with malate dehydrogenase. The assay system contained, in 3ml, 200ƒÊmol Tris-HCl buffer, ph 7.2, 11.4ƒÊmol PEP, 30ƒÊmol MgCl2, 40ƒÊmol KHCO3, 0.5ƒÊmol acetyl-coa, 0.6ƒÊmol NADH, 1mg bovin serum albumin, 7.5 units malate dehydrogenase and enzyme. After equilibration to 25 Ž PEP was added and the initial rate of NADH oxidation was measured at 366nm. The contribution of other enzymes to the rate of NADH oxidation may be estimated by omitting PEP from the assay system. (5) Oxaloacetate decarboxylase activity was measur ed by determining pyruvate and oxaloacetate with lac tate dehydrogenase and malate dehydrogenase, respec tively. The reaction mixture (1ml) contained: Tris- HCl buffer (ph 8.0), 100ƒÊmol; sodium oxaloacetate, 10ƒÊmol; NaCl, 20ƒÊmol ; and enzyme. Reactions were carried out at 30 Ž for 10min and terminated by addition of 0.2ml of 10% trichloroacetic acid. Samples of the supernatant fraction were assayed for the measurement of pyruvate and oxaloacetate. (6) Citrate synthase was assayed by the method of Srere.11 )(7) Isocitrate dehydrogenase activity was determined accordingly to the method of Daron et al.12 ) (8) Isocitrate lyase activity was measured by the method of Dixon et al.13) (9) Fumarase activity was determined by the method of Massey.14) (10) Malate dehydrogenase was assayed by the method of Ochoa.16) (11) Glutamate-oxaloacetate transaminase was assayed by measuring oxaloacetate production at 366 nm with an excess of malate dehydrogenase and NADH. Reaction mixture contained: (in micromoles) potassium phosphate buffer, ph 7.5, 100; L-aspartate, 50; NADH, 0.5; ƒ -ketoglutarate, 20; and also malate dehydrogenase, 7.5 units and enzyme in a total volume of 3ml. The reaction was started by the addition of 0.1ml of ƒ -ketoglutarate and the rate of decrease of absorbance at 366nm was recorded. (12) Aspartase activity was measured by deter mining the rate of aspartate formation from fumarate by coupling with glutamate-oxaloacetate transaminase and malate dehydrogenase. Concentrations in the reaction mixture (1.0ml) were as follows: Tris-HCl buffer (ph 8.5), 50ƒÊmol; ammonium fumarate (ph8.5), 50ƒÊmol; MnCl2, 10ƒÊmol and enzyme. Reaction was carried out at 35 Ž for 30min and stopped by boiling for 3min. Samples of the supernatant fraction were assayed for measurement of aspartate. (2) Pyruvate dehydrogenase complex activity 13was CO2 fixation assay. The reaction mixture (3ml) measured by the method of Reed et al.9) contained: sodium pyruvate, 2%; NaH13CO3 0.1 %; (3) Malic enzyme was assayed by the method of Zink et al.10) (4) PEP carboxylase activity was assayed spectro photometrically by measurement of oxaloacetate pro biotin, 500 tug/liter; KH2PO4, 0.1 %; MgSO4 E7142O, 0.04% and the washing cells, 1%. After 16hr of incubation at 30 Ž on a shaker, the reaction mixture was centrifuged at 55,000g for 10min. 13C-NMR

3 Biosynthesis of L-Lysine and L-Threonine in Brevibacterium 1515 spectra were measured using the XL-100A (Varian) spectrophotometer, working at 25.4 MHz. Chemicals. Nucleotides, pyruvate, oxaloacetate, isocitrate, acetyl-coa and most of enzymes were pur chased from Boehringer Manheim. Amino acid were the products of Ajinomoto Co., Inc. NaHr3CO3 and avidin were obtained from Merk Sharp and Dohne Canada and ICN pharmaceutical Inc., respectively. In Brev. lactofermentum, an absolute depen dence of growth exhibited upon the higher concentration of biotin (Fig. 2). On the other hand, strain AJ 3445 could grow on glucose RESULTS Effect of biotin on L-lysine production from pyruvate Preliminary experiment reveals that excess biotin plays a significant role for L-lysine for mation in Brev. lactofermentum. Figure 1 shows the time course of L-lysine production from pyruvate at different biotin concentrations. The rate and level of L- lysine formation was greatly increased upon addition of biotin. This result indicates that the factor responsible for the higher L-lysine production is tracted to a higher concentration of biotin present. FIG. 2. Growth of Brev. lactofermentum AJ 3445 on Pyruvate Medium Containing Various Levels of Biotin. A loopful of AJ 3445 cells grown on medium 1 agar slant for 2 days at 30 C was inoculated into 3ml of medium 2 containing pyruvate as sole carbon source, and incubated aerobically for 24hr., biotin alone; œ, biotin and 70mM of glutamate. at less than 20ƒÊg/liter of biotin. The same growth was obtained with the addition of TCA cycle members or glutamate to pyruvate medi um in the presence of 20ƒÊg/liter of biotin. Since higher biotin dependent pathway serves to supply the C4-dicarboxylic acids needed for the growth on pyruvate, its functioning would be unnecessary when intermediates of the tri carboxylic acid cycle are supplied in the growth medium. Requirements for growth of PEP carboxylase deficient mutant on glucose PEP carboxylase deficient mutant (ppc-2) FIG. 1. Time Course of L-Lysine Formation from Pyruvate by Washing Cells. AJ 3799 was cultured in medium 3 supplemented with 3ƒÊg/liter of biotin. Harvested cells were wished and suspended in reaction mixture containing 0.22M of pyruvate and the indicated concentration of biotin., biotin, 500ƒÊg/liter; ƒ, biotin, 0ƒÊg/liter. Requirements for growth on pyruvate as sole carbon source The relationship between biotin concentra tion and the growth on pyruvate was assayed. was derived from AJ 3445 using N-methyl-N Lnitro-N-nitrosoguanidine. The level of PEP carboxylase of ppc-2 was about 4% compared with the parental strain. The growth of ppc-2 was investigated in different media (Table I). Ppc-2 could not grow on glucose at less than 20ƒÊg/liter of biotin, while its growth proceeded normally at 200ƒÊg/liter of biotin. This biotin effect was replaced by addition of glutamate. These results suggest that the new anaplerotic pathway is present in addition to PEP carboxy-

4 TOSAKA, H. MORIOKA and K. TAKINAMI TABLE 1. GROWTH OF PEP CARBOXYLASE MUTANT ON GLUCOSE MEDIUM CONTAINING VARIOUS LEVELS OF BIOTIN A loopful of AJ 3445 or PPC-2 cells grown on medium 1 agar slant for 2 days at 30 C was inocu lated into 3ml of medium 2, and incubated aero bically for 24hr. With and without biotin (500ƒÊg/liter) in reaction mixture, the amount of L-lysine accumulated was 9.5 and 5.7mg/ml, respectively. The above growth experiments also excluded the possibility of PEP carboxylase activity in volved in this reaction. a 20ƒÊg per liter of biotin were added to basal medium. lase in Brev. lactofermentum. Incorporation of 13CO2 into L-lysine The previous finding that the stimulation of L-lysine formation by excess biotin was ac companied with the reduction of CO2 liberation has now been reinvestigated in more detailed with 13C-NMR. In Brev. lactofermentum, it is speculated that pyruvate is metabolized via oxaloacetate, and lysine is synthetized through the condensation of aspartate-ƒà-semialdehyde and pyruvate. Thus, carbon dioxide fixation into pyruvate is involved in the biosynthetic pathway to oxalo acetate and there is selective incorporation of CO, into r-ch, group of L-lysine (Fig. 3). FIG C-NMR Spectrum of L-Lysine Produced from Pyruvate by Washing Cells. Cultivation of Al 3799 and reaction were the same as those described in Fig. 1, except for the addition of 0.1% of NaH13CO3. 13C spectra of lysine accumulated in broth were measured using the XL-100A (Varian) spectrometer, working at 25.4 MHz. A) 13C spectrum of L-lysine produced in the presence of 500 Fig/liter of biotin and 0.1% of NaH13SCO3. B) 13C spectrum of natural L-lysine. Evidence for pyruvate carboxylase activity The above results indicated the possibility FIG. 3. Possible Pathway of L-Lysine Biosynthesis Involved in CO2 Fixation into Pyruvate. Actually, the washing cells of Brev. lacto fermentum AJ 3799 assimilated CO2 in the presence of pyruvate and 500ƒÊg/liter of biotin suggesting the involvement of pyruvate carbo xylase. The incorporation site of CO2 into L-lysine was identified as ƒá-ch2 of it by NMR (Fig. 4). of the presence of pyruvate carboxylase in Brev. lactofermentum. The activity of pyruvate carboxylase was assayed in cell free extracts obtained from late log cultures of Brev. lacto fermentum AJ 3445 which had been grown on lactate medium contained 500ƒÊg/liter of biotin and KHCO3. The enzyme was assayed by measurement of oxaloacetate production with MDH or citrate synthase. In the case of citrate synthase coupled method, L-leucine was

5 Biosynthesis of L-Lysine and L-Threonine in Brevibacterium 1517 added to the reaction mixture. Since leucine was found to be an inhibitor of reaction be tween acetyl-coa and pyruvate which inter fered with an assay of pyruvate carboxylase dependent on CoA formation with citrate synthase, the addition of leucine was useful in measurement of enzyme activity with ac curacy. The effects of the omission of various components from the assay mixture are shown in Table II. Oxaloacetate formation was com- TABLE II. REQUIREMENTS FOR THE REACTION CATALYZED BY PYRUVATE CARBOXYLASE FROM Brev. lactofermentum AJ 3445 The complete reaction mixture was that described in Methods, with the omissions or additions stated. A 0.1ml portion of cell free extract obtained cells grown on lactate and KHCO3 at 30 C was used for assay. TABLE HI. COMPARISON OF PYRUVATE CARBOXYLASE ACTIVITY IN Brev. lactofermentum AND Pseudomonas citronellolis Cells were grown in lactate medium and extracts were assayed using the complete reaction mixture (MDH coupled method) described in METHODS. TABLE IV. COMPARISON OF THE PROPERTIES OF PYRUVATE CARBOXYLASE AND PEP CARBOXYLASE FROM Brev. lactofermentum AJ 3799 A cell free extract was prepared from AJ 3799 grown on lactate. Pyruvate carboxylase activity was assayed by MDH coupled method. When avidin was present, cell free extract was preincubated for 20 min at 30 C with avidin. pletely dependent on the presence of pyruvate, ATP and KHCO3. Biotin stimulated the ac tivity significantly. Since pyruvate carboxylase had been studied in Pseudornonas citronellolis, we compared enzyme from Brev. lactofer mentum AJ 3445 with that from Ps. citronellolis ATCC The similar level of pyruvate carboxylase was observed in cell free extracts of both strains grown on lactate medium (Table III). Furthermore, pyruvate carboxy lase activity in the cell free extract was speciffi cally inactivated by incubation of an aliquot of the extract with avidin. This inactivation was prevented if excess biotin was added prior to addition of avidin. The presence of both pyruvate carboxylase and PEP carboxylase has also been reported in cell free extracts obtained from Ps. citronel lolis3) and Azotobacter vinelandii.5) The ac tivities of pyruvate carboxylase and PEP carbo- xylase were assayed in the cell free extract of Brev. lactofermentum AJ These data are presented in Table IV as specific activity. The cell free extract contained apparently both of pyruvate carboxylase and PEP carboxylase. Furthermore, addition of avidin did not affect the activity of PEP carboxylase in this extract. In contrast, addition of 0.5 U of avidin inhi bited 94% of pyruvate carboxylase. On the other hand, addition of 1mM of aspartate inhibited 73% of PEP carboxylase activity specifically. It is of interest that this pyruvate carboxylase was independent on acetyl-coa, which acts as an effector of this enzyme from Arthrobacter globiformis.16) The above results appeared that Brev. lacto fermentum possessed more than one enzyme

6 1518 O. TOSAKA, H. MORIOKA and K. TAKINAMI capable of catalyzing net oxaloacetate synthesis from three carbon precursors. One of the most important observations made in connec tion with the discovery of pyruvate carboxylase was that the stimulation of enzyme by excess biotin exhibits the increase in L-lysine for mation. Effect of biotin on the activity of various enzymes involved in pyruvate metabolism The activities of ten enzymes of pyruvate metabolism were assayed in the cell free extract from late log cultures of Brev. lacto fermentum AJ Table V shows the specific TABLE V. DEPENDENCE OF THE ENZYME RELATED TO THE METABOLISM OF PYRUVATE IN CELL FREE EXTRACT ON BIOTIN Preparation and assays were conducted as those described in METHODS. DISCUSSION In L-lysine fermentation, we found the pro motive effect of excess biotin on L-lysine pro duction. With glucose or pyruvate as the sole carbon source, L-lysine production was stimulated markedly in the presence of excess biotin. Little activation was observed with tricarboxylic acid cycle members and acetate, suggesting the presence of an activation site on the pathway from pyruvate to tricarboxylic acid cycle. Shiio17) have demonstrated that PEP carboxylase is present at significant levels in the cell free extract prepared from Brev. flavum grown on glucose as sole carbon source. However, pyruvate carboxylase could not detect in the cell free extract. Therefore, it seems that PEP carboxylase acts as a sole anaplerotic function in Brev. flavum, as in Escherichja coli18) and Salmonella typhimu rium.19) On the other hand, NADP-dependent malic enzyme in Brev. lactofermentum catalyzes a reaction: Malate+NADP Pyruvate + NADPH Therefore, the promotive effect of excess biotin on L-lysine formation could not be ex plained by the level of biotin-independent PEP carboxylase and malic enzyme. The presence of biotin-dependent pyruvate carboxylase seems to be necessary to explain the mechanism of this effect. Significant facts which was examined are (1) excess-biotin dependent of growth on pyruvate as sole carbon source: activity of those enzymes in the presence of 10ƒÊg of biotin or 0.5 U of avidin. Among these enzyme, malic enzyme and oxaloacetate decarboxylase catalyse a reaction of the type X pyruvate+co2. Oxaloacetate decarboxy lase from AJ 3799 was inactivated by incu bation with avidin, which appears to be de pendence on biotin for catalytic activity. No significant difference was shown in other enzyme with or without biotin. It suggests that promotive effect of excess biotin on L- lysine formation does not result from activation of these enzymes by biotin. (2) excess-biotin dependent of growth of PEP carboxylase deficient mutant on glucose as sole carbon source: (3) CO, fixation into ƒá-ch2 group of L-lysine produced from pyru vate in the presence of excess biotin: and (4) presence of pyruvate carboxylase in cell free extract of Brev. lactofermentum. These findings indicate that oxaloacetate production was stimulated by activation of pyruvate carboxylase by excess biotin and subsequently L-lysine production was pro moted. In fact, L-lysine production from glucose was strongly dependent on the amount of L-aspartate formed in lysine producer,

7 Biosynthesis of L-Lysine and L-Threonine in Brevibacterium 1519 since L-aspartate is a limiting precursor in lysine biosynthesis from glucose. Pyruvate carboxylase and PEP carboxylase were present simultaneously in Brev. lacto fermentum, as have been previously reported for Ps. citronellolis, Ps. fluorescens and A. vinelandii. The possibility that only one carbo xylase was present in Brev. lactofermentum was ruled out, since similar levels of the two enzymes were found and two carboxylases showed different regulatory properties. Pyru vate carboxylase was insensitive to acetyl-coa and L-aspartate, while PEP carboxylase was sensitive to L-aspartate. This regulatory dif ferences suggest that the two carboxylases may have different function in the living cells. At higher concentration of biotin in the culture medium, aspartate can not permeate through cell membrane and consequently intracellular levels of aspartate may be increased. Therefore, PEP carboxylase activity is strongly inhi bited by aspartate at less than 1 mm. These circumstances are unfavourable for the synthe sis of cell constituents. On the other hand, an apparent affinity of PEP for PEP carboxy lase is about one tenth of the affinity for pyruvate kinase,20) PEP is converted to pyru vate preferentially by pyruvate kinase and pyruvate may induce pyruvate carboxylase. Higher concentration of biotin was required for pyruvate carboxylase activity. These se- FIG. 5. Pathway and Regulation of Lysine Biosyn thesis from Glucose in Brev. lactofermentum._??_ tion., Feedback inhibition; _??_, Repression;, _??_:1 Acti quence involves a net conversion of pyruvate to oxaloacetate and permits growth on three carbon sources. These results were summari zed in Fig. 5. From the facts described above, we may conclude that Brev. lactofermentum contains pyruvate carboxylase which bring about stimu lation of L-lysine formation by higher concen tration of biotin. Acknowledgment. The authors are indebted to Drs. T. Shiro, H. Okada and Y. Hirose of our labora tories for their encouragement and to Dr. M. Kainosho for his help in the NMR analysis. They also wish to thank Mr. K. Kubota, Mr. Y. Yoshihara and M. Ishihara for helpful discussion. REFERENCES 1) O. Tosaka, H. Hirakawa and K. Takinami, Agric. Biol. Chem., 43(3), 491 (1979). 2) H. L. Kornberg, "Essays in Biochemistry," Vol. 2, Academic Press. Inc., New York, N. Y., 1966, p. 1. 3) R. W. O'Brien, B. L. Taylor and M. F. Utter, Proc. Aust. Biochem. Soc., 6, 34 (1973). 4) A. 1. Higa, S. R. Milard and J. J. Cazzulo, J. Gen. Microbiol., 93, 69 (1976). 5) M. C. Scrutton and B. L. Taylor, Arch. Biochem. Biophys., 164, 641 (1974). 6) O. Tosaka, K. Takinami and Y. Hirose, Agric. Biol. Chem., 42, 745 (1978). 7) O. H. Lowry, N. J. Rosebrough and R. J. Randal, J. Biol. Chem., 193, 265 (1951). 8) R. E. Barden, C. H. Fung, M. F. Utter and M. C. Scrutton, J. Biol. Chem., 247, 1323 (1972). 9) L. J. Reed and C. R. Willms, "Methods in Enzymology," Vol. IX, ed. by A. Wood, Academic Press Inc., New York, N. Y., 1966, p ) M. W. Zink, Can. J. Microbiol., 13, 1211 (1967). 11) P. A. Srere, Biochem. Biophys. Acta, 77, 693(1963). 12) H. H. Daron, W. J. Rutter and I. C. Gunsalus, Biochemistry, 5, 895 (1966). 13) G. H. Dixon and H. L. Kornberg, Biochem. J., 72, 3 (1959). 14) V. Massey, "Methods in Enzymology," Vol. I, ed. by S.P. Colowick and N.O. Kaplan, Academic Press Inc., New York, N. Y., 1955, p ) S. Ochoa, ibid., p ) E. S. Bridgeland and K. M. Jones, Biochem. J., 104, 9 (1967). 17) 1. Shiio, S. Otsuka and T. Tsunoda, J. Biochem., 48, 110 (1960). 18) J. M. Ashworth, H. L. Kornberg and R. L. Ward, Biochem. J., 94, 28 (1965). 19) T. S. Theodore and E. Engresberg, J. Bacteriol., 88, 946 (1964). 20) H. Ozaki and I. Shiio, J. Biochem., 66, 297 (1969).