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Phosphoglucose isomerase1 of green gram (Phaseolus radiatus)
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
62,
91-96
(1956)
Phosphoglucose Isomerase’ of Green Gram (Phaseolus radia tus) T. Ramasarma From
the Department
and K. V. Giri
of Biochemistry, Bangalore, Received
September
Indian India
Institute
of Science,
19, 1955
INTRODUCTION
Phosphoglucose isomerase which brings about equilibrium between glucose 6-phosphate (G-6-P) and fructose 6-phosphate (F-6-P) was first demonstrated by Lohmann (1) in 1933 in extracts prepared from yeast, muscle, heart, kidney, liver, and brain. Tanko (2) found that pea extracts with added inorganic phosphate formed a mixture of hexose phosphates from which he identified both G-6-P and F-6-P. He also showed that when pea extracts were incubated with F-6-P, a mixture of 62 % aldose and 38 % non-aldose esters was formed. Later, Somers and Cosby (3) added further evidence for the presence of the enzyme in pea extracts. Cori, Colowick, and Cori (4) showed that rabbit muscle extracts contain thii enzyme. Slein (5) reported the presence of phosphomannose isomerase in rabbit muscle. He also separated the phosphoglucose isomerase by fractionation with ammonium sulfate between 0.55 and 0.65 saturation (6). The widespread nature of the enzyme was indicated by Slein (5) and by Cori, Ochoa, and Slein (7) ; but it received very little attention for a long time until Bodansky (8) carried out a detailed study of the enzyme which he identified in the sera of several speciesincluding man. Sable and Calkins (9) found the enzyme in the tissue extracts of Molgula. Jagannathan and Singh (10) have identified the enzyme in the extracts of mycelium of Aspergillus niger. 1 Slein (6) has pointed out that the two isomerases which convert G-6-P and mannose 6-phosphate (M-6-P) into F-6-P, may now be more descriptively called phosphoglucose isomerase and phosphomannose isomerase, respectively, since it is now known that F-6-P is the product of M-6-P isomerization also. We propose to adopt this nomenclature. 91
92
T. RAMASARMA
AND K. V. GIRI
Evidence in support of the existence of an Embden-Meyerhof glycolytic pathway in green gram (Phasedus rczdiatus) has already been advanced in two earlier communications from this laboratory by Sri Ram and Giri (11) and by Ramasarma, Sri Ram, and Giri (12). The presence of phosphoglucose isomerase in green gram extracts was indicated by the increase in the concomitant fructose content in the reaction mixtures used for phosphoglucomutase assay. We have now been able to identify the enzyme using both G-6-P and F-6-P as substrates. In this paper the separation and characterization of phosphoglucose isomerase in green gram are reported. EXPERIMENTAL
Materials and Methods G-6-P (Ba salt) and F-6-P (Ba salt) were obtained from Nutritional Biochemicals. G-6-P (Ba salt) prepared enzymatically by the method of Colowiek and Sutherland (13) using sheep muscle as the source of phosphoglucomutase was also used. Fructose was determined by the method of Roe (14). The value for F-6-P was about 60% of that of equivalent free fructose which is in good agreement with the value reported by Umbreit and his associates (15).
Estimation of Enzyme Activity The reaction mixture always contained, unless otherwise stated, 2 amoles G-6-P, or 1 pmole of F-6-P wherever indicated, Verona1 buffer of pH 7.5-7.8 at a final concentration of 0.02 M, and enzyme solution containing 0.005 mg. protein N in a total volume of 0.25 ml. The reaction mixture was incubated at 37” for 2 min., and the reaction was stopped by the addition of hydrochloric acid used in the determination of fructose. The enzyme activity was expressed as micromoles of substrate converted per minute.
Partial Puri$.cation of the Enzyme The resting seeds of green gram were finely powdered, and the powder was extracted with five times its weight of water for 12 hr. under a layer of toluene in a refrigerator. One hundred milliliters of crude extract was adjusted to pH 7.0 with dilute alkali, and 100 ml. of cold acetone was slowly added with stirring. The precipitate obtained was centrifuged and discarded. Two hundred milliliters more of acetone was added to the supernatant. The precipitate obtained was dissolved in water and dialyzed against several changes of distilled water for 2 days. Any precipitate formed on dialysis was centrifuged and discarded. The temperature was maintained between 0 and 10” during these operations. A sixfold purification was achieved by this method, and the recovery was about 80% of the original activity. The enzyme preparations usually showed an activity of about 40 pmoles substrate conversion/min./mg. nitrogen.
PHOSPHOGLUCOSE
ISOMERASE
93
TABLE I of pH on Enzyme Activity The experimental conditions were the same as specified in the text, except the pH of the buffer used was varied. Acetate buffer at final concentration of 0.02 M was used to obtain pH 4.1-5.6. Erect
PH
4.1 4.6 5.1 5.6 6.5 7.0 7.4 7.8 8.3 8.7 9.0 9.2
Substrate G-6-P substrate p?nolcs/min.
0.00 0.02 0.05 0.13 0.13 0.15 0.18 0.25 0.20 0.20 0.19 0.18
converted F-6-P substrate pmoles/min.
0.03 0.04 0.11 0.14 0.14 0.18 0.20 0.17
These preparations did not liberate any phosphorus from G-6-P, F-6-P, and glucose l-phosphate. Phosphoglucomutase was found to be inactivated by acetone. These preparations were found to be completely free from fructose containing carbohydrates and inorganic phosphates which were present in large quantities in crude extracts.
RESULTS The enzyme was found to be active over a wide range of pH. The optimun was about 7.8 when G-6-P was used as the substrate and about 9.0 when F-6-P was used as the substrate (Table I). The reaction velocities were determined at various concentrations of G-6-P (1 X l!P to 10 X W3 M), and on plotting the reciprocals of the reaction velocities against the reciprocals of the substrate concentrations in accordance with the Lineweaver and Burk (16) modification of the Michaelis and Menten equation (17), a linear relationship was obtained. From the graph the value of the Michaelis constant was found to be 0.00345 moles/l. The enzyme activity was found to be proportional to the enzyme concentration until a change in substrate of about 15%. The temperature optimum for the enzyme activity was found to be 50” (Table II). The plot of the logarithm of activity against the reciprocal of absolute temperature gave a linear relationship. A value of 4800 cal./mole was obtained for the energy of activation calculated from the
94
T.
RAMASARMA
AND
TABLE E&cl
of Temperature
K.
V.
GIRI
II on Enzyme Activity
The experimental conditions were the same as specified in the text, except that the temperature was varied. The enzyme was incubated at the various temperatures for 10 min. before the substrate was added. Temperature “C.
20 25 30 35 40 45 48 50 52 55 60 70
Substrate converted pmoles/?dn.
0.07 0.11 0.16 0.18 0.20 0.23 0.25 0.27 0.25 0.21 0.07 0.00
graph. For the average temperature range between 35 and 45”, &IO was found to be 1.3. Equilibrium was approached with both G-6-P and F-6-P as substrates and, in both cases, the composition of the mixture at equilibrium was found to be about 60 % of G-6-P and 40 % of F-6-P (Fig. 1). Somers and Cosby (3) and Bhdansky (8) have observed that the phosphatase present in the crude preparations they used hydrolyzed F-6-P, which resulted in an increase in fructose color value. Bodansky has determined the amount, of phosphorus formed, and, assuming that the phosphorus was equally cleaved from both G-6-P and F-6-P, he calculated a value of 60% of G-6-P and 40% of F-6-P at, equilibrium. We have obtained the same figure using the purified preparation which did not hydrolyze the phosphoric esters. The enzyme was found to be stable for at least 4 weeks when kept frozen in a refrigerator, and the activity slowly decreased thereafter. Exposure of the enzyme to 0.02 N HCl or 0.02 N NaOH caused irreversible and complete inactivation of the enzyme. The enzyme was found to be rapidly inactivated when heated at temperatures above 45”. Incubation for 10 min. at 50, 55, and 60” caused a decrease in activity of about 20,33, and 90 %, respectively, at pH 7.5. The enzyme was found to be more resistant to heat denaturation in the alkaline range than in
PHOSPHOGLUCOSE
2
4
ISOMERASE
95
6 TIME,
MINUTES
1. Time course reaction of phosphoglucose isomerase activity and equilibrium between G-6-P and F-6-P. The experimental conditions were the same as specified in the text except that the reaction was stopped at different time intervals. 0, G-6-P conversion; 0, F-6-P conversion. FIG.
the acid range. The enzyme could be heated ‘at 50” at pH 8.0-9.0 for 10 min. with a lossin activity amounting to only about 6-8 %. Magnesium, manganese, and cobalt ions showed no activation of the enzyme between the concentrations 0.001 and 0.01 M. BaC12 (0.01 M), arsenate (0.01 M), and NaF (0.02 M) did not affect the enzyme at the concentrations tested. HgCL (0.002 M), KCN (0.02 M), and ZnSOc (0.001 M) inhibited the enzyme activity about 13, 20, and 18%, respectively. Iodoacetic acid (0.001X~002 M) did not affect the enzyme even after incubation for 10 min. at, 37”, indicating that the enzyme is not dependent upon the functional -SH groups for its activity. Glucose (0.1-0.8 mg.), fructose (0.1-0.2 mg.), and glucose l-phosphate (0.1-0.8 mg.) were not affected by the enzyme, and they showed very little effect either as activators or inhibitors when tested at the concentrations indicated above in the standard mixtures for the enzyme assay. Glucose 1,6-diphosphate (0.1 mg.) with F-6-P as the substrate and fructose 1,6-diphosphate (0.1 mg.) with G-6-P as the substrate did not, show any activation. Uridine diphosphoglucose, triphosphopyridine nucleotide, and diphosphopyridine nucleotide failed to activate the enzyme when tested in catalytic amounts. Aqueous extract of brewer’s
yeast, which is known to contain many of the nucleotides, also similarly failed to activate the enzyme.
96
T.
RAMASARMA
AKD
K.
V.
GIRI
ACKNOWLEDGMENT We are indebted to Prof. L. F. Leloir of the Institute of Biochemical Investigation, Julian Alvarez, Ruenos Aires, Argentina, for the gift of samples of glucose 1,6-diphosphate and uridine diphosphoglucose.
SUMMARY Phosphoglucoseisomeraseof green gram was partially purified by acetone fractionation. The enzyme was found to be rapidly inactivated when heated above 45”. The enzyme was found to be more resistant to heat denaturation in the alkaline range of pH. The optimum pH of the enzyme was around 7.8 with G-6-P as substrate and 9.0 with F-6-P as subst,rate. Temperature optimum was 50’. At equilibrium, about 60% of G-6-P and 40 % of F-6-P were found to exist. REFERENCES 1. LOHMANN, K., Biochem. 2. 262, 137 (1933). 2. TANKO, B., Biochem. J. 20, 692 (1936). 3. SOMERS, G. F., AND COBBY, E. L., Arch. Biochem. 6,295 (1945). 4. CORI, G. T., COLOWICK, S. P., AND CORI, C. F., J. Biol. Chem. l24,543 (1938). 5. SLEIN, M. W., J. Biol. Chem. 186, 753 (1950). 6. SLEIN, M. W., Federation Proc. 13, 988 (1954). 7. CORI, G. T., OCAOA, S., SLEIN, M. W., AND CORI, C. F., Biochim. et Biophys. Acta 7, 304 (1951). 8. BODANSKY, O., J. Biol. Chem. 202, 829 (1953). 9. SABLE, H. Z., AND CALKINS, C. W., JR., J. Biol. Chem. 204,695 (1963). 10. JAGANNATHAN, V., AND SINGH, K., Enzymologia 156, 150 (1953). 11. SRI RAM, J., AND GIRI, K. V., Arch. Biochem. and Biophys. 38,231 (1952). 12. RAMASARMA, T., SRI RA~I, J., AND GIRI, K. V., Arch. Rio&m. and Biophya. 63, 167 (1954). 13. COLOWICK, S. P., AND SUTHERLAND, W., J. Biol. Chem. 144,422 (1942). 14. ROE, J. H., J. Biol. Chem. 107, 15 (1934). 15. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. K., “Manometric Techniques and Related Methods for the Study of Tissue Metabolism,” p, 169. Burgess Publ. Co., Minneapolis, 1943. 16. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66,658 (1934). 17. MICHAELIS, I.., AND MENTEN, M. L., Biochem. 2. 49,333 (1913).
OF BIOCHEMISTRY
AND
BIOPHYSICS
62,
91-96
(1956)
Phosphoglucose Isomerase’ of Green Gram (Phaseolus radia tus) T. Ramasarma From
the Department
and K. V. Giri
of Biochemistry, Bangalore, Received
September
Indian India
Institute
of Science,
19, 1955
INTRODUCTION
Phosphoglucose isomerase which brings about equilibrium between glucose 6-phosphate (G-6-P) and fructose 6-phosphate (F-6-P) was first demonstrated by Lohmann (1) in 1933 in extracts prepared from yeast, muscle, heart, kidney, liver, and brain. Tanko (2) found that pea extracts with added inorganic phosphate formed a mixture of hexose phosphates from which he identified both G-6-P and F-6-P. He also showed that when pea extracts were incubated with F-6-P, a mixture of 62 % aldose and 38 % non-aldose esters was formed. Later, Somers and Cosby (3) added further evidence for the presence of the enzyme in pea extracts. Cori, Colowick, and Cori (4) showed that rabbit muscle extracts contain thii enzyme. Slein (5) reported the presence of phosphomannose isomerase in rabbit muscle. He also separated the phosphoglucose isomerase by fractionation with ammonium sulfate between 0.55 and 0.65 saturation (6). The widespread nature of the enzyme was indicated by Slein (5) and by Cori, Ochoa, and Slein (7) ; but it received very little attention for a long time until Bodansky (8) carried out a detailed study of the enzyme which he identified in the sera of several speciesincluding man. Sable and Calkins (9) found the enzyme in the tissue extracts of Molgula. Jagannathan and Singh (10) have identified the enzyme in the extracts of mycelium of Aspergillus niger. 1 Slein (6) has pointed out that the two isomerases which convert G-6-P and mannose 6-phosphate (M-6-P) into F-6-P, may now be more descriptively called phosphoglucose isomerase and phosphomannose isomerase, respectively, since it is now known that F-6-P is the product of M-6-P isomerization also. We propose to adopt this nomenclature. 91
92
T. RAMASARMA
AND K. V. GIRI
Evidence in support of the existence of an Embden-Meyerhof glycolytic pathway in green gram (Phasedus rczdiatus) has already been advanced in two earlier communications from this laboratory by Sri Ram and Giri (11) and by Ramasarma, Sri Ram, and Giri (12). The presence of phosphoglucose isomerase in green gram extracts was indicated by the increase in the concomitant fructose content in the reaction mixtures used for phosphoglucomutase assay. We have now been able to identify the enzyme using both G-6-P and F-6-P as substrates. In this paper the separation and characterization of phosphoglucose isomerase in green gram are reported. EXPERIMENTAL
Materials and Methods G-6-P (Ba salt) and F-6-P (Ba salt) were obtained from Nutritional Biochemicals. G-6-P (Ba salt) prepared enzymatically by the method of Colowiek and Sutherland (13) using sheep muscle as the source of phosphoglucomutase was also used. Fructose was determined by the method of Roe (14). The value for F-6-P was about 60% of that of equivalent free fructose which is in good agreement with the value reported by Umbreit and his associates (15).
Estimation of Enzyme Activity The reaction mixture always contained, unless otherwise stated, 2 amoles G-6-P, or 1 pmole of F-6-P wherever indicated, Verona1 buffer of pH 7.5-7.8 at a final concentration of 0.02 M, and enzyme solution containing 0.005 mg. protein N in a total volume of 0.25 ml. The reaction mixture was incubated at 37” for 2 min., and the reaction was stopped by the addition of hydrochloric acid used in the determination of fructose. The enzyme activity was expressed as micromoles of substrate converted per minute.
Partial Puri$.cation of the Enzyme The resting seeds of green gram were finely powdered, and the powder was extracted with five times its weight of water for 12 hr. under a layer of toluene in a refrigerator. One hundred milliliters of crude extract was adjusted to pH 7.0 with dilute alkali, and 100 ml. of cold acetone was slowly added with stirring. The precipitate obtained was centrifuged and discarded. Two hundred milliliters more of acetone was added to the supernatant. The precipitate obtained was dissolved in water and dialyzed against several changes of distilled water for 2 days. Any precipitate formed on dialysis was centrifuged and discarded. The temperature was maintained between 0 and 10” during these operations. A sixfold purification was achieved by this method, and the recovery was about 80% of the original activity. The enzyme preparations usually showed an activity of about 40 pmoles substrate conversion/min./mg. nitrogen.
PHOSPHOGLUCOSE
ISOMERASE
93
TABLE I of pH on Enzyme Activity The experimental conditions were the same as specified in the text, except the pH of the buffer used was varied. Acetate buffer at final concentration of 0.02 M was used to obtain pH 4.1-5.6. Erect
PH
4.1 4.6 5.1 5.6 6.5 7.0 7.4 7.8 8.3 8.7 9.0 9.2
Substrate G-6-P substrate p?nolcs/min.
0.00 0.02 0.05 0.13 0.13 0.15 0.18 0.25 0.20 0.20 0.19 0.18
converted F-6-P substrate pmoles/min.
0.03 0.04 0.11 0.14 0.14 0.18 0.20 0.17
These preparations did not liberate any phosphorus from G-6-P, F-6-P, and glucose l-phosphate. Phosphoglucomutase was found to be inactivated by acetone. These preparations were found to be completely free from fructose containing carbohydrates and inorganic phosphates which were present in large quantities in crude extracts.
RESULTS The enzyme was found to be active over a wide range of pH. The optimun was about 7.8 when G-6-P was used as the substrate and about 9.0 when F-6-P was used as the substrate (Table I). The reaction velocities were determined at various concentrations of G-6-P (1 X l!P to 10 X W3 M), and on plotting the reciprocals of the reaction velocities against the reciprocals of the substrate concentrations in accordance with the Lineweaver and Burk (16) modification of the Michaelis and Menten equation (17), a linear relationship was obtained. From the graph the value of the Michaelis constant was found to be 0.00345 moles/l. The enzyme activity was found to be proportional to the enzyme concentration until a change in substrate of about 15%. The temperature optimum for the enzyme activity was found to be 50” (Table II). The plot of the logarithm of activity against the reciprocal of absolute temperature gave a linear relationship. A value of 4800 cal./mole was obtained for the energy of activation calculated from the
94
T.
RAMASARMA
AND
TABLE E&cl
of Temperature
K.
V.
GIRI
II on Enzyme Activity
The experimental conditions were the same as specified in the text, except that the temperature was varied. The enzyme was incubated at the various temperatures for 10 min. before the substrate was added. Temperature “C.
20 25 30 35 40 45 48 50 52 55 60 70
Substrate converted pmoles/?dn.
0.07 0.11 0.16 0.18 0.20 0.23 0.25 0.27 0.25 0.21 0.07 0.00
graph. For the average temperature range between 35 and 45”, &IO was found to be 1.3. Equilibrium was approached with both G-6-P and F-6-P as substrates and, in both cases, the composition of the mixture at equilibrium was found to be about 60 % of G-6-P and 40 % of F-6-P (Fig. 1). Somers and Cosby (3) and Bhdansky (8) have observed that the phosphatase present in the crude preparations they used hydrolyzed F-6-P, which resulted in an increase in fructose color value. Bodansky has determined the amount, of phosphorus formed, and, assuming that the phosphorus was equally cleaved from both G-6-P and F-6-P, he calculated a value of 60% of G-6-P and 40% of F-6-P at, equilibrium. We have obtained the same figure using the purified preparation which did not hydrolyze the phosphoric esters. The enzyme was found to be stable for at least 4 weeks when kept frozen in a refrigerator, and the activity slowly decreased thereafter. Exposure of the enzyme to 0.02 N HCl or 0.02 N NaOH caused irreversible and complete inactivation of the enzyme. The enzyme was found to be rapidly inactivated when heated at temperatures above 45”. Incubation for 10 min. at 50, 55, and 60” caused a decrease in activity of about 20,33, and 90 %, respectively, at pH 7.5. The enzyme was found to be more resistant to heat denaturation in the alkaline range than in
PHOSPHOGLUCOSE
2
4
ISOMERASE
95
6 TIME,
MINUTES
1. Time course reaction of phosphoglucose isomerase activity and equilibrium between G-6-P and F-6-P. The experimental conditions were the same as specified in the text except that the reaction was stopped at different time intervals. 0, G-6-P conversion; 0, F-6-P conversion. FIG.
the acid range. The enzyme could be heated ‘at 50” at pH 8.0-9.0 for 10 min. with a lossin activity amounting to only about 6-8 %. Magnesium, manganese, and cobalt ions showed no activation of the enzyme between the concentrations 0.001 and 0.01 M. BaC12 (0.01 M), arsenate (0.01 M), and NaF (0.02 M) did not affect the enzyme at the concentrations tested. HgCL (0.002 M), KCN (0.02 M), and ZnSOc (0.001 M) inhibited the enzyme activity about 13, 20, and 18%, respectively. Iodoacetic acid (0.001X~002 M) did not affect the enzyme even after incubation for 10 min. at, 37”, indicating that the enzyme is not dependent upon the functional -SH groups for its activity. Glucose (0.1-0.8 mg.), fructose (0.1-0.2 mg.), and glucose l-phosphate (0.1-0.8 mg.) were not affected by the enzyme, and they showed very little effect either as activators or inhibitors when tested at the concentrations indicated above in the standard mixtures for the enzyme assay. Glucose 1,6-diphosphate (0.1 mg.) with F-6-P as the substrate and fructose 1,6-diphosphate (0.1 mg.) with G-6-P as the substrate did not, show any activation. Uridine diphosphoglucose, triphosphopyridine nucleotide, and diphosphopyridine nucleotide failed to activate the enzyme when tested in catalytic amounts. Aqueous extract of brewer’s
yeast, which is known to contain many of the nucleotides, also similarly failed to activate the enzyme.
96
T.
RAMASARMA
AKD
K.
V.
GIRI
ACKNOWLEDGMENT We are indebted to Prof. L. F. Leloir of the Institute of Biochemical Investigation, Julian Alvarez, Ruenos Aires, Argentina, for the gift of samples of glucose 1,6-diphosphate and uridine diphosphoglucose.
SUMMARY Phosphoglucoseisomeraseof green gram was partially purified by acetone fractionation. The enzyme was found to be rapidly inactivated when heated above 45”. The enzyme was found to be more resistant to heat denaturation in the alkaline range of pH. The optimum pH of the enzyme was around 7.8 with G-6-P as substrate and 9.0 with F-6-P as subst,rate. Temperature optimum was 50’. At equilibrium, about 60% of G-6-P and 40 % of F-6-P were found to exist. REFERENCES 1. LOHMANN, K., Biochem. 2. 262, 137 (1933). 2. TANKO, B., Biochem. J. 20, 692 (1936). 3. SOMERS, G. F., AND COBBY, E. L., Arch. Biochem. 6,295 (1945). 4. CORI, G. T., COLOWICK, S. P., AND CORI, C. F., J. Biol. Chem. l24,543 (1938). 5. SLEIN, M. W., J. Biol. Chem. 186, 753 (1950). 6. SLEIN, M. W., Federation Proc. 13, 988 (1954). 7. CORI, G. T., OCAOA, S., SLEIN, M. W., AND CORI, C. F., Biochim. et Biophys. Acta 7, 304 (1951). 8. BODANSKY, O., J. Biol. Chem. 202, 829 (1953). 9. SABLE, H. Z., AND CALKINS, C. W., JR., J. Biol. Chem. 204,695 (1963). 10. JAGANNATHAN, V., AND SINGH, K., Enzymologia 156, 150 (1953). 11. SRI RAM, J., AND GIRI, K. V., Arch. Biochem. and Biophys. 38,231 (1952). 12. RAMASARMA, T., SRI RA~I, J., AND GIRI, K. V., Arch. Rio&m. and Biophya. 63, 167 (1954). 13. COLOWICK, S. P., AND SUTHERLAND, W., J. Biol. Chem. 144,422 (1942). 14. ROE, J. H., J. Biol. Chem. 107, 15 (1934). 15. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. K., “Manometric Techniques and Related Methods for the Study of Tissue Metabolism,” p, 169. Burgess Publ. Co., Minneapolis, 1943. 16. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66,658 (1934). 17. MICHAELIS, I.., AND MENTEN, M. L., Biochem. 2. 49,333 (1913).