- Email: [email protected]
Pyrophosphatases of molds
230
LETTERS
TO
THE
EDITORS
These findings are in accord with those of Fries, Bergstroem, and Rottenberg (8), on a purine-less mutant of 0phiostomo.r Our experiments shed. no light on the question whether (I) or (II) has to be conjugated with ribose or desoxyribose in order to give the purine system (9,lO); experiments on the synthesis of the appropriate glycosides of (I) and (II) and their biological utilization are in hand. HzN-YO HsN-70 &NH
I 1. 2. 3. 4.
5. 6. 7. 8. 9. 10.
OHS
~--NH
II
REFERENCES STETTEN,M. R., AND Fox, C. L., J. Biol. Chem. 161,333 (1945). SHIVE, W., et al., J. Am. Chem. Sot. 69, 725 (1947). WOODS, D. D., Ann. N. Y. Ad. Sci. 52, 1206 (1950). BEN-ISHAI, R., VOLCANI, B., AND BEBQMANN,ERNST D., Ezper-i&ia 7,63 (1951); Bull. Research Council Israel 1, 160 (1951). GORDON, M., et al., J. Am. Chem. Sot. 70, 878 (1948). SHAW, E., J. Biol. Chem. 185, 439 (1950). DINNINQ, J. S., PAYNE, L. D., AND DAY, P. L., Arch. Biochem. 27, 467 (1950); STEKOL, J. A., AND WEISS, K., J. Biol. Chem. 186, 343 (1950). FFLIES, N., BEROBTROEM, S., AND ROTFENBEBQ, M., Physiobpia Plantarum 2,210 (1949). SHIVE, W., Ann. N. Y. Ad. Sci. 52, 1227 (1950). GREENBERG, G. R., Federation. Proc. 9,179 (1950).
Weizmann Inetitute of Science, Rehovoth, Israel Received October %,1960
RUTH BEN-ISWI BENJAMIN VOLCANI ERNST D. BERQMANN
Pyrophosphatases of Molds The existence of pyrophosphatases with diierent pH optima has not been so well established as that of the various phosphomonoesterases. Japanese workers (1) postulated three types of pyrophosphatases with respective activity optima near pH 4, 5, and 9. Bamann and Gall (2) reported the isolation from liver of three pyrophosphatases showing optimum activity at pH 4, 5.7, and 8. Very recently Norberg (3), working with homogenates of liver, showed the existence of four isodynamic pyrophosphatases, which differed with respect not only to pH optima but also substrate concentration and influence of added Mg ions. It was felt that molds would offer an ideal material for the search for pyrophosphatases of different pH optima. Some molds, growing on sugar media, produce large quantities of organic acids, whereas others are poor acid producers. Also, the isolation of pyrophosphate and a powerful phosphatase from molds (4,5) would indicate that 1After this Letter had been submitted for publication, Gets reported analogous observations on the same purine-less strain of E. wli [Arch. Biochem. 29,222 (1950)].
LETTERS
TO
THE
231
EDITORS
pyrophosphatases play an essential role in their metabolism. Two organisms were selected for study, one, a strain of AspergiUus niger, which was an excellent acid producer, and the second, a strain of PeniciUium chrysogenum, which produces comparatively little organic acids and in whose case the metabolism fluid actually turns alkaline toward the later stages of fermentation. The mycelial felt was harvested just prior to sporulation, ground with sand and water, and tested for pyrophosphatase activity by incubating with inorganic pyrophosphate. Both AspergiUus and Penicillium showed high pyrophosphatase activity, without the addition of Mg ions, over a wide range of pH, with an optimum toward the acidic side for the former and the neutral side for the latter. Addition of Mg, final concentration 0.01 M, inhibited the former but activated the latter. Aqueous extracts of the mats responded to the addition of Mg in a manner similar to the respective ground mat preparations. The enzymes were partially purified by fractional precipitation with ammonium sulfate. The determinations of pH-activity of the purified preparations showed that in the case of AspergiUus the optimum was in the region 3-4, whereas that for Penicillium was 6-7. When the precipitates obtained during fractionation with ammonium sulfate were taken up in water and assayed for pyrophosphatase activity, it was found that the enzyme from Penicillium was activated to the extent of 2300/, whereas the AspergiUua preparation was inhibited by 35%. These experiments would indicate the existence in molds of two different pyrophosphatases, one which has an optimum at pH 3-4 and which is inhibited by added Mg ions, and a second one which has a pH optimum near about neutrality and which is activated by Mg ions. REFERENCES
1. 2. 3. 4.
Quoted from: FOLLEY, S. J., AND KAY, H. D., Ergeb. Enzymjorsch. BAMANW, E., AND GALL, H., Biochem. 2. 383, 1 (1937). NORBERG, B., A& Chem. Stand. 4, 601 (1950). MANN, T., Biochem. J. 38, 345 (1944). 5. MANN, T., Biochem. J. 38, 339 (1944).
Division of Biochemistry, Nationnl Chemical Labor&y, Received March IS, 1951
The “Dicarboxylic
5, 159 (1936).
P. S. KRISHNAN Poona, India
Acid Cycle” in Bacterial Metabolism
Barron, Ardao, and Hearon (1) have recently shown that oxidation of acetate by creatinovorans probably proceeds through a cyclic process in which one of the steps is an oxidative condensation of two molecules of acetate to give succbte. Using aerated, nonproliferating suspensions of Aerobacter aerogcncswe have completed a study of the kinetics of formation of pyruvate from succinic, fumaric, malic, and acetic acids (2), and we have also rejected the tricarboxylic acid cycle as the mechanism of oxidation of acetate by this organism. Unless the bacteria have been previously trained to grow on acetate as the sole carbon source, nitrogen being supplied as the ammonium salt, little or no pyruvate can be detected in an acetate-phosphate buffer medium in which the cells are aerated. Thus, nonproliferating suspensions of cells grown on malate and succinate and adjusted to the same turbidity show an initial
Corynebackrium
LETTERS
TO
THE
EDITORS
These findings are in accord with those of Fries, Bergstroem, and Rottenberg (8), on a purine-less mutant of 0phiostomo.r Our experiments shed. no light on the question whether (I) or (II) has to be conjugated with ribose or desoxyribose in order to give the purine system (9,lO); experiments on the synthesis of the appropriate glycosides of (I) and (II) and their biological utilization are in hand. HzN-YO HsN-70 &NH
I 1. 2. 3. 4.
5. 6. 7. 8. 9. 10.
OHS
~--NH
II
REFERENCES STETTEN,M. R., AND Fox, C. L., J. Biol. Chem. 161,333 (1945). SHIVE, W., et al., J. Am. Chem. Sot. 69, 725 (1947). WOODS, D. D., Ann. N. Y. Ad. Sci. 52, 1206 (1950). BEN-ISHAI, R., VOLCANI, B., AND BEBQMANN,ERNST D., Ezper-i&ia 7,63 (1951); Bull. Research Council Israel 1, 160 (1951). GORDON, M., et al., J. Am. Chem. Sot. 70, 878 (1948). SHAW, E., J. Biol. Chem. 185, 439 (1950). DINNINQ, J. S., PAYNE, L. D., AND DAY, P. L., Arch. Biochem. 27, 467 (1950); STEKOL, J. A., AND WEISS, K., J. Biol. Chem. 186, 343 (1950). FFLIES, N., BEROBTROEM, S., AND ROTFENBEBQ, M., Physiobpia Plantarum 2,210 (1949). SHIVE, W., Ann. N. Y. Ad. Sci. 52, 1227 (1950). GREENBERG, G. R., Federation. Proc. 9,179 (1950).
Weizmann Inetitute of Science, Rehovoth, Israel Received October %,1960
RUTH BEN-ISWI BENJAMIN VOLCANI ERNST D. BERQMANN
Pyrophosphatases of Molds The existence of pyrophosphatases with diierent pH optima has not been so well established as that of the various phosphomonoesterases. Japanese workers (1) postulated three types of pyrophosphatases with respective activity optima near pH 4, 5, and 9. Bamann and Gall (2) reported the isolation from liver of three pyrophosphatases showing optimum activity at pH 4, 5.7, and 8. Very recently Norberg (3), working with homogenates of liver, showed the existence of four isodynamic pyrophosphatases, which differed with respect not only to pH optima but also substrate concentration and influence of added Mg ions. It was felt that molds would offer an ideal material for the search for pyrophosphatases of different pH optima. Some molds, growing on sugar media, produce large quantities of organic acids, whereas others are poor acid producers. Also, the isolation of pyrophosphate and a powerful phosphatase from molds (4,5) would indicate that 1After this Letter had been submitted for publication, Gets reported analogous observations on the same purine-less strain of E. wli [Arch. Biochem. 29,222 (1950)].
LETTERS
TO
THE
231
EDITORS
pyrophosphatases play an essential role in their metabolism. Two organisms were selected for study, one, a strain of AspergiUus niger, which was an excellent acid producer, and the second, a strain of PeniciUium chrysogenum, which produces comparatively little organic acids and in whose case the metabolism fluid actually turns alkaline toward the later stages of fermentation. The mycelial felt was harvested just prior to sporulation, ground with sand and water, and tested for pyrophosphatase activity by incubating with inorganic pyrophosphate. Both AspergiUus and Penicillium showed high pyrophosphatase activity, without the addition of Mg ions, over a wide range of pH, with an optimum toward the acidic side for the former and the neutral side for the latter. Addition of Mg, final concentration 0.01 M, inhibited the former but activated the latter. Aqueous extracts of the mats responded to the addition of Mg in a manner similar to the respective ground mat preparations. The enzymes were partially purified by fractional precipitation with ammonium sulfate. The determinations of pH-activity of the purified preparations showed that in the case of AspergiUus the optimum was in the region 3-4, whereas that for Penicillium was 6-7. When the precipitates obtained during fractionation with ammonium sulfate were taken up in water and assayed for pyrophosphatase activity, it was found that the enzyme from Penicillium was activated to the extent of 2300/, whereas the AspergiUua preparation was inhibited by 35%. These experiments would indicate the existence in molds of two different pyrophosphatases, one which has an optimum at pH 3-4 and which is inhibited by added Mg ions, and a second one which has a pH optimum near about neutrality and which is activated by Mg ions. REFERENCES
1. 2. 3. 4.
Quoted from: FOLLEY, S. J., AND KAY, H. D., Ergeb. Enzymjorsch. BAMANW, E., AND GALL, H., Biochem. 2. 383, 1 (1937). NORBERG, B., A& Chem. Stand. 4, 601 (1950). MANN, T., Biochem. J. 38, 345 (1944). 5. MANN, T., Biochem. J. 38, 339 (1944).
Division of Biochemistry, Nationnl Chemical Labor&y, Received March IS, 1951
The “Dicarboxylic
5, 159 (1936).
P. S. KRISHNAN Poona, India
Acid Cycle” in Bacterial Metabolism
Barron, Ardao, and Hearon (1) have recently shown that oxidation of acetate by creatinovorans probably proceeds through a cyclic process in which one of the steps is an oxidative condensation of two molecules of acetate to give succbte. Using aerated, nonproliferating suspensions of Aerobacter aerogcncswe have completed a study of the kinetics of formation of pyruvate from succinic, fumaric, malic, and acetic acids (2), and we have also rejected the tricarboxylic acid cycle as the mechanism of oxidation of acetate by this organism. Unless the bacteria have been previously trained to grow on acetate as the sole carbon source, nitrogen being supplied as the ammonium salt, little or no pyruvate can be detected in an acetate-phosphate buffer medium in which the cells are aerated. Thus, nonproliferating suspensions of cells grown on malate and succinate and adjusted to the same turbidity show an initial
Corynebackrium