GENETIC AND BIOCHEMICAL STUDIES ON BACTERIAL FORMATION OF L-GLUTAMATE

J. Gen. App!. Microbiol., 15, 27-40 (1969) GENETIC AND BIOCHEMICAL STUDIES ON BACTERIAL FORMATION OF L-GLUTAMATE I. RELATIONSHIP BETWEEN ISOCITRATE LYASE, ACETATE KINASE, AND PHOSPHATE ACETYLTRANSFERASE LEVELS AND GLUTAMATE PRODUCTION IN BREVIBAGTERI UM FLA V UM ISAMU SHIIO, HARUO MOMOSE AND AKIKO OYAMA Central Research Laboratories of Ajinomoto Co., Inc., Kawasaki, Japan (Received August 16, 1968) 1. One acetate kinase-negative, 53 phosphate acetyltransferase-negative, and seven isocitrate lyase-negative strains were found among 73 mutants of Brevibacterium fiavum, which grew on glucose but not on acetate. The result indicates that these enzymes are essential for the utilization of acetate in B. flavum, and also that the participation of acetyl-coa synthetase is absent in this strain. 2. The genetic defect of any one of these three enzymes had no effect on the production of L-glutamate from glucose. 3. Many mutants possessing a variety of isocitrate lyase activities were obtained by deriving revertants grown on acetate from four isocitrate lyasenegative strains. By an experiment using such revertants, it was concluded that isocitrate lyase had an important effect on the production of L-glutamate from acetate, which was shown as a positive correlation between the activity of this enzyme and L-glutamate productivity in the cells grown in an acetate medium. It was also shown that the change of this enzyme activity had no effect on the production of L-glutamate from glucose. 4. Mutants possessing a variety of phosphate acetyltransferase activities were obtained by deriving revertants grown on acetate from four strains lacking this enzyme. In this case, too, a positive correlation was observed between the activity of this enzyme and L-glutamate productivity in the cells grown in an acetate medium. 5. In connection with the present study, it was pointed out that genetic elevation of the levels of key enzymes was important for the improvement of microbial strains for industrial purposes. Byevibacterium flavum strain 2247 is one of the typical glutamate-producing bacteria which require biotin for the growth and produce a large amount of L-glutamate when the concentration of biotin in the culture medium is limited 27

28 SHIIO, MOMOsE ANI) OYAMA VOL. 15 (1). Srio et al. investigated, in detail, the mechanism of L-glutamate production in this strain, and showed that L-glutamate was formed from glucose and acetate via pathways indicated in Fig. 1(2-10). One of the characteristics of this strain is that it synthesizes several enzymes specific for the acetate metabolism even when grown in a glucose medium (8). From the point of yield of the glutamate production, therefore, it is of interest to know whether the presence of these enzymes affect the production of L-glutamate from glucose or not. On the other hand, the production from acetate seems to depend on the levels of these enzymes because these are believed to be essential for the utilization of acetate as a source of carbon. However, there has been no published report which dealt with this relationship. Fig. L L-Glutamate formation from glucose and acetate observed in Brevibacterium lavum strain 2247 (2-10). Enzymes: 1, acetate kinase ; 2, phosphate acetyltransferase ; 3, citrate synthase ; 4, isocitrate lyase ; 5, malate synthase ; 6, succinate dehydrogenase ; 7, fumarate hydratase ; 8, malate dehydrogenase.

1969 Enzyme Levels and Glutamate Production 29 The present authors derived mutants of B, flavum strain 2247 lacking isocitrate lyase [EC 4.1.3.1, Ls-isocitrate glyoxylate lyase], acetate kinase [EC 2.7.2.1, ATP : acetate phosphotransferase], or phosphate acetyltransferase [EC 2.3.1.8, acetyl-coa : orthophosphate acetyl transferase], and investigated the production of L-glutamate from glucose by these mutants in order to answer the above-mentioned question. Moreover, from these isocitrate lyasenegative mutants as well as phosphate acetyltransferase-negative mutants, revertants were derived which possessed various levels of their respective enzymes, and the relationship between these enzyme levels and the glutamate production from acetate was investigated using these revertants. The present paper describes these results. MATERIALS AND METHODS Chemicals. CoA, glutathione, sodium glyoxylate, L-malic acid, and citrate synthase were purchased from Sigma Chemical Company, thiamine hydrochloride and DL-isocitric acid from Nutritional Biochemical Corp., dilithium acetyl-phosphate, ATP, and NADH2 from Boehringer & Soehne GmbH, oxaloacetic acid and biotin from California Corp, for Biochemical Research, ethyl methanesulfonate from K & K Laboratories Inc., and N-methyl-N'-nitro-Nnitrosoguanidine from the Aldrich Chemical Company. Acetyl-CoA was prepared chemically from CoA and anhydroacetic acid according to SIMoN and SHEMIN (11). Bacterial strains. Brevibacterium flavum strain 2247 (wild type) and its mutant strains were used. Strain 2247 requires biotin for its growth and produces L-glutamate in biotin-limited media such as the G2 or A0.5 medium (see below) (12). Media. Six media were employed. The complete medium contained 10 g polypeptone, 10 g yeast extract, and 5 g NaCI in 1,000 ml distilled water (ph 7.2). The GM medium contained 5 g glucose, 1.5 g urea, 1.5 g (NH4)2S02, 1 g KH2PO4, 3 g K2HPO4, 0.1 g Mg5O4.7H2O, 1 mg CaCl2.2H2O, 30 pg biotin, 100 pg thiamine hydrochloride, and 1 ml trace element solution (13) in 1,000 ml distilled water (ph 7.2). The AM medium contained 50.6 g CH3000Na 3H2O, 17.4 g CH3COONH4, 2 g KH2PO4, 0.8 g MgSO4.7H2O, 2 ppm Fee, 2 ppm Mn2+, 30 pg biotin, 100 pg thiamine hydrochloride, 0.5 ml Mi-eki (solution of amino acid mixture prepared from soybean hydrolysate ; total nitrogen, 24 g/ liter) in 1,000 ml of distilled water. The ph was adjusted to 8.0. The G30 medium was comprised of 36 g glucose, 10 g urea, 1 g KH2PO4, 0.4 g MgSO4 7H2O, 2 ppm Fee, 2 ppm Mn2+, 30 pg biotin, 100,gig thiamine hydrochloride, and 1 ml Mi-eki in 1,000 ml of distilled water. The ph was adjusted to 6.8. The G2 medium is the same as the G30 medium except that the concentration of biotin was 2 'g/liter. The A0.5 medium is the same as the AM medium except that it contained 0.5 pg/liter of biotin, 1 ml/liter of Mi-eki, and 5 mg/ liter of Cresol Red as a ph indicator. Agar plates were prepared by adding

30 SHIIO, MOMOSE AND OYAMA VOL. 15 2% of agar to the composition of complete, GM, or AM medium. Derivation of mutants. For the derivation of mutants which grow on glucose but not on acetate, ethyl methanesulfonate (EMS) was used as a mutagen (14). The cells of B. flavum strain 2247 grown in the complete medium were washed and treated with 0.4 M EMS in 0.1 M phosphate buffer (ph 7.0) for 40 min under shaking at 30. After the EMS-treated cells were first grown on the complete plates, mutant colonies which grew on the GM plate but not on the AM plate were selected by the replica method. In order to isolate revertants from these mutants, cells were treated with 2,000,eg/ml of N-methyl-N'-nitro-N-nitrosoguanidine (15) in the same buffer for 30 min in an ice bath. The treated cells were plated on the AM plate supplemented with 0.1% casein hydrolysate, and revertant colonies that appeared after 3-6 days at 30 were isolated. Cultivation of organisms. The organisms were grown on a shaker at 30 in 500-ml flasks containing 50 ml of the G;30, G2, or A0,5 medium for 24, 48, or 72 hr, respectively. When grown in A05 medium, ph was adjusted to 7.8 by the addition of 5 N HCl at 24-hr intervals. These cultures served for the preparation of enzyme extract and for the measurement of L-glutamate productivity. Preparation of enzyme extract. The cells were harvested, washed twice with 0.2% KCI, and suspended in 0.05 M Triis buffer (ph 7.5). The suspended cells were disrupted by treatment with a 10-kc sonic oscillator (Toyo-Riko Type 50-5, 80 W) for 20 min. The cell debris was removed by centrifugation at 13,000 X g or at 600 X g (for succinate dehydrogenase only). These crude extracts were used for the enzyme assay after appropriate dilution. All experimental processes after the harvesting of cells were carried out at a low temperature. Enzyme assay. Acetate kinase activity was determined by the method of ROSE et al. (16). Phosphate acetyl transferase activity was determined by coupling the reaction with citrate synthase [EC 4.1.3.7, citrate oxaloacetate lyase (CoA-acetylating)] (17, 18). The reaction mixture contained 10 pmoles of dilithium acetyl phosphate, 65 mi moles of CoA, 10 moles of dipotassium oxaloacetate, 5 ~~moles of MgCl2, 8.25 pmoles of cysteine, 100 moles of Tris buffer (ph 8.0), excess amount of citrate synthase (0.1 ml of 10 times dilution of original solution containing 70 units of the enzyme per mg protein), and 0.1 ml of the enzyme extract in a final volume of 1 ml. The reaction was carried out at 25 for 30 min and stopped with 1 ml of 10 o trichloroacetic acid. The amount of citrate formed was measured according to NATELSON et al. (19). The specific activity was defined as the amount of citrate formed (,mole)/mg protein/min. In experiments in which phosphate acetyltransferasenegative mutants were screened, the addition of commercial citrate synthase was omitted (see later section). Isocitrate lyase was assayed by measuring glyoxylate formed from isocitrate according to the method of OZAKI and SHIIO (20). Malate synthase [EC 4.1.3.2, L-malate glyoxylate-lyase (CoA-

1969 Enzyme Levels and Glutamate Production 31 acetylating)] activity was assayed by observing the decrease in optical density at 232 mr due to the consumption of acetyl-coa (21). Succinate dehydrogenase [EC 1.3.99.1, succinate : (acceptor) oxi.dor&uctase] activity was assayed manometrically by measuring oxygen consumption in the presence of KCN and Methylene Blue (22). Fumarate hydratase [EC 4.2.1.2, L-malate hydrolyase] activity was assayed by measuring the increase in optical density at 240 mp due to the formation of fumarate from malate (23). Malate dehydrogenase [EC 1.1.1.37, L-malate : NAD oxidoreductase] was assayed spectrophotometrically according to the method of OCHOA (24). Citrate synthase activity was assayed according to GILVARG and DAVIS (17). Measurement of L-glutamate productivity. L-Glutamate accumulated in the culture medium (G2 or A0.5) was determined by the manometric method (25) using acetone-dried cells of E. coli " Crookes " strain, a specific L-glutamate decarboxylase [EC 4.1.1.15, L-glutamate 1-carboxy-lyase] preparation. RESULTS Derivation of mutants and their genetic blocks 73 mutant strains, which grew on glucose but not on acetate, were derived from B, flavum strain 2247 by EMS treatment. The frequency of mutants obtained was approximately 4 X 10-3. In Fig. 2, the growth responses of a typical strain, 16-44, to acetate and glucose are indicated in comparison with those of its parent strain. The other mutant strains, also showed almost the same response pattern as did strain 16-44, except strain 17-5 alone which underwent to show weak response to glucose after several transfers on bouillon slants. The genetic blocks in these 73 mutant strains were then analyzed by measuring the following enzyme activities in the cells grown in G30 medium ; acetate kinase, phosphate acetyltransferase, isocitrate lyase, malate synthase, succinate dehydrogenase, f umarate hydratase, and malate dehydrogenase (see Fig. 1). As a result, 63 strains were found to be completely deficient in one or two of these enzyme activities, and the other ten strains had normal activities of these enzymes tested. The 63 strains were composed of one acetate kinase-negative, 53 phosphate acetyltransferase-negative, seven isocitrate lyase-negative, one acetate kinase-phosphate acetyltransferase-double negative, and one phosphate acetyltransferase-isocitrate lyase-double negative mutants (Table 1). Mutants deficient in malate synthase, succinate dehydrogenase, fumarate hydratase, or malate dehydrogenase were not obtained. It should be noted that more than 70% of the isolated mutants incapable of growing on acetate were phosphate acetyltransferase-negative mutants, although the reason for it is not clear. Although the phosphate acetyltransferase assay was done in this experiment by measuring the formation of citrate from acetyl phosphate, CoA, and oxaloacetate without the addition of commercial citrate synthase (see Materials and Methods), it seems unlikely that some of these

32 SHIIo, MOMOSE AND OYAMA VOL. 15 Fig. 2. Growth responses of strain 16-44 and its parent strain 2247 to glucose and acetate. Washed cells were inoculated to 50 ml each of G30 medium (containing glucose) and AM medium (containing acetate), and cultivation was carried out at 30. At appropriate intervals, aliquot of each culture was withdrawn, diluted 26 times and cell density measured at 562 m/c. 0-0 2247 in G30 medium; A -A 2247 in AM medium; O- ~ 16-44 in G30 medium; A -A 16-44 in AM medium. Table 1. Genetic blocks in mutants isolated.

1969 Enzyme Levels and Glutamate Production 33 mutants might be deficient in citrate synthase, because all these mutants can grow normally in GM medium. In fact, eight strains randomly selected had the same level of citrate synthase as that of the wild strain. Two pathways are known to exist for acetyl-coa synthesis from acetate ; one involves the above-mentioned two enzymes, acetate kinase (26) and phosphate acetyltransferase (27), and the other involves acetyl-coa synthetase [EC 6.2.1.1, acetate : CoA ligase (AMP)] (28). To test the possibility that B, flavum possesses the latter enzyme, citrate formation from acetate, ATP, CoA, and oxaloacetate was determined in several strains. These results are shown in Table 2. Citrate was formed by an enzyme extract from the wild strain, but not by enzyme extracts prepared from either a phosphate acetyltransferase-negative mutant 16-28, or an acetate kinase-phosphate acetyltransferase-double negative mutant 16-47. This indicates that B. flavum has no activity of acetyl-coa synthetase and that phosphate acetyltransferase, which catalyzes acetyl-coa formation from acetyl-phosphate, is essential for the citrate formation. Therefore, B, flavum is believed not to possess the alternative pathway catalyzed by acetyl-coa synthetase (see Discussion). Table 2. Citrate formation of acetate, ATP, by enzyme CoA, and extract in the oxaloacetate presence Significance of acetate kinase, phosphate acetyltransferase, and isocitrate lyase on L -glutamate production in glucose medium Several strains were selected from each type of mutant strains (Table 1) which lacked acetate kinase, phosphate acetyltransferase, or isocitrate lyase, cultivated for 48 hr in G2 medium, and L-glutamate accumulated in the culture was measured (see Materials and Methods). As shown in Table 3, it was found that almost all the mutant strains had the same productivity of glutamate (110-120 r moles/ml) as the wild strain except strain 16-47, a mutant deficient in both acetate kinase and phosphate acetyltransferase, and which had fairly lower productivity than the wild strain.

34 SHIIo, MOMosE AND OYAMA VOL. 15 Table 3. Production deficient in of L-glutamate in a enzymes essential for glucose acetate medium metabolism. by mutants Effect of isocitrate lyase and phosphate acetyltransferase activity on glutamate production in acetate medium To obtain mutants possessing various isocitrate lyase activities, revertants which can grow on acetate were derived from four isocitrate lyase-negative mutants 16-14, 16-46, 16-54, and 17-4 (see Materials and Methods). The frequency of the appearance of these revertants was approximately 10-6, and 50-100 revertant colonies were isolated from each mutant. Among them, revertants which showed the same growth level in A0.5 medium as strain 2247 were selected, and the glutamate productivity in this medium was examined. As a result, 19 revertants from 16-46 and 23 revertants from 17-4 showed a fairly wide distribution of glutamate productivity between 0 and 90 pmoles/ml. On the other hand, 32 revertants from 16-54 showed a close distribution around the average productivity of the wild strain 2247 (65 pmoles/ ml), and six revertants from 16-14 showed almost no productivity. From each group, several revertants were further selected which represented a variety of glutamate productivities, and the relationship between isocitrate lyase activity and L-glutamate productivity in A0.5 medium was examined. As shown in Fig. 3, it was found that there was an apparent positive correlation between these two parameters. It should be noted that revertants from 17-4 showed a wide distribution of the enzyme activity (4-101) as well as glutamate productivity (9-86 pmoles/ml), and the plots of glutamate productivity against enzyme activity formed part of a hyperbola. The plots from the other groups of revertants were also distributed along this line. Similar experiments were also made for the relationship between isocitrate

1969 Enzyme Levels and Glutamate Production 35 Fig. 3. Correlation between isocitrate lyase level and L-glutamate production from acetate. Enzyme activity and L-glutamate produced were estimated after 72-hr cultivation in Ao ; medium. Strains tested: Revertants from 16-14 ( ), 16-46 (~ ), 16-54 (x), 17-4 (s), and isolates from strain 2247 (s). lyase level and glutamate productivity with 11 clones randomly selected from single colonies of strain 2247. In this case, too, the plots were distributed along the above-mentioned curve, although these were considerably clustered compared with the case of revertants from strain 17-4. Table 4 shows the effect of isocitrate lyase activity on glutamate productivity in glucose medium G2. In this medium, too, various degrees of enzyme activity were observed in these revertants although these enzyme levels were, in general, lower than those in acetate medium. The amount of glutamate accumulated in G2 medium, however, showed almost a constant level, having no correlation to the enzyme activity. This result is compatible with that in the previous section. Effect of phosphate acetyltransferase on glutamate production in acetate medium was also examined by using revertants from four phosphate acetyltransferase-negative mutants 16-18, 16-22, 16-28, and 16-33. As shown in Fig. 4, it seems that there is a positive correlation but with the exception of the revertant 1633-22 which shows very high enzyme activity in spite of its low level of glutamate productivity. DISCUSSION The fact that several isocitrate lyase-negative strains were obtained from Brevibacterium flavum as mutants which grew on glucose but not on acetate

36 SHno, MOMOSE AND OYAMA VOL. 15 Table 4. Relation productivity between isocitrate lyase in revertants grown in a level and L-glutamate glucose medium. indicates that, in B. }lavum as well as in the case of B. coli, the enzyme is essential for the utilization of acetate but not for that of glucose. It is also the case with acetate kinase and phosphate acetyltransferase. Acetate kinase and phosphate acetyltransferase have been shown to be widely distributed among many bacteria (29), and it has been suggested that acetyl-coa is synthesized from acetate by these enzymes, although it has not been completely verified yet. On the other hand, acetyl-coa synthetase is known to exist in higher plants and animals, which catalyzes the direct formation of acetyl-coa from acetate. In the present study, the assay method for acetate kinase is based on the formation of a colored ferric hydroxamate complex from acetyl-

1969 Enzyme Levels and Glutamate Production 37 Fig. 4. Correlation between phosphate acetyltransferase level and L-glutamate production from acetate. Enzyme activity and L-glutamate produced were estimated after 72-hr cultivation in A0.5 medium. Strains tested: Revertants from 16-18 (0), 16-22 (0), 16-28 (x), 16-33 (A), and strain 2247 (u). phosphate synthesized. Moreover, this method cannot distinguish between the reaction catalyzed by acetate kinase and that by acetyl-coa synthetase, if a trace amount of CoA is present in a crude enzyme extract, because the colored ferric hydroxamate complex is also formed from acetyl-coa which is synthesized by the latter enzyme. It was previously suggested that, in B. iavum, acetyl-coa was formed via acetyl phosphate because the hydroxamate formation from acetate was not stimulated by the addition of CoA, but citrate and malate formation from acetate required CoA (8). However, these facts cannot rule out a possibility that a smaller amount of acetyl-coa synthetase is present in the crude extract together with a greater amount of acetate kinase, and only the former enzyme takes part in the formation of acetyl-coa when the amount of phosphate acetyltransferase is far less than that of acetyl-coa synthetase. In the present study, however, many phosphate acetyltransferase-negative strains, which still possessed the ability for hydroxamate formation from acetate, were obtained as mutants incapable of growing on acetate and which were derived by one mutational event, and no citrate was formed by the crude extract of these mutant strains from acetate, ATP, CoA, and oxaloacetate (Table 2). These facts strongly indicate that phosphate acetyltransferase is essential for the formation of acetyl-coa. Therefore, it will be concluded that, in B. flavum, the acetyl-coa formation from acetate is catalyzed successively by acetate kinase and by phosphate acetyltransferase, but not by acetyl-coa synthetase. Next, the problem of whether these above-mentioned three enzyme levels affect the production of glutamate from glucose and from acetate will be

38 SHIIO, MOMOSE AND OYAMA VOL. 15 discussed. As indicated in Table 3, genetic defect of either one of these three enzymes had no effect on the production of glutamate from glucose. Strain 16-47 alone showed 74% of the glutamate productivity of the wild strain 2247. Since this strain is an acetate kinase- and phosphate acetyltransferase-double negative mutant, it is considered that the formation of two or more enzymes of the wild strain was simultaneously affected by an EMSinduced mutation. Furthermore, taking it into consideration that many other mutants lacking either one of these two enzymes showed the same glutamate productivity as the wild strain, it is likely that the mutation simultaneously reduced activity of some other enzyme(s) involved in the glutamate formation pathway from glucose, directly or indirectly, and that this caused the low glutamate productivity in strain 16-47 rather than the defects of both acetate kinase and phosphate acetyltransferase did directly. Table 4 shows glutamate production from glucose by revertants which have a variety of isocitrate lyase activities ; the enzyme activities show a wide distribution from nearly 0 to 7 times higher activity than that in the wild strain. Nevertheless, all revertants except two have almost the same level of glutamate productivity as the wild strain. Two strains, 174-5 and 174-12, have fairly lower productivity, but this fact is considered tc be independent of the enzyme level, because strain 174-5 has no detectable amount of isocitrate lyase activity and strain 174-12 has about 6 times higher activity than the wild strain. From these results, it will be concluded that no genetic change in the intracellular level of at least acetate kinase, phosphate acetyltransferase, or isocitrate lyase can directly affect the production of glutamate from glucose. This conclusion is not compatible with a suggestion of KIMURA et al. that isocitrate lyase level, depending on the concentration of biotin in the culture medium, directly affects the glutamate production from glucose in Micrococcus glutamicus (30). It is possible that, under the condition of the glutamate production from glucose, isocitrate lyase has been completely inhibited inside the cell, and, as a result, the lyase level, if any, has no effect on the glutamate productivity. In fact, it has been found by OZAKI and SHIIO that the activity of isocitrate lyase partially purified from B. flavum is specifically inhibited by various organic acids related to the TCA cycle and glyoxylate bypass (20). In the production of glutamate from acetate, on the other hand, the importance of isocitrate lyase level was clearly shown by an experiment using revertants possessing various levels of this enzyme activity (Fig. 3). It is expected that there may be an optimum level of isocitrate lyase for glutamate production, because this enzyme has dual roles, positive and negative, for the glutamate production ; one is that it takes part in the net synthesis from acetate of organic acids in the TCA cycle, which are precursors for the glutamate formation, and the other is that it catalyzes the reaction branching out from the glutamate pathway at isocitrate, one of the precursors. However, the present experimental result is that glutamate productivity rapidly increased in the r 'inge of lower level of isocitrate lyase than that of strain

1969 Enzyme Levels and Glutamate Production 39 2247, and showed a saturation curve in the range of higher level. The rapid increase observed is not based on an increased amount of cells formed from acetate, the main source of carbon, because all revertants tested in this experiment had the same growth level as the wild strain 2247 in A0.5 medium. The saturation in productivity suggests that the above-mentioned feed-back inhibition of isocitrate lyase operates significantly in the range of the higher lyase level, which causes the elevation of intracellular pool of various organic acids related to the TCA cycle and glyoxylate bypass. It is probable that the mutation alters an enzyme not only in the activity itself but also in the regulatory mechanism. Thus, it can be imagined that such mutants, if any, are included in the revertants which deviate far from the average curve indicating the lyase level-glutamate productivity relationship in Fig. 3. It was also shown that phosphate acetyltransferase activity has a positive correlation to the glutamate production from acetate. This seems quite natural because this enzyme is directly involved in the glutamate pathway in a simple meaning, compared with the case of isocitrate lyase which has a dual role as described above. In the field of industrial microbiology, various attempts have been made for derivation of mutants producing a specific metabolite or metabolites, in which genetic defects of enzymes or genetic alterations of regulatory mechanisms were applied. The present study demonstrates that the genetic elevation of the levels of some specific key enzymes can also be applied for the improvement of various microbial strains for industrial purposes. The authors are indebted to Dr. T. Yoshida, Dr. T. Tsunoda, and Dr. N. Katsuya of the laboratories for their encouragement. The authors are also indebted to Mr. T. Iwasawa of the laboratory for his technical assistance. This study was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, held on March 31, 1967, and on April 1, 1968. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) I. SHIIO, S. OTSUKA and M. TAKAHASHI, J. Biochem., 51, 56 (1962). I. SHIIO, S. OTSUKA and T. TSUNODA, J. Biochem., 46, 1303 (1959). I. SHIIO, S. OTSUKA and T. TSUNODA, J. Biochem., 46, 1597 (1959). I. SHIIO, S. OTSUKA and T. TSUNODA, J. Biochem., 47, 414 (1960). I. SHIIO, S. OTSUKA and T. TSUNODA, J. Biochem., 48, 110 (1960). I. SHIIO, S. OTSUKA and M. TAKAHASHI, J. Biochem., 49, 397 (1961). I. SHIIO, S. OTSUKA and M. TAKAHASHI, J. Biochem., 50, 34 (1961). I. SHIIO, J. Biochem., 47, 273 (1960). I. SHIIO and T. TSUNODA, J. Biochem., 49, 141 (1961). I. SHIIO and T. TSUNODA, J. Biochem., 49, 148 (1961). El. SIMoN and D. SHEMIN, J. Am. Chem. Soc., 75, 2520 (1953). I. SHIIO, S. OTSUKA and M. TAKAHASHI, J. Biochem., 51, 56 (1962).

40 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) 26) 27) 28) 29) 30) SHIIO, MOMOSE AND OYAMA VOL. 15 H. MoMosE, J. Gen. Appl. Microbiol., 7, 359 (1961). A. LOVELESS and S. HOWARTH, Nature, 184, 1780 (1959). A. EISENSTARK, R. EISENSTARK and R. VAN SICKLE, Mutation Research, 2, 1(1965). IA. ROSE, M. GRUNBERG-MANA ;0, SR. KoREY and S. OCHOA, J. Biol. Chem., 211, 737 (1954). C. GILVARG and B.D. DAVIS, J. Biol. Chem., 222, 307 (1956). ER. STADTMAN, G.D. NOVELLI and F. LIPMANN, J. Biol. Chem., 191, 365 (1951). S. NATELSON, J.B. PINCUS and J.K. LUGOVOY, J. Biol. Chem., 175, 745 (1948). H. OZAKI and I. SHIIO, Symposia on Enzyme Chemistry (Japan), 18, 58 (1966). E. VANDERWINKEL, P. LIARD, F. RAMOS and J.M. WIAME, Biochem. Biophys. Res. Commun., 12, 157 (1963). E.C. SLATER, Biochem. J., 45, 1 (1949). E.C. BACKER, Biochim. Biophys. Acta, 4, 211 (1950). S. OCHOA, Methods in Enzymology, Academic Press Inc., New York, Vol. I, p. 735 (1955). I. SHHO, J. Biochem.. 44, 175 (1957). IA. ROSE, Methods in Enzymology, Academic Press Inc., New York, Vol. I, p. 591 (1955). ER. STADTMAN, Methods in Enzymology, Academic Press Inc., New York, Vol. I, p. 596 (1955). ME. JONES and F. LIPMANN, Methods in Enzymology, Academic Press Inc., New York, Vol. I, p. 585 (1955). IA. ROSE, The Enzymes, Academic Press Inc., New York, Vol. 6, 115 (1962). K. KIMURA, K. TANAKA and S. KINOSHITA, Nippon Nogei Kagaku Kaishi, 36, 754 (1962).