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[19] GDPM Pyrophosphorylase
[19]
GDPM PYROPHOSPHORYLASE
171
ammonium sulfate. The pooled extracts are dialyzed overnight. The precipitate is washed and extracted again with ammonium sulfate solution. The precipitation by dialysis and the extraction are repeated if necessary in order to obtain a preparation with a low content in phosphatase and in UDPG-fructose transglucosylase.
Properties Even the best preparation obtained by the method described still contains some phosphatase, and hence the reaction product was usually a mixture of sucrose phosphate and free fructose. pH optimum. The optimum pH is 6.4 (0.15M Tris or acetate buffers). Kinetics Properties. The apparent Michaelis constant at different concentrations of F-6-P is 2.2 X 10-'~.
[ 19]
GDPM
Pyrophosphorylase
GDPM q- PP ~ GTP 4- M-1-P
By AGNETE MUNCH-PETERSEN
Assay Method Principle. Two different assay methods have been used for following the pyrophosphorolytic cleavage of GDPM. For estimation of specific enzyme activity in crude tissue extracts, both methods require a substrate free of other nueleotide phosphoric esters, since these may also undergo pyrophosphorolysis. The first method is based on the formation of P32-1abeled GTP when P32-1abeled pyrophosphate is used in the reaction. The GTP formed may be isolated by adsorption on charcoal and estimated through the radioactivity contained in the charcoal. In the second method, which is described below, the formation of GTP is measured spectrophotometrically in the following sequence of enzymatic reactions : GDPM 4- PP GTP 4- ADP ATP q- glucose Glueose-6-phosphate 4- TPN 4- H20
--~ GTP 4- mannose-l-phosphate ~ GDP ~ ATP --~ ADP 4- glueose-6-phosphate -~ 6-Phosphoglueonic acid 4- TPNH
The rate of pyrophosphorolysis is measured by the increase in optical density at 340 m~ due to the formation of TPNH.
172
ENZYMES OF CARBOHYI)RATE METABOLISM
[19]
Reagents GDPM, 0.1 micromole. PP, 1 micromole. ADP, 0.05 micromole. Glucose, 2 micromoles. Magnesium chloride, 5 micromoles. 0.05 M Tris-HC1 buffer, pH 7.5. Hexokinase, 25 Ixl. (10 mg./ml.). Glucose-6-phosphate dehydrogenase, 15 Ixl. (1 mg./ml.). GDPM pyrophosphorylase. The amounts of hexokinase and glucose-6-phosphate dehydrogenase depend on their specific activity. Both enzymes should be in excess so that the GDPM pyrophosphorylase is the limiting factor. Procedure. The reagents except pyrophosphate are mixed in a 1-ml. quartz cuvette to give a final volume of 1 ml. To avoid interference from pyrophosphatases, sodium fluoride in a concentration of 5 X 10-s M is added if crude tissue extracts are examined. The optical density at 340 mix is read in a spectrophotometer, and when PP is added the rise in optical density per minute, at 340 mix is registered. This rise is proportional to the rate of pyrophosphorolysis of GDPM. An extinction coefficient of 6.22 X l0 Gcm. 2 per mole T P N H was used in the calculations.1 Definition o] Unit and Specific Activity. One unit of enzyme is defined as the amount causing the pyrophosphorolysis of 1 micromole of GDPM per minute. Specific activity is defined as units per milligram of protein. Protein concentrations have been determined by the nephelometric method described by Biicher. 2 Purification Procedure All operations, unless specially indicated, were carried out at 2 ° to 5 ° .
Step 1. Preparation of Crude Extract. Twenty grams of dried brewer's yeast was suspended in 60 ml. of 0.1 M sodium bicarbonate and autolyzed for 5 hours at 37 °. The flask was shaken mechanically during the autolysis. The mixture was centrifuged for 20 minutes at 7000 X g, and 36 ml. of supernatant was obtained. Step 2. Ammonium SulJate Fractionation. The supernatant was diluted with the same volume of distilled water, and 72 ml. of saturated ammonium sulfate solution was added. After 30 minutes at 2 ° the mixture was centrifuged and the precipitate discarded. Then 86.7 ml. of saturated ammonium sulfate solution was added dropwise, with stirring, lB. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). 2 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).
[19]
GDPM PYROPHOSPHORYLASE
173
to 130 ml. of supernatant. The mixture was left overnight at 2 ° to complete precipitation. Step 3. Ethanol Fractionation. Next day the precipitate was separated by centrifugation and dissolved in 25 ml. of 0.05 M Tris buffer, pH 8.3. The solution was dialyzed for 2 hours against tap water. The 40 ml. of dialyzate, obtained in this way, was diluted with 40 ml. of 0.01 M phosphate buffer, pH 7.0, and cooled to 0 ° in dry ice-alcohol bath. Then 65 ml. of 50% ethanol (by volume) was added dropwise with mechanical stirring. The temperature of the bath was gradually lowered to --10 ° and kept there during the fractionation procedure. The precipitate was separated by centrifugation at --10 ° and discarded. A further 50 ml. of 50% ethanol (by volume) was added to the supernatant. The precipitate was centrifuged down at --10 °, the supernatant was discarded, and the centrifuge tubes with the precipitates were immediately placed at --16 ° and kept at this temperature overnight. Step 5. Calcium Phosphate Gel Adsorption. The precipitate was dissolved in 5 ml. of 0.02 M phosphate buffer, pH 6.5. The solution was centrifuged, and the opalescent supernatant was diluted with the same volume of distilled water before it was subjected to calcium phosphate gel adsorption. Ten milliliters of solution was treated twice with 0.6 ml. of calcium phosphate gel (dry weight 31 mg./ml.). The calcium phosphate gel precipitates were discarded. The supernatant was then treated with 0.8 ml. of calcium phosphate gel. The gel was centrifuged down, washed with 2 ml. of distilled water, and eluted twice with 1 ml. of 0.1M phosphate buffer, pH 7.5. This fraction was clear and colorless and had a protein concentration which varied from 3 to 6 mg./ml. This fraction represents a purification of seventy- to eightyfold as compared to the autolyzate and an over-all yield of 17 to 18%. For unknown reasons different batches of dried yeasts were found to be extremely variable in GDPM pyrophosphorylase content. The figures shown in the table represent a yeast of high pyrophosphorylase content and were reproducible for this yeast. SUMMARY OF PURIFICATION PROCEDURE
Fraction
Autolyzate Ammonium sulfate 22-30% ethanol Calcium phosphate gel eluate
Volume, ml.
Total activity, units
Yield, %
Specific activity, units/mg. protein
36 40 5 2
34 8 39 4 11.0 6.1
100 (113) 31.6 17 .'5
0. 0167 0.0296 0.12l 1.32
174
ENZYMES OF CARBOHYDRATE METABOLISM
[20]
Properties p H Ef]ect. The curve representing the p H dependence of the reaction shows t h a t the m a x i m u m velocity is reached between p H 8.1 and p H 8.5. At p H 7.5 and at p H 9.2 the reaction velocities are 70% of t h a t at p H 8.5. Specificity. T h e purified fraction was free of inorganic pyrophosphatase. I t contained traces of U D P G and U D P A G pyrophosphorylases. Activator. A divalent metal ion such as M g ++ is required in the reaction. A M g ++ ion concentration of 5 X 10 - 3 M has been used throughout. Dissociation Constant. The Michaelis constant for G D P M has been found to be 1.1 X 10 -5 M at p H 7.5.
[20]
Enzymes
of the Leloir Pathway
I-4
B y ELIZABETH S. MAXWELL, KIYOSHI KURAHASHI, and HERMAN M. t~ALCKAR
I. Q u a n t i t a t i v e D e t e r m i n a t i o n of E n z y m a t i c A c t i v i t y in B r o k e n Cell Preparations The following methods are specific enzymatic assays designed especially for use with crude bacterial extracts. In cases where less rigorous controls are required the assays described later in the sections on purification of enzymes are more suitable. Galactokinase 5-7 Principle. The enzyme catalyzes the reaction A T P + G a l - ~ A D P +a-Gal-l-P. The G a l - I - P produced in a preincubation mixture is quantitatively determined. Fluoride is added in order to protect A T P and G a l - I - P from being dephosphorylated by phosphatase. Addition of magnesium ions serves to ensure t h a t the reaction, which depends on
1L. F. Leloir, Arch. Biochem. Biophys. 33, 186 (1951), see also Vol. I [35]. R. Caputto, L. F. Leloir, R. E. Trucco, C. E. Cardini, and A. J. Paladini, .l. Biol. Chem. 179, 497 (1949). I-I. M. Kalckar, B. Braganca, and A. Munch-Petersen, Nature 172, 1036 (1953). 4A. Muneh-Petersen, H. M. Kalckar, and E. E. B. Smith, Kgl. Da~ske Videnslcab. Selslcab, Biol. Medd. 22, 3 (1955). 5 t:i. W. Kosterlitz, B~ochem. J. 37, 322 (1943). ~R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittelman, Arch. Biochem. 18, 137 (1948). 'C. E. Cardini and L. F. Leloir, Arch. Biochem. Biophys. 45, 55 (1953).
GDPM PYROPHOSPHORYLASE
171
ammonium sulfate. The pooled extracts are dialyzed overnight. The precipitate is washed and extracted again with ammonium sulfate solution. The precipitation by dialysis and the extraction are repeated if necessary in order to obtain a preparation with a low content in phosphatase and in UDPG-fructose transglucosylase.
Properties Even the best preparation obtained by the method described still contains some phosphatase, and hence the reaction product was usually a mixture of sucrose phosphate and free fructose. pH optimum. The optimum pH is 6.4 (0.15M Tris or acetate buffers). Kinetics Properties. The apparent Michaelis constant at different concentrations of F-6-P is 2.2 X 10-'~.
[ 19]
GDPM
Pyrophosphorylase
GDPM q- PP ~ GTP 4- M-1-P
By AGNETE MUNCH-PETERSEN
Assay Method Principle. Two different assay methods have been used for following the pyrophosphorolytic cleavage of GDPM. For estimation of specific enzyme activity in crude tissue extracts, both methods require a substrate free of other nueleotide phosphoric esters, since these may also undergo pyrophosphorolysis. The first method is based on the formation of P32-1abeled GTP when P32-1abeled pyrophosphate is used in the reaction. The GTP formed may be isolated by adsorption on charcoal and estimated through the radioactivity contained in the charcoal. In the second method, which is described below, the formation of GTP is measured spectrophotometrically in the following sequence of enzymatic reactions : GDPM 4- PP GTP 4- ADP ATP q- glucose Glueose-6-phosphate 4- TPN 4- H20
--~ GTP 4- mannose-l-phosphate ~ GDP ~ ATP --~ ADP 4- glueose-6-phosphate -~ 6-Phosphoglueonic acid 4- TPNH
The rate of pyrophosphorolysis is measured by the increase in optical density at 340 m~ due to the formation of TPNH.
172
ENZYMES OF CARBOHYI)RATE METABOLISM
[19]
Reagents GDPM, 0.1 micromole. PP, 1 micromole. ADP, 0.05 micromole. Glucose, 2 micromoles. Magnesium chloride, 5 micromoles. 0.05 M Tris-HC1 buffer, pH 7.5. Hexokinase, 25 Ixl. (10 mg./ml.). Glucose-6-phosphate dehydrogenase, 15 Ixl. (1 mg./ml.). GDPM pyrophosphorylase. The amounts of hexokinase and glucose-6-phosphate dehydrogenase depend on their specific activity. Both enzymes should be in excess so that the GDPM pyrophosphorylase is the limiting factor. Procedure. The reagents except pyrophosphate are mixed in a 1-ml. quartz cuvette to give a final volume of 1 ml. To avoid interference from pyrophosphatases, sodium fluoride in a concentration of 5 X 10-s M is added if crude tissue extracts are examined. The optical density at 340 mix is read in a spectrophotometer, and when PP is added the rise in optical density per minute, at 340 mix is registered. This rise is proportional to the rate of pyrophosphorolysis of GDPM. An extinction coefficient of 6.22 X l0 Gcm. 2 per mole T P N H was used in the calculations.1 Definition o] Unit and Specific Activity. One unit of enzyme is defined as the amount causing the pyrophosphorolysis of 1 micromole of GDPM per minute. Specific activity is defined as units per milligram of protein. Protein concentrations have been determined by the nephelometric method described by Biicher. 2 Purification Procedure All operations, unless specially indicated, were carried out at 2 ° to 5 ° .
Step 1. Preparation of Crude Extract. Twenty grams of dried brewer's yeast was suspended in 60 ml. of 0.1 M sodium bicarbonate and autolyzed for 5 hours at 37 °. The flask was shaken mechanically during the autolysis. The mixture was centrifuged for 20 minutes at 7000 X g, and 36 ml. of supernatant was obtained. Step 2. Ammonium SulJate Fractionation. The supernatant was diluted with the same volume of distilled water, and 72 ml. of saturated ammonium sulfate solution was added. After 30 minutes at 2 ° the mixture was centrifuged and the precipitate discarded. Then 86.7 ml. of saturated ammonium sulfate solution was added dropwise, with stirring, lB. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948). 2 T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).
[19]
GDPM PYROPHOSPHORYLASE
173
to 130 ml. of supernatant. The mixture was left overnight at 2 ° to complete precipitation. Step 3. Ethanol Fractionation. Next day the precipitate was separated by centrifugation and dissolved in 25 ml. of 0.05 M Tris buffer, pH 8.3. The solution was dialyzed for 2 hours against tap water. The 40 ml. of dialyzate, obtained in this way, was diluted with 40 ml. of 0.01 M phosphate buffer, pH 7.0, and cooled to 0 ° in dry ice-alcohol bath. Then 65 ml. of 50% ethanol (by volume) was added dropwise with mechanical stirring. The temperature of the bath was gradually lowered to --10 ° and kept there during the fractionation procedure. The precipitate was separated by centrifugation at --10 ° and discarded. A further 50 ml. of 50% ethanol (by volume) was added to the supernatant. The precipitate was centrifuged down at --10 °, the supernatant was discarded, and the centrifuge tubes with the precipitates were immediately placed at --16 ° and kept at this temperature overnight. Step 5. Calcium Phosphate Gel Adsorption. The precipitate was dissolved in 5 ml. of 0.02 M phosphate buffer, pH 6.5. The solution was centrifuged, and the opalescent supernatant was diluted with the same volume of distilled water before it was subjected to calcium phosphate gel adsorption. Ten milliliters of solution was treated twice with 0.6 ml. of calcium phosphate gel (dry weight 31 mg./ml.). The calcium phosphate gel precipitates were discarded. The supernatant was then treated with 0.8 ml. of calcium phosphate gel. The gel was centrifuged down, washed with 2 ml. of distilled water, and eluted twice with 1 ml. of 0.1M phosphate buffer, pH 7.5. This fraction was clear and colorless and had a protein concentration which varied from 3 to 6 mg./ml. This fraction represents a purification of seventy- to eightyfold as compared to the autolyzate and an over-all yield of 17 to 18%. For unknown reasons different batches of dried yeasts were found to be extremely variable in GDPM pyrophosphorylase content. The figures shown in the table represent a yeast of high pyrophosphorylase content and were reproducible for this yeast. SUMMARY OF PURIFICATION PROCEDURE
Fraction
Autolyzate Ammonium sulfate 22-30% ethanol Calcium phosphate gel eluate
Volume, ml.
Total activity, units
Yield, %
Specific activity, units/mg. protein
36 40 5 2
34 8 39 4 11.0 6.1
100 (113) 31.6 17 .'5
0. 0167 0.0296 0.12l 1.32
174
ENZYMES OF CARBOHYDRATE METABOLISM
[20]
Properties p H Ef]ect. The curve representing the p H dependence of the reaction shows t h a t the m a x i m u m velocity is reached between p H 8.1 and p H 8.5. At p H 7.5 and at p H 9.2 the reaction velocities are 70% of t h a t at p H 8.5. Specificity. T h e purified fraction was free of inorganic pyrophosphatase. I t contained traces of U D P G and U D P A G pyrophosphorylases. Activator. A divalent metal ion such as M g ++ is required in the reaction. A M g ++ ion concentration of 5 X 10 - 3 M has been used throughout. Dissociation Constant. The Michaelis constant for G D P M has been found to be 1.1 X 10 -5 M at p H 7.5.
[20]
Enzymes
of the Leloir Pathway
I-4
B y ELIZABETH S. MAXWELL, KIYOSHI KURAHASHI, and HERMAN M. t~ALCKAR
I. Q u a n t i t a t i v e D e t e r m i n a t i o n of E n z y m a t i c A c t i v i t y in B r o k e n Cell Preparations The following methods are specific enzymatic assays designed especially for use with crude bacterial extracts. In cases where less rigorous controls are required the assays described later in the sections on purification of enzymes are more suitable. Galactokinase 5-7 Principle. The enzyme catalyzes the reaction A T P + G a l - ~ A D P +a-Gal-l-P. The G a l - I - P produced in a preincubation mixture is quantitatively determined. Fluoride is added in order to protect A T P and G a l - I - P from being dephosphorylated by phosphatase. Addition of magnesium ions serves to ensure t h a t the reaction, which depends on
1L. F. Leloir, Arch. Biochem. Biophys. 33, 186 (1951), see also Vol. I [35]. R. Caputto, L. F. Leloir, R. E. Trucco, C. E. Cardini, and A. J. Paladini, .l. Biol. Chem. 179, 497 (1949). I-I. M. Kalckar, B. Braganca, and A. Munch-Petersen, Nature 172, 1036 (1953). 4A. Muneh-Petersen, H. M. Kalckar, and E. E. B. Smith, Kgl. Da~ske Videnslcab. Selslcab, Biol. Medd. 22, 3 (1955). 5 t:i. W. Kosterlitz, B~ochem. J. 37, 322 (1943). ~R. E. Trucco, R. Caputto, L. F. Leloir, and N. Mittelman, Arch. Biochem. 18, 137 (1948). 'C. E. Cardini and L. F. Leloir, Arch. Biochem. Biophys. 45, 55 (1953).