- Email: [email protected]
[67] Pyruvate dehydrogenase phosphatase from bovine heart
402
PHOSPHATASES
[67]
(Activity in presence of DOC) - (activity without DOC) × 100 activity in presence of DOC Liver microsomes have a much smaller latency (i.e., ca. 30%) toward glucose 6-phosphate, but latency toward mannose 6-phosphate is virtually total. 14 2, Brain microsome preparations did not exhibit sensitivity to diisothiocyanostilbene disulfonate (DIDS) under conditions is that inhibited the glucose-6-phosphatase of intact liver microsomes. The sensitivity to DIDS of liver enzyme system has been used in support of the concept that the microsomes have a transport protein to convey glucose 6-phosphate from the cytoplasmic side of the endoplasmic reticulum to the lumenal side. 14 The action of DIDS is blocked by the simultaneous presence of glucose 6-phosphate, but not of mannose 6-phosphate. 15 The apparent lack of a transport protein for glucose 6-phosphate distinguishes the brain enzyme from the liver preparation. 14 A. J. Lange, W. J. Arion, and A. L. Beaudet, J. Biol. Chem. 255, 8381 (1980). 1.5 M. A. Zoccoli and M. L. K a r n o v s k y , J. Biol. Chem. 255, 1113 (1980).
[67] P y r u v a t e D e h y d r o g e n a s e P h o s p h a t a s e f r o m Bovine Heart
By F L O R A
H. PETTIT, W. MARTIN TEAGUE, and
(Phospho),,pyruvate dehydrogenase(El) + ~1 H20
~
L E S T E R J. R E E D
pyruvate dehydrogenase+ n P,
Pyruvate dehydrogenase phosphatase is a mitochondrial enzyme that catalyzes dephosphorylation and concomitant reactivation of the phosphorylated, inactive pyruvate dehydrogenase component (El) of the pyruvate dehydrogenase complex. It is a Mg2+-dependent and Ca2+-stimulated phosphatase. Assay Method
Principle. Assay of pyruvate dehydrogenase phosphatase activity is based on measurement of the initial rate of reactivation of phosphorylated, inactive pyruvate dehydrogenase complex from bovine kidney or
METHODS IN ENZYMOLOGY. VOL. 90
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
[67]
PYRUVATE DEHYDROGENASE PHOSPHATASE
403
heart. The method is modified from that of Linnet al. 1 Phosphatase activity may also be determined by measuring the rate of release of 32p-labeled phosphoryl groups from enzyme complex that has been phosphorylated with [y-32P]ATE2"~ These assay methods may not be applicable to whole tissue extracts in view of reports 4'5 of the existence of substantial amounts of extramitochondrial pyruvate dehydrogenase phosphatase-like activity. It seems likely that this activity is due to a cytosolic phosphoprotein phosphatase of broad substrate specificity.6 Reagents Assay buffer: 0.01 M sodium 2-(N-morpholino)propane sulfonate (MOPS), pH 7.0, 0.1 mM CaCI2, 0.5 mM MgC12, 0.5 mM dithiothreitol MgCI2, 0.1 M ATP, 0.01 M Glucose, 0.1 M Hexokinase (Boehfinger), 1 mg/ml Preparation of Substrate. Phosphorylated, inactive pyruvate dehydrogenase complex is prepared by incubating a solution containing 1 mg of highly purified complex from bovine kidney, 7 essentially free of pyruvate dehydrogenase phosphatase, in 1 ml of assay buffer with 0.002 ml of 0.01 M ATP for 15 min at 30°. A 0.01-ml aliquot of the incubation mixture is assayed for pyruvate dehydrogenase complex activity, 7 which should be less than 5% of the activity of the untreated complex. One-hundredth milliliter of 0. ! M glucose and 0.001 ml of hexokinase solution are added to scavenge the remaining ATE After 1 rain at 30°, the preparation is placed in an ice bath. Procedure. To 0.08 ml of phosphorylated, inactive pyruvate dehydrogenase complex (80 t~g) is added 0.01 ml of phosphatase solution containT. C. Linn, J. W. Pelley, F. H. Pettit, F. Hucho, D. D. Randall, and L. J. Reed, Arch. Biochem. Biophys. 148, 327 (1972). 2 E. A. Siess and O. H. Wieland, E,r. J. Biochern. 26, 96 (1972). :~ R. M. Denton, E J. Randle. and B. R. Martin, Biochem. J. 128, 161 (1972). 4 D. Stansbie, R. M. Denton, B. J. Bridges, H. T. Pask, and P. J. Randle, Biochem. J. 154, 225 (1976). '~ E. A. Siess and O. H. Wieland, FEBS Lett. 65, 163 (1976). A highly purified preparation of a low-molecular-weight (32,000) phosphorylase phosphatase (designated protein phosphatase C-I), kindly furnished by S. R. Silberman and E. Y. C. Lee, exhibited activity toward phosphorylated pyruvate dehydrogenase complex comparable to that of the homogeneous pyruvate dehydrogenase phosphatase described in this article. 7 F. H. Pettit and L. J. Reed, this series, Vol. 89 [651.
404
PnOSPHATASES
[6 7]
ing 0.1-0.4 u n i t ? The solution is incubated at 30 ° for 1 rain, and the reaction is started by addition of 0.01 ml of 0.1 M MgC12. The control contains buffer instead of phosphatase. After 2 min, a 0.02-ml aliquot is withdrawn for assay of pyruvate dehydrogenase complex activity. 7 Units. One unit of pyruvate dehydrogenase phosphatase activity is defined as the amount of e n z y m e that reactivates one unit of p y r u v a t e dehydrogenase complex activity per minute at 30 °. Specific activity is expressed as units per milligram of protein. Pyruvate dehydrogenase complex activity is expressed as micromoles of N A D H produced per minute at 30°. 7 Purification Procedure The procedure is applicable to purification of p y r u v a t e dehydrogenase phosphatase from both heart and kidney mitochondria. H o w e v e r , heart mitochondria contain at least 3 times as much phosphatase activity as kidney mitochondria and is the preferred source for isolation of the phosphatase. Forty to sixty percent of the phosphatase is associated with the m e m b r a n e fraction after extraction of the pyruvate dehydrogenase complex from mitochondria. The remainder of the phosphatase copurifies with the complex. The phosphatase binds to the dihydrolipoyl transacetylase (Ez) component of the complex in the presence of Ca2+2 The phosphatase is separated from the complex by ultracentrifugation in the presence of the CaZ+-chelating agent ethylene g l y c o l - b i s ( 2 - a m i n o e t h y l ) - N , N ' - t e t r a a c e t a t e (EGTA). This portion of the total phosphatase is combined with that extracted from the m e m b r a n e fraction. A key step in the purification procedure is affinity c h r o m a t o g r a p h y on E2 coupled to Sepharose 4B. In the presence of Ca 2+ the phosphatase binds to the absorbent and is subsequently released in the presence of EGTA. The purification procedure is summarized in the table. Unless specified otherwise, all operations are performed at 4 ° . Preparation of E2-Sepharose 4B. A mixture of 15 g of CNBr-activated Sepharose 4B (Sigma Chemical Co., St. Louis, Missouri) and 100 ml of 0.2 M NaHCO3 is adjusted to p H 9.2 with N a O H . Highly purified dihydrolipoyl transacetylase (80 mg) from bovine heart '° is added, and the mixture is stirred gently for 20 hr. The gel is washed on a sintered-glass When possible, phosphatase is added to the assay mixture from concentrated solution to minimize dilution of the enzyme. If dilution is necessary, assay buffer containing 0.3% bovine serum albumin is used. 9 F. H. Pettit, T. E. Roche, and L. J. Reed, Biochem. Biophys. Res. Commun. 49, 563 (1972). ,0 F. H. Pettit, S. J. Yeaman, and L. J. Reed, this volume [30]. Dihydrolipoyl transacetylase obtained from step 2 of the purification procedure is used.
[67]
PYRUVATE DEHYDROGENASE PHOSPHATASE
405
PURIFICATION OF PYRUVATE DEHYDROGENASE PHOSPHATASE FROM BOVINE HEART ~z
Fraction M e m b r a n e extract First protamine precipitation Ultracentrifuge s u p e r n a t a n t ~ Combined fractions 2 and 3 Second protamine precipitation Ultracentrifugation Affinity c h r o m a t o g r a p h y concentrate
Volume (ml)
Protein (mg)
Specific activity t'
Recovery (9~)
1830 283 260 543 292 280 1.8
6240 1190 923 2114 742 403 5.8
2.1 9.5 8.5 9.0 22.6 36.3 1655
---100 78 69 45
'~ From about 80 p o u n d s of heart. t, Units of pyruvate d e h y d r o g e n a s e complex reactivated per minute per milligram of protein. " P h o s p h a t a s e released from partially purified pyruvate d e h y d r o g e n a s e complex by ultracentrifugation in the presence of EGTA.
funnel with 0.2 M NaHCO3 and then with deionized water. The gel is treated with a solution of 250 mg of bovine serum albumin in 100 ml of 0.2 M NaHCO3 for 4 hr and then washed well with 0.2 M NaHCO3 and deionized water. Step 1. Extraction qf Mitochondrial Membrane Fraction. The membrane fraction obtained after extraction of the pyruvate dehydrogenase complex from bovine heart mitochondria 7 is suspended in 2 liters of 0.1 M MOPS, pH 7.0, at 23 °. The suspension is stirred for 1.5 hr and then centrifuged at 30,000 g for 30 min at 20°; the pellets are discarded. Step 2. First Protamine Precipitation. The supernatant fluid from step 1 is cooled to 4 °, and 0.011 volume of 2% protamine sulfate (Elanco Products 1°") is added dropwise with stirring. After 15 min the precipitate is collected by centrifugation at 30,000 g for 15 min; the supernatant fluid is discarded. The protamine precipitate is resuspended, by means of a large glass homogenizer equipped with a motor-driven Teflon pestle, in 300 ml of buffer A (0.02 M MOPS, pH 7.0; 5 m M MgC12; 10% (v/v) glycerol; 0.5 m M dithiothreitol) containing 0.16% yeast sodium ribonucleate (ICN Pharmaceuticalsl°~). The suspension is stirred overnight, then 0.01 volume of 0.2 M EGTA is added. The mixture is warmed to 23 ° for 20 min and then centrifuged at 30,000 g for 15 min at 20 °. The clear, yellow supernatant fluid is cooled to 4°; the pellets are discarded. Step 3. Second Protamine Precipitation. The supernatant fluid from step 2 is combined with the supernatant fluid from the first ultracentrifuga10,, Elanco Products, Indianapolis, Indiana; ICN Pharmaceuticals, Irvine, California; BioMolecular Dynamics, Beaverton, Oregon.
406
PHOSPHATASES
[6 7]
tion step in the purification of the pyruvate dehydrogenase complex. 7 To this solution is added dropwise, with stirring, 0.01 volume of 2% protamine sulfate. After 15 min, the precipitate is collected by centrifugation for 15 min. The protamine precipitate is extracted exactly as described in step 2. Step 4. Ultracentrifitgation. The supernatant fluid from step 3 is centrifuged at 105,000 g for 3.5 hr; the pellets are discarded. To the supernatant fluid is added 0.05 volume of 0.1 M CaC12. Step 5. Affinity Chromatography. The phosphatase solution from step 4 is applied slowly to a column (1.5 × 26 cm) of dihydrolipoyl transacetylase (E~)-Sepharose 4B that has been equilibrated with buffer A containing 2 mM CaC12. The column is washed with 150 ml of this equilibration buffer. The phosphatase is eluted with buffer A containing 2 mM EGTA and 0.02% sodium azide. The active fractions are combined and concentrated by vacuum dialysis against buffer A in a Bio-Molecular Dynamics l°a concentrator. Properties Enzyme obtained from step 5, from both heart and kidney mitochondria, gives one band on polyacrylamide gel electrophoresis with Tris(hydroxymethyl)aminomethane-glycine buffer in the absence of sodium dodecyl sulfate (SDS). 11 The phosphatase has a molecular weight by sedimentation equilibrium of about 150,000 and a sedimentation coefficient (s20.w) of about 7.4 S. 12 It consists of two subunits with molecular weights of about 97,000 and 50,000 as estimated by SDS-gel electrophoresis.13 Phosphatase activity appears to reside in the latter subunit, which is sensitive to proteolysis. Pyruvate dehydrogenase phosphatase preparations from bovine heart and kidney I and pig heart 2 were reported to have a molecular weight of 95,000-I00,000 as estimated by SDSpolyacrylamide gel electrophoresis and by filtration through a calibrated column of Sephadex G-100. The activity of these phosphatase preparations was less than 10% of the activity of the homogeneous preparation described in this chapter. It appears that the earlier preparations were deficient in the protease-sensitive subunit of molecular weight 50,000. Catalytic Properties. Pyruvate dehydrogenase phosphatase catalyzes dephosphorylation at all three phosphorylation sites on El. The phosphatase requires a divalent cation (Mg 2+ or Mn2+). The apparent Km for 11 B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). 12 F. H. Pettit, W. M. Teague, and L. J. Reed, unpublished results. 13 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[68]
SWINE KIDNEY PHOSPHOPROTEIN PHOSPHATASE
407
Mg 2+ is about 2 mM. 14 Ca2+ markedly stimulates phosphatase activity 3,9 in the presence, but not in the absence, of dihydrolipoyl transacetylase (E2). 9 The apparent Km for phosphorylated E, is about 58 #M. In the presence of Ca 2+ and E2 this value is decreased to about 2.9/~M. There is some uncertainty as to the apparent Km for Ca 2+. Values in the range of 1 /xM to 100 # M have been reported. '5'16 Highly purified pyruvate dehydrogenase phosphatase from bovine heart binds one Ca 2+ with a dissociation constant (Kd) of about 8 IzM.'7 In the presence of E2, the phosphatase binds a second Ca 2+ with a Kd of about 5 #M. It appears that Ca 2+ may play a structural role. In the presence of Ca 2+, the phosphatase binds to E2, thereby facilitating the Mg~+-dependent dephosphorylation of phosphorylated E,. Phosphatase activity is inhibited by NADH, and this inhibition is reversed by NAD +.'8 Phosphatase activity is inhibited by fluoride ion, by inorganic orthophosphate, and by increasing the ionic strength. 2'14 Pyruvate dehydrogenase phosphatase exhibits only slight activity toward phosphorylase a, about 10% of the activity observed with phosphorylated pyruvate dehydrogenase complex as substrate. The phosphatase is inactive toward p-nitrophenyl phosphate. It is not inhibited by protein phosphatase inhibitor-1 or inhibitor-2 (kindly furnished by Dr. Philip Cohen). ,4 F. Hucho, D. D. Randall, T. E. Roche, M. W. Burgett, J. W. Pelley, and L. J. Reed,Arch. Biochem. Biophys. 151, 328 (1972). ,s A. L. Kerbey and P. J. Randle, FEBS Lett. 108, 485 (1979). ,6 R. M. Denton and J. G. McCormack, FEBS Lett. 119, 1 (1980). ,7 T-L. Wu and L. J. Reed, unpublished results. ,s F. H. Pettit, J. W. Pelley, and L. J. Reed, Biochem. Biophys. Res. Commun. 65, 575 (1975).
[68] By K.
Phosphoprotein Phosphatase from Swine Kidney MUNIYAPPA, FREDRICH LEIBACH,
and
JOSEPH M E N D I C I N O
Phosphoprotein phosphatase (EC 3.1.3.16) catalyzes the reaction Phosphoenzyme + ,7 H20
phosphoprotein ) dephosphoenzyme + n P~ phosphatase
The homogeneous phosphoprotein phosphatase isolated from swine kidney extracts catalyzes the dephosphorylation of the phosphorylated forms of glycogen synthase, phosphorylase, phosphofructokinase,
METHODS IN ENZYMOLOGY,VOL. 90
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 13,-12-181990-6
PHOSPHATASES
[67]
(Activity in presence of DOC) - (activity without DOC) × 100 activity in presence of DOC Liver microsomes have a much smaller latency (i.e., ca. 30%) toward glucose 6-phosphate, but latency toward mannose 6-phosphate is virtually total. 14 2, Brain microsome preparations did not exhibit sensitivity to diisothiocyanostilbene disulfonate (DIDS) under conditions is that inhibited the glucose-6-phosphatase of intact liver microsomes. The sensitivity to DIDS of liver enzyme system has been used in support of the concept that the microsomes have a transport protein to convey glucose 6-phosphate from the cytoplasmic side of the endoplasmic reticulum to the lumenal side. 14 The action of DIDS is blocked by the simultaneous presence of glucose 6-phosphate, but not of mannose 6-phosphate. 15 The apparent lack of a transport protein for glucose 6-phosphate distinguishes the brain enzyme from the liver preparation. 14 A. J. Lange, W. J. Arion, and A. L. Beaudet, J. Biol. Chem. 255, 8381 (1980). 1.5 M. A. Zoccoli and M. L. K a r n o v s k y , J. Biol. Chem. 255, 1113 (1980).
[67] P y r u v a t e D e h y d r o g e n a s e P h o s p h a t a s e f r o m Bovine Heart
By F L O R A
H. PETTIT, W. MARTIN TEAGUE, and
(Phospho),,pyruvate dehydrogenase(El) + ~1 H20
~
L E S T E R J. R E E D
pyruvate dehydrogenase+ n P,
Pyruvate dehydrogenase phosphatase is a mitochondrial enzyme that catalyzes dephosphorylation and concomitant reactivation of the phosphorylated, inactive pyruvate dehydrogenase component (El) of the pyruvate dehydrogenase complex. It is a Mg2+-dependent and Ca2+-stimulated phosphatase. Assay Method
Principle. Assay of pyruvate dehydrogenase phosphatase activity is based on measurement of the initial rate of reactivation of phosphorylated, inactive pyruvate dehydrogenase complex from bovine kidney or
METHODS IN ENZYMOLOGY. VOL. 90
Copyright © 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181990-6
[67]
PYRUVATE DEHYDROGENASE PHOSPHATASE
403
heart. The method is modified from that of Linnet al. 1 Phosphatase activity may also be determined by measuring the rate of release of 32p-labeled phosphoryl groups from enzyme complex that has been phosphorylated with [y-32P]ATE2"~ These assay methods may not be applicable to whole tissue extracts in view of reports 4'5 of the existence of substantial amounts of extramitochondrial pyruvate dehydrogenase phosphatase-like activity. It seems likely that this activity is due to a cytosolic phosphoprotein phosphatase of broad substrate specificity.6 Reagents Assay buffer: 0.01 M sodium 2-(N-morpholino)propane sulfonate (MOPS), pH 7.0, 0.1 mM CaCI2, 0.5 mM MgC12, 0.5 mM dithiothreitol MgCI2, 0.1 M ATP, 0.01 M Glucose, 0.1 M Hexokinase (Boehfinger), 1 mg/ml Preparation of Substrate. Phosphorylated, inactive pyruvate dehydrogenase complex is prepared by incubating a solution containing 1 mg of highly purified complex from bovine kidney, 7 essentially free of pyruvate dehydrogenase phosphatase, in 1 ml of assay buffer with 0.002 ml of 0.01 M ATP for 15 min at 30°. A 0.01-ml aliquot of the incubation mixture is assayed for pyruvate dehydrogenase complex activity, 7 which should be less than 5% of the activity of the untreated complex. One-hundredth milliliter of 0. ! M glucose and 0.001 ml of hexokinase solution are added to scavenge the remaining ATE After 1 rain at 30°, the preparation is placed in an ice bath. Procedure. To 0.08 ml of phosphorylated, inactive pyruvate dehydrogenase complex (80 t~g) is added 0.01 ml of phosphatase solution containT. C. Linn, J. W. Pelley, F. H. Pettit, F. Hucho, D. D. Randall, and L. J. Reed, Arch. Biochem. Biophys. 148, 327 (1972). 2 E. A. Siess and O. H. Wieland, E,r. J. Biochern. 26, 96 (1972). :~ R. M. Denton, E J. Randle. and B. R. Martin, Biochem. J. 128, 161 (1972). 4 D. Stansbie, R. M. Denton, B. J. Bridges, H. T. Pask, and P. J. Randle, Biochem. J. 154, 225 (1976). '~ E. A. Siess and O. H. Wieland, FEBS Lett. 65, 163 (1976). A highly purified preparation of a low-molecular-weight (32,000) phosphorylase phosphatase (designated protein phosphatase C-I), kindly furnished by S. R. Silberman and E. Y. C. Lee, exhibited activity toward phosphorylated pyruvate dehydrogenase complex comparable to that of the homogeneous pyruvate dehydrogenase phosphatase described in this article. 7 F. H. Pettit and L. J. Reed, this series, Vol. 89 [651.
404
PnOSPHATASES
[6 7]
ing 0.1-0.4 u n i t ? The solution is incubated at 30 ° for 1 rain, and the reaction is started by addition of 0.01 ml of 0.1 M MgC12. The control contains buffer instead of phosphatase. After 2 min, a 0.02-ml aliquot is withdrawn for assay of pyruvate dehydrogenase complex activity. 7 Units. One unit of pyruvate dehydrogenase phosphatase activity is defined as the amount of e n z y m e that reactivates one unit of p y r u v a t e dehydrogenase complex activity per minute at 30 °. Specific activity is expressed as units per milligram of protein. Pyruvate dehydrogenase complex activity is expressed as micromoles of N A D H produced per minute at 30°. 7 Purification Procedure The procedure is applicable to purification of p y r u v a t e dehydrogenase phosphatase from both heart and kidney mitochondria. H o w e v e r , heart mitochondria contain at least 3 times as much phosphatase activity as kidney mitochondria and is the preferred source for isolation of the phosphatase. Forty to sixty percent of the phosphatase is associated with the m e m b r a n e fraction after extraction of the pyruvate dehydrogenase complex from mitochondria. The remainder of the phosphatase copurifies with the complex. The phosphatase binds to the dihydrolipoyl transacetylase (Ez) component of the complex in the presence of Ca2+2 The phosphatase is separated from the complex by ultracentrifugation in the presence of the CaZ+-chelating agent ethylene g l y c o l - b i s ( 2 - a m i n o e t h y l ) - N , N ' - t e t r a a c e t a t e (EGTA). This portion of the total phosphatase is combined with that extracted from the m e m b r a n e fraction. A key step in the purification procedure is affinity c h r o m a t o g r a p h y on E2 coupled to Sepharose 4B. In the presence of Ca 2+ the phosphatase binds to the absorbent and is subsequently released in the presence of EGTA. The purification procedure is summarized in the table. Unless specified otherwise, all operations are performed at 4 ° . Preparation of E2-Sepharose 4B. A mixture of 15 g of CNBr-activated Sepharose 4B (Sigma Chemical Co., St. Louis, Missouri) and 100 ml of 0.2 M NaHCO3 is adjusted to p H 9.2 with N a O H . Highly purified dihydrolipoyl transacetylase (80 mg) from bovine heart '° is added, and the mixture is stirred gently for 20 hr. The gel is washed on a sintered-glass When possible, phosphatase is added to the assay mixture from concentrated solution to minimize dilution of the enzyme. If dilution is necessary, assay buffer containing 0.3% bovine serum albumin is used. 9 F. H. Pettit, T. E. Roche, and L. J. Reed, Biochem. Biophys. Res. Commun. 49, 563 (1972). ,0 F. H. Pettit, S. J. Yeaman, and L. J. Reed, this volume [30]. Dihydrolipoyl transacetylase obtained from step 2 of the purification procedure is used.
[67]
PYRUVATE DEHYDROGENASE PHOSPHATASE
405
PURIFICATION OF PYRUVATE DEHYDROGENASE PHOSPHATASE FROM BOVINE HEART ~z
Fraction M e m b r a n e extract First protamine precipitation Ultracentrifuge s u p e r n a t a n t ~ Combined fractions 2 and 3 Second protamine precipitation Ultracentrifugation Affinity c h r o m a t o g r a p h y concentrate
Volume (ml)
Protein (mg)
Specific activity t'
Recovery (9~)
1830 283 260 543 292 280 1.8
6240 1190 923 2114 742 403 5.8
2.1 9.5 8.5 9.0 22.6 36.3 1655
---100 78 69 45
'~ From about 80 p o u n d s of heart. t, Units of pyruvate d e h y d r o g e n a s e complex reactivated per minute per milligram of protein. " P h o s p h a t a s e released from partially purified pyruvate d e h y d r o g e n a s e complex by ultracentrifugation in the presence of EGTA.
funnel with 0.2 M NaHCO3 and then with deionized water. The gel is treated with a solution of 250 mg of bovine serum albumin in 100 ml of 0.2 M NaHCO3 for 4 hr and then washed well with 0.2 M NaHCO3 and deionized water. Step 1. Extraction qf Mitochondrial Membrane Fraction. The membrane fraction obtained after extraction of the pyruvate dehydrogenase complex from bovine heart mitochondria 7 is suspended in 2 liters of 0.1 M MOPS, pH 7.0, at 23 °. The suspension is stirred for 1.5 hr and then centrifuged at 30,000 g for 30 min at 20°; the pellets are discarded. Step 2. First Protamine Precipitation. The supernatant fluid from step 1 is cooled to 4 °, and 0.011 volume of 2% protamine sulfate (Elanco Products 1°") is added dropwise with stirring. After 15 min the precipitate is collected by centrifugation at 30,000 g for 15 min; the supernatant fluid is discarded. The protamine precipitate is resuspended, by means of a large glass homogenizer equipped with a motor-driven Teflon pestle, in 300 ml of buffer A (0.02 M MOPS, pH 7.0; 5 m M MgC12; 10% (v/v) glycerol; 0.5 m M dithiothreitol) containing 0.16% yeast sodium ribonucleate (ICN Pharmaceuticalsl°~). The suspension is stirred overnight, then 0.01 volume of 0.2 M EGTA is added. The mixture is warmed to 23 ° for 20 min and then centrifuged at 30,000 g for 15 min at 20 °. The clear, yellow supernatant fluid is cooled to 4°; the pellets are discarded. Step 3. Second Protamine Precipitation. The supernatant fluid from step 2 is combined with the supernatant fluid from the first ultracentrifuga10,, Elanco Products, Indianapolis, Indiana; ICN Pharmaceuticals, Irvine, California; BioMolecular Dynamics, Beaverton, Oregon.
406
PHOSPHATASES
[6 7]
tion step in the purification of the pyruvate dehydrogenase complex. 7 To this solution is added dropwise, with stirring, 0.01 volume of 2% protamine sulfate. After 15 min, the precipitate is collected by centrifugation for 15 min. The protamine precipitate is extracted exactly as described in step 2. Step 4. Ultracentrifitgation. The supernatant fluid from step 3 is centrifuged at 105,000 g for 3.5 hr; the pellets are discarded. To the supernatant fluid is added 0.05 volume of 0.1 M CaC12. Step 5. Affinity Chromatography. The phosphatase solution from step 4 is applied slowly to a column (1.5 × 26 cm) of dihydrolipoyl transacetylase (E~)-Sepharose 4B that has been equilibrated with buffer A containing 2 mM CaC12. The column is washed with 150 ml of this equilibration buffer. The phosphatase is eluted with buffer A containing 2 mM EGTA and 0.02% sodium azide. The active fractions are combined and concentrated by vacuum dialysis against buffer A in a Bio-Molecular Dynamics l°a concentrator. Properties Enzyme obtained from step 5, from both heart and kidney mitochondria, gives one band on polyacrylamide gel electrophoresis with Tris(hydroxymethyl)aminomethane-glycine buffer in the absence of sodium dodecyl sulfate (SDS). 11 The phosphatase has a molecular weight by sedimentation equilibrium of about 150,000 and a sedimentation coefficient (s20.w) of about 7.4 S. 12 It consists of two subunits with molecular weights of about 97,000 and 50,000 as estimated by SDS-gel electrophoresis.13 Phosphatase activity appears to reside in the latter subunit, which is sensitive to proteolysis. Pyruvate dehydrogenase phosphatase preparations from bovine heart and kidney I and pig heart 2 were reported to have a molecular weight of 95,000-I00,000 as estimated by SDSpolyacrylamide gel electrophoresis and by filtration through a calibrated column of Sephadex G-100. The activity of these phosphatase preparations was less than 10% of the activity of the homogeneous preparation described in this chapter. It appears that the earlier preparations were deficient in the protease-sensitive subunit of molecular weight 50,000. Catalytic Properties. Pyruvate dehydrogenase phosphatase catalyzes dephosphorylation at all three phosphorylation sites on El. The phosphatase requires a divalent cation (Mg 2+ or Mn2+). The apparent Km for 11 B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404 (1964). 12 F. H. Pettit, W. M. Teague, and L. J. Reed, unpublished results. 13 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[68]
SWINE KIDNEY PHOSPHOPROTEIN PHOSPHATASE
407
Mg 2+ is about 2 mM. 14 Ca2+ markedly stimulates phosphatase activity 3,9 in the presence, but not in the absence, of dihydrolipoyl transacetylase (E2). 9 The apparent Km for phosphorylated E, is about 58 #M. In the presence of Ca 2+ and E2 this value is decreased to about 2.9/~M. There is some uncertainty as to the apparent Km for Ca 2+. Values in the range of 1 /xM to 100 # M have been reported. '5'16 Highly purified pyruvate dehydrogenase phosphatase from bovine heart binds one Ca 2+ with a dissociation constant (Kd) of about 8 IzM.'7 In the presence of E2, the phosphatase binds a second Ca 2+ with a Kd of about 5 #M. It appears that Ca 2+ may play a structural role. In the presence of Ca 2+, the phosphatase binds to E2, thereby facilitating the Mg~+-dependent dephosphorylation of phosphorylated E,. Phosphatase activity is inhibited by NADH, and this inhibition is reversed by NAD +.'8 Phosphatase activity is inhibited by fluoride ion, by inorganic orthophosphate, and by increasing the ionic strength. 2'14 Pyruvate dehydrogenase phosphatase exhibits only slight activity toward phosphorylase a, about 10% of the activity observed with phosphorylated pyruvate dehydrogenase complex as substrate. The phosphatase is inactive toward p-nitrophenyl phosphate. It is not inhibited by protein phosphatase inhibitor-1 or inhibitor-2 (kindly furnished by Dr. Philip Cohen). ,4 F. Hucho, D. D. Randall, T. E. Roche, M. W. Burgett, J. W. Pelley, and L. J. Reed,Arch. Biochem. Biophys. 151, 328 (1972). ,s A. L. Kerbey and P. J. Randle, FEBS Lett. 108, 485 (1979). ,6 R. M. Denton and J. G. McCormack, FEBS Lett. 119, 1 (1980). ,7 T-L. Wu and L. J. Reed, unpublished results. ,s F. H. Pettit, J. W. Pelley, and L. J. Reed, Biochem. Biophys. Res. Commun. 65, 575 (1975).
[68] By K.
Phosphoprotein Phosphatase from Swine Kidney MUNIYAPPA, FREDRICH LEIBACH,
and
JOSEPH M E N D I C I N O
Phosphoprotein phosphatase (EC 3.1.3.16) catalyzes the reaction Phosphoenzyme + ,7 H20
phosphoprotein ) dephosphoenzyme + n P~ phosphatase
The homogeneous phosphoprotein phosphatase isolated from swine kidney extracts catalyzes the dephosphorylation of the phosphorylated forms of glycogen synthase, phosphorylase, phosphofructokinase,
METHODS IN ENZYMOLOGY,VOL. 90
Copyright © 1982by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 13,-12-181990-6