- Email: [email protected]
[38] Isolation of an oligomycin-sensitive ATPase complex from rat liver mitochondria
328
ATP SYNTHESISAND REGULATION
[38]
gave values of 354,000 and 362,000. An independent determination of molecular weight by polyacrylamide gel chromatography on Bio-Gel P300 with phosphofructokinase, pyruvate kinase, and yeast alcohol dehydrogenase as reference proteins a gave a value of 355,000, in excellent agreement with the ultracentrifuge data. As in the case of the heart mitochondrial ATPase, 4 the e n z y m e from rat liver is readily dissociated into its subunits when held at 5 ° in the K C l - p h o s p h a t e buffer described at the beginning of this section. 3 C a t a l y t i c P r o p e r t i e s . With Mg-ATP as the variable substrate and with Tris-chloride or Tris-sulfate as the buffer, mitochondrial ATPase exhibits negative cooperativity; the Hill coefficient is approximately 0.5. In the presence of physiological concentrations (20 mM) of bicarbonate, the velocity is greatly enhanced, and double reciprocal plots are linear. Many different anions are capable of stimulating the activity of ATPase but, except for bromide, all are dianions and the monoionized form is the active species. 2° With Mg-GTP or Mg-ITP as the variable substrate, L i n e w e a v e r - B u r k plots are linear. There is evidence 21"22for two types of nucleotide-binding sites on mitochondrial ATPase. The catalytic site(s) binds Mg-ATP, Mg-GTP, and Mg-ITP; the regulatory site(s) binds MgATP, Mg-ADP, and Mg-AMP-PNP with an increasing order of affinity. Binding of these nucleotides at the regulatory site decreases the affinity of the catalytic site for ATP. Both G T P and ITP bind to the regulatory site with much less affinity and with little or no effect on A T P binding to the catalytic site. 22
Acknowledgment T h e r e s e a r c h o f W . A . C a t t e r a l l a n d P. L . P e d e r s o n w a s s u p p o r t e d b y N I H g r a n t C A 10951.
z0 R. E. Ebel and H. A. Lardy, J. Biol. Chem. 250, 191 (1975). zl H. A. Lardy, S. M. Schuster, and R. E. Ebel, J. Supramol. Struct. 3, 214 (1975). ~2S. M. Schuster, R. E. Ebel, and H. A. Lardy, J. Biol. Chem. 250, 7848 (1975).
[38] Isolation of an Oligomycin-Sensitive ATPase Complex from Rat Liver Mitochondria B y JOHN W. SOPER and PETER L. PEDERSEN
Although the antibiotic oligomycin simultaneously blocks the terminal step in oxidative phosphorylation (phosphoryl transfer to ADP) and inhibits ATPase activity of submitochondrial particles, 1 purified F1 ATPase 1 H . A . L a r d y , D. J o h n s o n , a n d W . C .
METHODS IN ENZYMOLOGY, VOL. LV
McMurray,Arch. Biochem. Biophys. 78, 587 (1958). copyright (~ 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
[38]
RAT LIVEROLIGOMYCIN-SENSITIVEATPase
329
preparations isolated from rat liver mitochondria *'3 are insensitive to oligomycin. Since the ATP synthetic and hydrolytic activities of this enzyme in situ are affected by oligomycin, a more complete understanding of its physiological role requires that the isolated enzyme retain inhibitor sensitivities similar to those seen in mitochondria. We describe here a procedure for isolating the ATPase complex of rat liver mitochondria in a form that is detergent soluble and sensitive to all inhibitors of the terminal steps of oxidative phosphorylation. Solutions
H medium: 220 mM mannitol, 70 mM sucrose, 2.5 mM H E PE S buffer, pH 7.4, containing 0.5 mg/ml defatted bovine serum albumin P buffer: 300 mM K2HPO4, 50 mM EDTA, pH 7.9 PA buffer: 300 mM K2HPO4, 5 mM EDTA, 2 mM ATP, 10% ethylene glycol, 0.5 mM dithiothreitol, pH 7.9 Deoxycholate (sodium salt): 2% solution, recrystallized three times from 70% ethanol (5 ml/g) Millipore Filters
Thirteen-millimeter diameter, 250-A pore size, catalogue VSWP from Millipore (Bedford, Massachusetts); treated via filtration with 10 ml of 1 mg/ml bovine serum albumin in PA buffer Analytical P r o c e d u r e s ATPase Assays
ATPase assays are carried out by coupling the hydrolysis of ATP to the loss of absorbance of NADH at 340 nm in a Giiford spectrophotometer (model 222) equipped with a Sargent SRL recorder. The assay buffer is composed of 40 mM Tris-HCO3, 5 mM MgSO4, 5 mM KCN, pH 7.9. Before the assay, the following components, dissolved in the assay buffer, are added at the indicated final concentrations: ATP (4 raM), NADH (0.3 raM), and phosphoenolpyruvate (0.4 raM). One microliter of lactate dehydrogenase (6.2 IU) and 2/.~1 of pyruvate kinase (8 IU), used as commercially obtained, are then added per assay. The final volume of the complete assay medium is 1.0 ml. One unit of activity is equivalent to the hydrolysis of 1 /zmol ATPase per minute at 37°. Inhibitor Assays
Alcoholic solutions of oligomycin, venturicidin, and dicyclohexylcarbodiimide (DCCD) are diluted in methanol to the desired final concenw. A. Catterall and P. L. Pedersen.J. Biol. Chem. 246, 4987 (1971). 3 D. O. Lambeth and H. A. Lardy, Eur. J. Biochem. 22, 355 (1971).
330
ATP SYNTHESISAND REGULATION
[38]
trations. A quantity of ATPase sufficient to hydrolyze approximately 2 nmol ATP per minute is then added to 1.25 ml of 40 mM Tris-HCO3, pH 7.9. Exactly 0.25 ml of the ATPase-HCO3 mixture is added to each of four tubes. Fifty microliters of the various methanolic inhibitor solutions are then added to the above Tris-HCO3 buffer to give a final volume of 0.25 ml. These inhibitor solutions are immediately added to the above enzyme solutions, and the enzyme-inhibitor mixture are allowed to preincubate for 20 min. An enzyme-control tube containing only the appropriate quantity of methanol is included in every set of assays. At the end of the preincubation period, 0.5 ml of a solution containing a twofold concentration of all ATPase assay components in 40 mM Tris-HCO3, 10 mM MgSO4, 10 mM KCI, pH 7.9, is added to each tube and incubated for 1 min. All procedures to this point are carried out on ice. Tubes are then incubated at 37° for 1 min and immediately assayed for ATPase activity at 37° as described above.
Protein Determinations All determinations of protein for the purpose of calculating protein concentrations during the treatment of mitochondria with digitonin and Lubrol are based on the biuret procedure, with bovine serum albumin as a standard. All protein determinations for the calculation of specific activity are based on a micromodification of the Lowry procedure? Potentially interfering materials are removed by precipitation of protein before Lowry determinations. Membranous samples are precipitated with ice-cold 10% trichloracetic acid (TCA) and then centrifuged at 23,000 g for 30 min. Detergent supernatants are first precipitated with ice-cold acetone, followed by dissolution in 50/A of 1 N NaOH and reprecipitation with 1.5 ml of ice-cold 10% TCA. All Lowry determinations are standardized against solutions of bovine serum albumin taken through identical procedures. Isolation P r o c e d u r e s
Isolation of Mitochondria. Rat liver mitochondria are prepared according to a high-yield procedure involving repeated nuclear extractions as described elsewhere. 5"6 4 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 5 p. L. Pedersen, J. W. Greenawalt, B. Reynafarje, J. Hullihen, G. Decker, J. W. Soper, and E. Bustamante, Methods Cell Biol.20, 411 (1978). 6 E. Bustamante, J. Soper, and P. L. Pedersen, Anal. Biochem. 80, 401 (1977).
[38]
RAT L|VER OLIGOMYCIN-SENSITIVEATPase
331
Preparation of Phosphate- Washed Inner Membrane Vesicles Inner membrane vesicles that exhibit a high ATPase specific activity are used for the isolation of soluble oligomycin-sensitive ATPase. These membrane vesicles are prepared according to the following procedureT:
Step 1. Freshly isolated mitochondria, prepared as described above, are treated with a solution of digitonin in H medium equivalent to 0.12 mg per milligram of mitochondrial protein, s Immediately after addition of digitonin, the protein concentration is adjusted to 50 mg/ml, and the suspension is incubated at 0° for 20 min. At the end of this time the reaction mixture is quickly diluted fivefold with H medium and centrifuged at 23,600 g for 20 rain in a Sorvall SS-34 rotor. The mitoplast (inner membrane plus matrix) fraction is resuspended and washed twice in H medium. A low-speed fraction sedimenting at 3000 g is discarded before the final wash. Step 2. Lubrol WX sufficient to give 0.16 mg per milligram protein is added to the mitoplast suspension from a 4% stock solution in H20, and the final protein concentration is immediately adjusted to 35 mg/ml. After 20 min of incubation on ice the mitoplast suspension is diluted with an equal volume of P buffer. The diluted suspension is then centrifuged at 201,000 g for 90 min in a Spinco type 65 rotor. The resultant sediment, resuspended in 40 ml of P buffer, is centrifuged in the Sorvall SS-34 rotor at 4300 g for 10 rain. The low-speed sediment is discarded. Step 3. The step 2 supernatant fraction containing purified inner membrane vesicles is centrifuged at 201,000 g for 60 min and resuspended in a minimal volume of PA buffer. Membranes are then rapidly frozen in a Dry I c e - a c e t o n e bath and stored at - 2 0 ° for approximately 1 week. Step 4. Membranes are thawed, diluted to approximately 80 ml with P buffer, and recentrifuged at 4300 g for 10 min. The low-speed sediment is discarded, and the supernatant membranes are washed three times in P buffer by centrifuging at 201,000 g for 60 min. The resultant sediment is resuspended in PA buffer, and material sedimenting after centrifugation for 20 min at 12,300g in the Sorvall SS-24 rotor is discarded. Finally, the membranes remaining in the supernatant are adjusted to a protein concentration of 5 mg/ml, and 2-ml aliquots are frozed in Dry I c e - a c e t o n e and stored at - 2 0 ° for up to 2 weeks. 7 j. W. Soper and P. L. Pedersen, Biochemisto' 15, 2682 (1976). 8 T. L. Chan, J. W. Greenawalt, and P. L. Pedersen, .I. ('ell Biol. 45,291 (1970).
332
A T P SYNTHESIS AND REGULATION
[38]
Extraction of Oligomycin-Sensitive A TPase from Phosphate-Washed Membranes 7 P h o s p h a t e - w a s h e d m e m b r a n e s , as described a b o v e , are thawed and diluted to 1 mg/ml with PA buffer. D e o x y c h o l a t e , recrystallized from 70% ethanol, is added at a concentration of 0.1 mg/ml and incubated with the m e m b r a n e s for 15 min at 4 °. It is absolutely critical that the deoxycholate used has been extensively repurified and that detergent concentrations do not exceed those specified above. A t t e m p t s to increase the a m o u n t of detergent used m a y cause an apparent increase in the a m o u n t of A T P a s e activity extracted, but this is achieved at the e x p e n s e of d e c r e a s e d sensitivity to oligomycin. 7 The m e m b r a n e suspension is then subjected to centrifugation at 201,000 g for 90 min in the Spinco type 65 rotor. The sediment is discarded. Soluble oligomycin-sensitive A T P a s e in the supernatant is freed of any residual m e m b r a n e contamination by passing the supernatant fractions through 250 A Millipore filters that have been pretreated with bovine s e r u m albumin to prevent nonspecific adsorption of A T P a s e to filters. The supernatant fraction (10 ml, 50/xg/ml) is passed through the filters contained in Swinnex filter holders attached to a 3-ml syringe. All p r o c e d u r e s are carried out at 4 ° . The specific activity of the e n z y m e is 12 /zmol/min per milligram protein (Table I). The yield of activity is a p p r o x i m a t e l y 5-10% of the A T P a s e activity of the starting m e m b r a n e fraction. Stability of O l i g o m y c i n - S e n s i t i v e A T P a s e The e n z y m e is stored in PA buffer at 4 °. Retention of activity is greater than 75% after 4 days. 7 TABLE I PURIFICATION OF OLIGOMYCIN-SENSITIVE ATPAsE OF RAT LIVER
Fraction Mitochondria Mitoplasts PrWashed membranes Oligomycin-sensitive ATPase
ATPase specific activitya
Yield (%)
2.0 3.2 21.6 12.0
(100) 65-70 68 5-10b
a Expressed as micromoles ATP hydrolyzed per minute and milligram protein. b Yield from P~-washed membranes.
[39]
CRYSTALLIZATION OF MITOCHONDRIALATPase
333
P r o p e r t i e s of O l i g o m y c i n - S e n s i t i v e A T P a s e The e n z y m e preparation is characterized by 13 different Coomassiestainable bands upon S D S - p o l y a c r y l a m i d e gel electrophoresis carried out by the procedure of W e b e r and O s b o r n 2 Six of these 13 bands can be attributed to the subunits of the F1 complex. Gels of the m e m b r a n e s used for the extraction are characterized by 17 different Coomassiestainable bands, TM and Lubrol inner m e m b r a n e s by 23 bands. 11 When assays are carried out as described in the section on analytical procedures, the A T P a s e activity is inhibited a p p r o x i m a t e l y 60-70% by oligomycin, venturicidin, and D C C D at concentrations of 2.5 × 10-2, 1.3 × 10 -1, and 1 p M inhibitor, respectively. 7 When a s s a y e d in Tris-bicarbonate buffer the e n z y m e exhibits biphasic kinetics ( E a d i e - H o f s t e e plots) with Km values of 29 and 313 pM. 7 Acknowledgments This work was supported by NIH grant CA 10951 and a postdoctral fellowship (CA 05055) to JWS. 9 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). 10j. W. Soper and P. L. Pedersen, unpublished results (1978). it p. L. Pedersen, T. Eska, H. P. Morris, and W. A. Catterall, Proc. Natl. Acad. Sci. (USA) 68, 1079 (1971).
[39] Crystallization
o f F1 A T P a s e Mitochondria
from
Rat
Liver
By L. MARIO AMZEL and PETER L. PEDERSEN
P u r i f i c a t i o n a n d Crystallization A T P a s e (F1) was obtained from rat liver mitochondria and purified through the D E A E step following the p r o c e d u r e of Catterall and Pedersen.1 Before crystallization a sample of the purified e n z y m e was subjected to electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (SDS). The pattern of electrophoretic mobilities obtained was indistinguishable from those of previously purified preparations. 2 F o r crystallization p u r p o s e s the purified e n z y m e is dissolved at r o o m t e m p e r a t u r e in a buffer containing 200 mM p o t a s s i u m p h o s p h a t e and 5 1 w. A. Catterall and P. L. Pedersen, J. Biol. Chem. 246, 4987 (1971). 2 W. A. Canerall, W. A. Coty, and P. L. Pedersen, J. Biol. Chem. 248, 7427 (1973). M E T H O D S IN E N Z Y M O L O G Y . VOL. LV
Copyright ~) 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
ATP SYNTHESISAND REGULATION
[38]
gave values of 354,000 and 362,000. An independent determination of molecular weight by polyacrylamide gel chromatography on Bio-Gel P300 with phosphofructokinase, pyruvate kinase, and yeast alcohol dehydrogenase as reference proteins a gave a value of 355,000, in excellent agreement with the ultracentrifuge data. As in the case of the heart mitochondrial ATPase, 4 the e n z y m e from rat liver is readily dissociated into its subunits when held at 5 ° in the K C l - p h o s p h a t e buffer described at the beginning of this section. 3 C a t a l y t i c P r o p e r t i e s . With Mg-ATP as the variable substrate and with Tris-chloride or Tris-sulfate as the buffer, mitochondrial ATPase exhibits negative cooperativity; the Hill coefficient is approximately 0.5. In the presence of physiological concentrations (20 mM) of bicarbonate, the velocity is greatly enhanced, and double reciprocal plots are linear. Many different anions are capable of stimulating the activity of ATPase but, except for bromide, all are dianions and the monoionized form is the active species. 2° With Mg-GTP or Mg-ITP as the variable substrate, L i n e w e a v e r - B u r k plots are linear. There is evidence 21"22for two types of nucleotide-binding sites on mitochondrial ATPase. The catalytic site(s) binds Mg-ATP, Mg-GTP, and Mg-ITP; the regulatory site(s) binds MgATP, Mg-ADP, and Mg-AMP-PNP with an increasing order of affinity. Binding of these nucleotides at the regulatory site decreases the affinity of the catalytic site for ATP. Both G T P and ITP bind to the regulatory site with much less affinity and with little or no effect on A T P binding to the catalytic site. 22
Acknowledgment T h e r e s e a r c h o f W . A . C a t t e r a l l a n d P. L . P e d e r s o n w a s s u p p o r t e d b y N I H g r a n t C A 10951.
z0 R. E. Ebel and H. A. Lardy, J. Biol. Chem. 250, 191 (1975). zl H. A. Lardy, S. M. Schuster, and R. E. Ebel, J. Supramol. Struct. 3, 214 (1975). ~2S. M. Schuster, R. E. Ebel, and H. A. Lardy, J. Biol. Chem. 250, 7848 (1975).
[38] Isolation of an Oligomycin-Sensitive ATPase Complex from Rat Liver Mitochondria B y JOHN W. SOPER and PETER L. PEDERSEN
Although the antibiotic oligomycin simultaneously blocks the terminal step in oxidative phosphorylation (phosphoryl transfer to ADP) and inhibits ATPase activity of submitochondrial particles, 1 purified F1 ATPase 1 H . A . L a r d y , D. J o h n s o n , a n d W . C .
METHODS IN ENZYMOLOGY, VOL. LV
McMurray,Arch. Biochem. Biophys. 78, 587 (1958). copyright (~ 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
[38]
RAT LIVEROLIGOMYCIN-SENSITIVEATPase
329
preparations isolated from rat liver mitochondria *'3 are insensitive to oligomycin. Since the ATP synthetic and hydrolytic activities of this enzyme in situ are affected by oligomycin, a more complete understanding of its physiological role requires that the isolated enzyme retain inhibitor sensitivities similar to those seen in mitochondria. We describe here a procedure for isolating the ATPase complex of rat liver mitochondria in a form that is detergent soluble and sensitive to all inhibitors of the terminal steps of oxidative phosphorylation. Solutions
H medium: 220 mM mannitol, 70 mM sucrose, 2.5 mM H E PE S buffer, pH 7.4, containing 0.5 mg/ml defatted bovine serum albumin P buffer: 300 mM K2HPO4, 50 mM EDTA, pH 7.9 PA buffer: 300 mM K2HPO4, 5 mM EDTA, 2 mM ATP, 10% ethylene glycol, 0.5 mM dithiothreitol, pH 7.9 Deoxycholate (sodium salt): 2% solution, recrystallized three times from 70% ethanol (5 ml/g) Millipore Filters
Thirteen-millimeter diameter, 250-A pore size, catalogue VSWP from Millipore (Bedford, Massachusetts); treated via filtration with 10 ml of 1 mg/ml bovine serum albumin in PA buffer Analytical P r o c e d u r e s ATPase Assays
ATPase assays are carried out by coupling the hydrolysis of ATP to the loss of absorbance of NADH at 340 nm in a Giiford spectrophotometer (model 222) equipped with a Sargent SRL recorder. The assay buffer is composed of 40 mM Tris-HCO3, 5 mM MgSO4, 5 mM KCN, pH 7.9. Before the assay, the following components, dissolved in the assay buffer, are added at the indicated final concentrations: ATP (4 raM), NADH (0.3 raM), and phosphoenolpyruvate (0.4 raM). One microliter of lactate dehydrogenase (6.2 IU) and 2/.~1 of pyruvate kinase (8 IU), used as commercially obtained, are then added per assay. The final volume of the complete assay medium is 1.0 ml. One unit of activity is equivalent to the hydrolysis of 1 /zmol ATPase per minute at 37°. Inhibitor Assays
Alcoholic solutions of oligomycin, venturicidin, and dicyclohexylcarbodiimide (DCCD) are diluted in methanol to the desired final concenw. A. Catterall and P. L. Pedersen.J. Biol. Chem. 246, 4987 (1971). 3 D. O. Lambeth and H. A. Lardy, Eur. J. Biochem. 22, 355 (1971).
330
ATP SYNTHESISAND REGULATION
[38]
trations. A quantity of ATPase sufficient to hydrolyze approximately 2 nmol ATP per minute is then added to 1.25 ml of 40 mM Tris-HCO3, pH 7.9. Exactly 0.25 ml of the ATPase-HCO3 mixture is added to each of four tubes. Fifty microliters of the various methanolic inhibitor solutions are then added to the above Tris-HCO3 buffer to give a final volume of 0.25 ml. These inhibitor solutions are immediately added to the above enzyme solutions, and the enzyme-inhibitor mixture are allowed to preincubate for 20 min. An enzyme-control tube containing only the appropriate quantity of methanol is included in every set of assays. At the end of the preincubation period, 0.5 ml of a solution containing a twofold concentration of all ATPase assay components in 40 mM Tris-HCO3, 10 mM MgSO4, 10 mM KCI, pH 7.9, is added to each tube and incubated for 1 min. All procedures to this point are carried out on ice. Tubes are then incubated at 37° for 1 min and immediately assayed for ATPase activity at 37° as described above.
Protein Determinations All determinations of protein for the purpose of calculating protein concentrations during the treatment of mitochondria with digitonin and Lubrol are based on the biuret procedure, with bovine serum albumin as a standard. All protein determinations for the calculation of specific activity are based on a micromodification of the Lowry procedure? Potentially interfering materials are removed by precipitation of protein before Lowry determinations. Membranous samples are precipitated with ice-cold 10% trichloracetic acid (TCA) and then centrifuged at 23,000 g for 30 min. Detergent supernatants are first precipitated with ice-cold acetone, followed by dissolution in 50/A of 1 N NaOH and reprecipitation with 1.5 ml of ice-cold 10% TCA. All Lowry determinations are standardized against solutions of bovine serum albumin taken through identical procedures. Isolation P r o c e d u r e s
Isolation of Mitochondria. Rat liver mitochondria are prepared according to a high-yield procedure involving repeated nuclear extractions as described elsewhere. 5"6 4 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 5 p. L. Pedersen, J. W. Greenawalt, B. Reynafarje, J. Hullihen, G. Decker, J. W. Soper, and E. Bustamante, Methods Cell Biol.20, 411 (1978). 6 E. Bustamante, J. Soper, and P. L. Pedersen, Anal. Biochem. 80, 401 (1977).
[38]
RAT L|VER OLIGOMYCIN-SENSITIVEATPase
331
Preparation of Phosphate- Washed Inner Membrane Vesicles Inner membrane vesicles that exhibit a high ATPase specific activity are used for the isolation of soluble oligomycin-sensitive ATPase. These membrane vesicles are prepared according to the following procedureT:
Step 1. Freshly isolated mitochondria, prepared as described above, are treated with a solution of digitonin in H medium equivalent to 0.12 mg per milligram of mitochondrial protein, s Immediately after addition of digitonin, the protein concentration is adjusted to 50 mg/ml, and the suspension is incubated at 0° for 20 min. At the end of this time the reaction mixture is quickly diluted fivefold with H medium and centrifuged at 23,600 g for 20 rain in a Sorvall SS-34 rotor. The mitoplast (inner membrane plus matrix) fraction is resuspended and washed twice in H medium. A low-speed fraction sedimenting at 3000 g is discarded before the final wash. Step 2. Lubrol WX sufficient to give 0.16 mg per milligram protein is added to the mitoplast suspension from a 4% stock solution in H20, and the final protein concentration is immediately adjusted to 35 mg/ml. After 20 min of incubation on ice the mitoplast suspension is diluted with an equal volume of P buffer. The diluted suspension is then centrifuged at 201,000 g for 90 min in a Spinco type 65 rotor. The resultant sediment, resuspended in 40 ml of P buffer, is centrifuged in the Sorvall SS-34 rotor at 4300 g for 10 rain. The low-speed sediment is discarded. Step 3. The step 2 supernatant fraction containing purified inner membrane vesicles is centrifuged at 201,000 g for 60 min and resuspended in a minimal volume of PA buffer. Membranes are then rapidly frozen in a Dry I c e - a c e t o n e bath and stored at - 2 0 ° for approximately 1 week. Step 4. Membranes are thawed, diluted to approximately 80 ml with P buffer, and recentrifuged at 4300 g for 10 min. The low-speed sediment is discarded, and the supernatant membranes are washed three times in P buffer by centrifuging at 201,000 g for 60 min. The resultant sediment is resuspended in PA buffer, and material sedimenting after centrifugation for 20 min at 12,300g in the Sorvall SS-24 rotor is discarded. Finally, the membranes remaining in the supernatant are adjusted to a protein concentration of 5 mg/ml, and 2-ml aliquots are frozed in Dry I c e - a c e t o n e and stored at - 2 0 ° for up to 2 weeks. 7 j. W. Soper and P. L. Pedersen, Biochemisto' 15, 2682 (1976). 8 T. L. Chan, J. W. Greenawalt, and P. L. Pedersen, .I. ('ell Biol. 45,291 (1970).
332
A T P SYNTHESIS AND REGULATION
[38]
Extraction of Oligomycin-Sensitive A TPase from Phosphate-Washed Membranes 7 P h o s p h a t e - w a s h e d m e m b r a n e s , as described a b o v e , are thawed and diluted to 1 mg/ml with PA buffer. D e o x y c h o l a t e , recrystallized from 70% ethanol, is added at a concentration of 0.1 mg/ml and incubated with the m e m b r a n e s for 15 min at 4 °. It is absolutely critical that the deoxycholate used has been extensively repurified and that detergent concentrations do not exceed those specified above. A t t e m p t s to increase the a m o u n t of detergent used m a y cause an apparent increase in the a m o u n t of A T P a s e activity extracted, but this is achieved at the e x p e n s e of d e c r e a s e d sensitivity to oligomycin. 7 The m e m b r a n e suspension is then subjected to centrifugation at 201,000 g for 90 min in the Spinco type 65 rotor. The sediment is discarded. Soluble oligomycin-sensitive A T P a s e in the supernatant is freed of any residual m e m b r a n e contamination by passing the supernatant fractions through 250 A Millipore filters that have been pretreated with bovine s e r u m albumin to prevent nonspecific adsorption of A T P a s e to filters. The supernatant fraction (10 ml, 50/xg/ml) is passed through the filters contained in Swinnex filter holders attached to a 3-ml syringe. All p r o c e d u r e s are carried out at 4 ° . The specific activity of the e n z y m e is 12 /zmol/min per milligram protein (Table I). The yield of activity is a p p r o x i m a t e l y 5-10% of the A T P a s e activity of the starting m e m b r a n e fraction. Stability of O l i g o m y c i n - S e n s i t i v e A T P a s e The e n z y m e is stored in PA buffer at 4 °. Retention of activity is greater than 75% after 4 days. 7 TABLE I PURIFICATION OF OLIGOMYCIN-SENSITIVE ATPAsE OF RAT LIVER
Fraction Mitochondria Mitoplasts PrWashed membranes Oligomycin-sensitive ATPase
ATPase specific activitya
Yield (%)
2.0 3.2 21.6 12.0
(100) 65-70 68 5-10b
a Expressed as micromoles ATP hydrolyzed per minute and milligram protein. b Yield from P~-washed membranes.
[39]
CRYSTALLIZATION OF MITOCHONDRIALATPase
333
P r o p e r t i e s of O l i g o m y c i n - S e n s i t i v e A T P a s e The e n z y m e preparation is characterized by 13 different Coomassiestainable bands upon S D S - p o l y a c r y l a m i d e gel electrophoresis carried out by the procedure of W e b e r and O s b o r n 2 Six of these 13 bands can be attributed to the subunits of the F1 complex. Gels of the m e m b r a n e s used for the extraction are characterized by 17 different Coomassiestainable bands, TM and Lubrol inner m e m b r a n e s by 23 bands. 11 When assays are carried out as described in the section on analytical procedures, the A T P a s e activity is inhibited a p p r o x i m a t e l y 60-70% by oligomycin, venturicidin, and D C C D at concentrations of 2.5 × 10-2, 1.3 × 10 -1, and 1 p M inhibitor, respectively. 7 When a s s a y e d in Tris-bicarbonate buffer the e n z y m e exhibits biphasic kinetics ( E a d i e - H o f s t e e plots) with Km values of 29 and 313 pM. 7 Acknowledgments This work was supported by NIH grant CA 10951 and a postdoctral fellowship (CA 05055) to JWS. 9 K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). 10j. W. Soper and P. L. Pedersen, unpublished results (1978). it p. L. Pedersen, T. Eska, H. P. Morris, and W. A. Catterall, Proc. Natl. Acad. Sci. (USA) 68, 1079 (1971).
[39] Crystallization
o f F1 A T P a s e Mitochondria
from
Rat
Liver
By L. MARIO AMZEL and PETER L. PEDERSEN
P u r i f i c a t i o n a n d Crystallization A T P a s e (F1) was obtained from rat liver mitochondria and purified through the D E A E step following the p r o c e d u r e of Catterall and Pedersen.1 Before crystallization a sample of the purified e n z y m e was subjected to electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate (SDS). The pattern of electrophoretic mobilities obtained was indistinguishable from those of previously purified preparations. 2 F o r crystallization p u r p o s e s the purified e n z y m e is dissolved at r o o m t e m p e r a t u r e in a buffer containing 200 mM p o t a s s i u m p h o s p h a t e and 5 1 w. A. Catterall and P. L. Pedersen, J. Biol. Chem. 246, 4987 (1971). 2 W. A. Canerall, W. A. Coty, and P. L. Pedersen, J. Biol. Chem. 248, 7427 (1973). M E T H O D S IN E N Z Y M O L O G Y . VOL. LV
Copyright ~) 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8