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[82] Preparation and properties of mitochondrial ATPase (coupling factor 1)
522
COUPLING FAC~aS
[82]
ADP exchange reaction2 It is stable after both DEAE-cellulose and polyacrylamide elution in liquid N~ for at least 3 weeks. The absorption spectrum shows a maximum at 278 m~, a minimum at 250 m~, and a shoulder at 290 n~. It appears as a single component in the ultracentrifuge (tool. wt. = 124,000), but two minor bands appear in disc electrophoresis.
[82] P r e p a r a t i o n a n d Properties of M i t o c h o n d r i a l A T P a s e ( C o u p l i n g F a c t o r 1) B y HARVEY S. PENEFSKY 1
General Principles The soluble ATPase of beef heart mitochondria also serves as a factor which couples the esterification of inorganic phosphate to the oxidation of suhstrates catalyzed by deficient submitochondrial particles. Ia,2 The enzyme is solubilized when beef heart mitochondria are disrupted by shaking with glass beads in a high speed reciprocal shaker. This method is based on one already presented s and introduces changes which permit preparation of the enzyme on a larger scale. Definition o] Unit and Specific Activity. A unit of activity is defined as that amount of enzyme which catalyzes the turnover of 1 micromole of substrate per minute under the specified conditions of assay2 Specific activity is expressed as units per milligram of dry weight of protein. Measurement .o] Protein Co~centr~tio~. The protein concentration of the mitochondrial suspension and of the step 1 and 2 fractions is determined by a biuret method modified for mitochondria2 Soluble protein is measured in a Brice-Phoenix differential refractometer model BP2000. The specific refractive index increment of bovine serum albumin, 188 >( 10-~ dl/g, 5 is used to calculate protein concentration from the difference in refractive index between protein and solvent. Protein con1This manuscript was prepared while the author was at the Department of Biochemistry, The Public Health Research Institute of the City of New York, Inc., New York. la M. E. Pullman, H. S. Penefsky, A. Datta, and E. Racker, J. Biol. Chem. 235, 3322 (1960). H. S. Penefsky, M. E. Pullman, A. Datta, and E. Racker, J. Biol. Chem. 235, 3330 (1960). M. E. Pullman and H. S. Penefsky, Vol. VI [34]. ' E. E. Jacobs, M. Jacob, D. R. Sanadi, and L. B. Bradley, J. Biol. Chem. ~'~3, 147 (1956). ~G, E. Perlmann and L. G. Longsworth, J. Am. Chem. ~oc. 70, 2719, (1948).
[82]
MITOCHONDRIAL ATPASE (COUPLING FACTOR 1)
523
centration determined by refractometry agrees to within 5% with values based on the weight of dialyzed and dried enzyme. Values of protein concentration in step 3 and 4 fractions also may be determined by ultraviolet absorption e and are corrected to dry weight when multiplied by 1.87. 7 Purification Procedure Step 1. Disintegration of the Mitochondria. "Light layer" mitochondria (6.75 g) prepared according to the method of Green and Ziegler, 8 is suspended in ice cold 0.25 M sucrose containing 2 mM EDTA, pH 7.4, and 2 mM ATP to give a final volume of 225 ml. Fifteen milliliters of this suspension and 7 ml of glass beads 9 are placed in the stainless steel cup of a Nossal 1° type reciprocal shaker 1~ and evacuated as described previously. 3 During the shaking period of 2 minutes, ice water is circulated around the cup and associated bearing and water at 25 ° is circulated around the main bearing. Following disruption of the mitochondria the contents of the cups are pooled and warmed to room temperature 12; the supernatant solution is removed after allowing the beads to settle. The beads are washed once by resuspension with 7(~ ml of 0.25 M sucrose, 2 mM EDTA, pH 7.4, and 2 mM ATP. The extract and bead washings are combined and centrifuged at room. temperature in the number 30 rotor of the Spinco Model L centrifuge. The packed brown pellet is discarded and the clear yellow supernatant solution is collected. The crude extract containing 1280 mg of protein is allowed to stand overnight at room temperature before the start of step 2. Step ~. pH Fractionation. The pH of the crude extract is adjusted to pH 5.4 by addition of 1.9 ml of 1 N acetic acid at room temperature with mechanical stirring. The solution is centrifuged at room temperature for 5 minutes at 5000 g, and the precipitate is discarded. The pH of the supernatant solution is adjusted to pH 6.7 by addition of 0.7 ml of 2 M Tris-S04, pH 10.7. Step 8. Prota~ine Fractionation. To prepare 0.5% protamine sulfate solution suspend 1.0 g protamine sulfate (Eli Lilly and Company) in 52 6See Vol. III [73]. ~H. S. Penefsky and R. C. Warner, J. BioL Chem. 240, 4694 (1965). 8D. E. Green and D. M. Ziegler, Vol. VI [58]. t Purchased from Minnesota Mining and Manufacturing Co., Ridgefield, New Jersey, 3M "Superbrite," catalog number 090, 0.0110-inch diameter. lop. M. Nossal, Australian J. Expel Biol. Med. Sci. 31, 583 (1953). 11Model RS, manufactured by the Lourdes Instrument Corporation, Brooklyn, New York. WAll subsequent manipulations are carried out at room temperature (25°) unless otherwise indicated.
524
COUPLING FACTORS
[82]
ml of distilled water. Adjust the pH to 7.{) with 0.1 M acetic acid, and bring the volume to 100 ml with water. Let stand in ice for 2{) minutes. Centrifuge in the cold at 18,000 g for 5 minutes and discard the residue. To the supernatant solution add an equal volume of water, and allow the solution to warm to room temperature before use. A 5.6-ml sample of 0.5% protamine sulfate solution is slowely added with mechanical stirring at room temperature to 224 ml of the step 2 fraction. 13 Stirring is continued for 15 minutes after the addition of protamine, and the light precipitate which forms is removed by centrifugation for 10 minutes at 15,000 g. All the ATPase activity remains in the clear supernatant solution. To the supernatant solution 73 ml of protamine sulfate solution is added as before. A heavy yellow precipitate forms and is collected by centrifugation. The supernatant solution is discarded, and the precipitate is dissolved by adding 6 ml of 0.25M sucrose, 2 mM EDTA, 0.01 M Tris-S04, pH 7.4, 0.4 M (NI~)~S04, pH 7.4. The resulting slightly turbid, yellow solution is clarified by centrifugation, and the small amount of insoluble residue is discarded. To the clear yellow supernatant solution is added with gentle stirring an equal volume of saturated ammonium sulfate, pH 5.5. The suspension is kept in ice for 15 minutes to ensure complete precipitation of the enzyme and then is centrifuged. The precipitate is dissoIved in 4 ml of 0.25M sucrose-0.01 M Tris-S04 pH 7.4-2 mM EDTA ~4 at room temperature. The dissolved enzyme is again precipitated by adding an equal volume of saturated ammonium sulfate solution. If desired the enzyme may be stored at this point at 4 ° before proceeding to the next step. Step ~,. Temperature Fraction~tion. The ammonium sulfate suspension of the enzyme from step 3 is centrifuged and dissolved in Sucrose-Tris-EDTA to give a final protein concentration of 2{) mg/ml. This solution is made 8 mM in ATP and heated in 12-ml conical glass centrifuge tubes at 65 ° for 4 minutes. Immediately thereafter the tubes are cooled in a 25 ° bath and centrifuged to remove denatured protein. The enzyme is precipitated from the supernatant solution by the addition of an equal volume of saturated ammonium sulfat~ solution. The enzyme is dissolved in Sucrose-Tris-EDTA and once again precipitated 13Since the exact amount of protamine required varies slightly for different preparations, a preliminary fractionation with protamine is carried out on a 4-ml sample of the step 2 fraction and scaled up appropriately. Usually 0.1 ml of 0.5% protamine solution is sufficient to precipitate inactive protein and lipid without reducing enzymatic activity in the supernatant solution. The subsequent addition of approximately 12, ml of protamine to the 4-ml sample results in precipitation of the bulk of the enzyme. ~4This solution will be referred to as Sucrose-Tris-EDTA.
[82]
MITOCHONDRIAL ATPASE (COUPLING FACTOR 1)
525
PURIFICATIONPROCEDURE FOR ATPAsE
Fraction
Volume (ml)
Step 1. Crude extract
237
Step 2. pH fractionation Step 3. Protamine fractionation Step 4. Temperature fractionation
224 10 8
Units 2760• 4950b 3860 3350 3240
Protein (rag)
Specific activity (units/rag Yield protein) (%)
1280 620 93.5 58
2.2 3.9 6.2 35.9 74.8
100 179 140 121 157
a Activity measured on the day of preparation. Activity measured on the following day. The specific activity of the crude extract increased about 2-fold upon standing overnight. with ammonium sulfate. The purified enzyme is stored at 4 ° as a suspension in ammonium sulfate. Samples of the enzyme m a y be prepared for assay by the centrifugation of desired aliquots of the ammonium sulfate suspension followed by removal of ammonium sulfate and solution of the pellet in appropriate buffers. Properties
Stability. The enzyme is stable for several months when stored at 4 ° at a suspension in half-saturated ammonium sulfate solution. The activity of the dissolved enzyme decreases slowly over a period of several days at room temperature in S u c r o s e - T r i s - E D T A , 4 m M ATP. The enzyme is stable for about 1 day in S u c r o s e - T r i s - E D T A and in buffers which do not contain sucrose provided A T P is present. Aqueous solutions of the A T P a s e are stable for at least 5 days when stored at --70 ° if frozen quickly in liquid nitrogen and thawed rapidly at 300. 7 Cold Lability. ATPase activity and coupling factor activity of the purified enzyme are cold labile in aqueous buffers, la,2 The rate of cold inactivation is greater at low protein concentrations (the half-time at 0 ° is about 7 minutes at 0.05 m g / m l and about 21 minutes at 3 m g / m l ) , is accelerated by anions (the order of effectiveness of anions in accelerating cold inactivation is I- > NO3- > Br- > C1- > S O ( - ) and is more labile at 0 ° (half-time about 7 minutes) than at 10 ° (half-time about 30 minutes). 7 The enzyme is stabilized in the cold by factors derived from beef heart mitochondria such as a heat stable dialyzable inhibitor, 1~ a heat stable protein inhibitor, 1G mitochondrial phospholipid, 1~ and Fo, an insoluble protein containing phospholipid which confers sensitivity to E. Racker, Federation Proc. 21, 54 (1962). le M. E. Pullman and G. Monroy, J. Biol. Chem. 238, 3762 (1963).
526
COUPLING FACTORS
[83]
oligomycin on the ATPase. 17 The enzyme also is stabilized in the cold by ethylene glycol, methanol, glycerol, and ethanolJ The cold-inactivated enzyme may be partially reactivated by rewarming at concentrations greater than 0.1 mg/ml. Physical Properties. At 25 ° in 20 mM P~, pH 7.2, 2 mM EDTA, 4 mM ATP, 0.1 M KCI, ~0.w is 12.9 °, the molecular weight determined by equilibrium ultracentrifugation is 284,000, the intrinsic viscosity is 3.2 ml/g, and the partial specific volume is 0.74 ml/gJ When incubated in the cold, the enzyme dissociates forming an equilibrium mixture of 3.4 S, 9.2 S, and 11.9 S components. Rewarming of the cold-treated enzyme under conditions which restore enzymatic activity results in the formation of a single sedimenting species with a sedimentation coefficient identical with that of the native enzyme. The ATPase dissociates at 25 ° in 1% sodium dodecyl sulfate to form a single sedimenting species with an S2o,w of 2.6 S and approximate molecular weight of 29,000. Chemical Analysis. The minimum molecular weight based on the half-cystine content is 26,100. TM Tryptophan, galactosamine, glucosamine, and fatty acidsTM were not detected. Sulfhydryl group analyses indicate 10 or 11 SH per mole. There is no evidence for disulfide bridges in the moleculeJ Other Praperties. The specificity of the ATPase, activators, and inhibitors of the enzyme and the pH optima have been described2 ,7 E. Racker, Biochem. Biophya. Res. Commun. 10, 435 (1963). The amino acid analyses were kindly performed by Dr. S. Moore. Fatty acid analyses were carried out by Dr. S. Fleischer and Dr. I. Mossbach.
[83] P r e p a r a t i o n of 3 H - A c e t y l - A T P a s e ( C o u p l i n g F a c t o r 1)
By Yasuo ICCGAWA1 General larinciple ATPase TM containing 5-6 moles of 3H-acetyl groups per mole of enzyme is prepared by reaction with SH-acetie anhydride. The labeled preparations appear to retain all the properties of the native enzyme and are useful in studies of the binding of the soluble ATPase to insoluble mitochondrial fractions. 1See footnote 1, page 505. ,I See this volume [82].
COUPLING FAC~aS
[82]
ADP exchange reaction2 It is stable after both DEAE-cellulose and polyacrylamide elution in liquid N~ for at least 3 weeks. The absorption spectrum shows a maximum at 278 m~, a minimum at 250 m~, and a shoulder at 290 n~. It appears as a single component in the ultracentrifuge (tool. wt. = 124,000), but two minor bands appear in disc electrophoresis.
[82] P r e p a r a t i o n a n d Properties of M i t o c h o n d r i a l A T P a s e ( C o u p l i n g F a c t o r 1) B y HARVEY S. PENEFSKY 1
General Principles The soluble ATPase of beef heart mitochondria also serves as a factor which couples the esterification of inorganic phosphate to the oxidation of suhstrates catalyzed by deficient submitochondrial particles. Ia,2 The enzyme is solubilized when beef heart mitochondria are disrupted by shaking with glass beads in a high speed reciprocal shaker. This method is based on one already presented s and introduces changes which permit preparation of the enzyme on a larger scale. Definition o] Unit and Specific Activity. A unit of activity is defined as that amount of enzyme which catalyzes the turnover of 1 micromole of substrate per minute under the specified conditions of assay2 Specific activity is expressed as units per milligram of dry weight of protein. Measurement .o] Protein Co~centr~tio~. The protein concentration of the mitochondrial suspension and of the step 1 and 2 fractions is determined by a biuret method modified for mitochondria2 Soluble protein is measured in a Brice-Phoenix differential refractometer model BP2000. The specific refractive index increment of bovine serum albumin, 188 >( 10-~ dl/g, 5 is used to calculate protein concentration from the difference in refractive index between protein and solvent. Protein con1This manuscript was prepared while the author was at the Department of Biochemistry, The Public Health Research Institute of the City of New York, Inc., New York. la M. E. Pullman, H. S. Penefsky, A. Datta, and E. Racker, J. Biol. Chem. 235, 3322 (1960). H. S. Penefsky, M. E. Pullman, A. Datta, and E. Racker, J. Biol. Chem. 235, 3330 (1960). M. E. Pullman and H. S. Penefsky, Vol. VI [34]. ' E. E. Jacobs, M. Jacob, D. R. Sanadi, and L. B. Bradley, J. Biol. Chem. ~'~3, 147 (1956). ~G, E. Perlmann and L. G. Longsworth, J. Am. Chem. ~oc. 70, 2719, (1948).
[82]
MITOCHONDRIAL ATPASE (COUPLING FACTOR 1)
523
centration determined by refractometry agrees to within 5% with values based on the weight of dialyzed and dried enzyme. Values of protein concentration in step 3 and 4 fractions also may be determined by ultraviolet absorption e and are corrected to dry weight when multiplied by 1.87. 7 Purification Procedure Step 1. Disintegration of the Mitochondria. "Light layer" mitochondria (6.75 g) prepared according to the method of Green and Ziegler, 8 is suspended in ice cold 0.25 M sucrose containing 2 mM EDTA, pH 7.4, and 2 mM ATP to give a final volume of 225 ml. Fifteen milliliters of this suspension and 7 ml of glass beads 9 are placed in the stainless steel cup of a Nossal 1° type reciprocal shaker 1~ and evacuated as described previously. 3 During the shaking period of 2 minutes, ice water is circulated around the cup and associated bearing and water at 25 ° is circulated around the main bearing. Following disruption of the mitochondria the contents of the cups are pooled and warmed to room temperature 12; the supernatant solution is removed after allowing the beads to settle. The beads are washed once by resuspension with 7(~ ml of 0.25 M sucrose, 2 mM EDTA, pH 7.4, and 2 mM ATP. The extract and bead washings are combined and centrifuged at room. temperature in the number 30 rotor of the Spinco Model L centrifuge. The packed brown pellet is discarded and the clear yellow supernatant solution is collected. The crude extract containing 1280 mg of protein is allowed to stand overnight at room temperature before the start of step 2. Step ~. pH Fractionation. The pH of the crude extract is adjusted to pH 5.4 by addition of 1.9 ml of 1 N acetic acid at room temperature with mechanical stirring. The solution is centrifuged at room temperature for 5 minutes at 5000 g, and the precipitate is discarded. The pH of the supernatant solution is adjusted to pH 6.7 by addition of 0.7 ml of 2 M Tris-S04, pH 10.7. Step 8. Prota~ine Fractionation. To prepare 0.5% protamine sulfate solution suspend 1.0 g protamine sulfate (Eli Lilly and Company) in 52 6See Vol. III [73]. ~H. S. Penefsky and R. C. Warner, J. BioL Chem. 240, 4694 (1965). 8D. E. Green and D. M. Ziegler, Vol. VI [58]. t Purchased from Minnesota Mining and Manufacturing Co., Ridgefield, New Jersey, 3M "Superbrite," catalog number 090, 0.0110-inch diameter. lop. M. Nossal, Australian J. Expel Biol. Med. Sci. 31, 583 (1953). 11Model RS, manufactured by the Lourdes Instrument Corporation, Brooklyn, New York. WAll subsequent manipulations are carried out at room temperature (25°) unless otherwise indicated.
524
COUPLING FACTORS
[82]
ml of distilled water. Adjust the pH to 7.{) with 0.1 M acetic acid, and bring the volume to 100 ml with water. Let stand in ice for 2{) minutes. Centrifuge in the cold at 18,000 g for 5 minutes and discard the residue. To the supernatant solution add an equal volume of water, and allow the solution to warm to room temperature before use. A 5.6-ml sample of 0.5% protamine sulfate solution is slowely added with mechanical stirring at room temperature to 224 ml of the step 2 fraction. 13 Stirring is continued for 15 minutes after the addition of protamine, and the light precipitate which forms is removed by centrifugation for 10 minutes at 15,000 g. All the ATPase activity remains in the clear supernatant solution. To the supernatant solution 73 ml of protamine sulfate solution is added as before. A heavy yellow precipitate forms and is collected by centrifugation. The supernatant solution is discarded, and the precipitate is dissolved by adding 6 ml of 0.25M sucrose, 2 mM EDTA, 0.01 M Tris-S04, pH 7.4, 0.4 M (NI~)~S04, pH 7.4. The resulting slightly turbid, yellow solution is clarified by centrifugation, and the small amount of insoluble residue is discarded. To the clear yellow supernatant solution is added with gentle stirring an equal volume of saturated ammonium sulfate, pH 5.5. The suspension is kept in ice for 15 minutes to ensure complete precipitation of the enzyme and then is centrifuged. The precipitate is dissoIved in 4 ml of 0.25M sucrose-0.01 M Tris-S04 pH 7.4-2 mM EDTA ~4 at room temperature. The dissolved enzyme is again precipitated by adding an equal volume of saturated ammonium sulfate solution. If desired the enzyme may be stored at this point at 4 ° before proceeding to the next step. Step ~,. Temperature Fraction~tion. The ammonium sulfate suspension of the enzyme from step 3 is centrifuged and dissolved in Sucrose-Tris-EDTA to give a final protein concentration of 2{) mg/ml. This solution is made 8 mM in ATP and heated in 12-ml conical glass centrifuge tubes at 65 ° for 4 minutes. Immediately thereafter the tubes are cooled in a 25 ° bath and centrifuged to remove denatured protein. The enzyme is precipitated from the supernatant solution by the addition of an equal volume of saturated ammonium sulfat~ solution. The enzyme is dissolved in Sucrose-Tris-EDTA and once again precipitated 13Since the exact amount of protamine required varies slightly for different preparations, a preliminary fractionation with protamine is carried out on a 4-ml sample of the step 2 fraction and scaled up appropriately. Usually 0.1 ml of 0.5% protamine solution is sufficient to precipitate inactive protein and lipid without reducing enzymatic activity in the supernatant solution. The subsequent addition of approximately 12, ml of protamine to the 4-ml sample results in precipitation of the bulk of the enzyme. ~4This solution will be referred to as Sucrose-Tris-EDTA.
[82]
MITOCHONDRIAL ATPASE (COUPLING FACTOR 1)
525
PURIFICATIONPROCEDURE FOR ATPAsE
Fraction
Volume (ml)
Step 1. Crude extract
237
Step 2. pH fractionation Step 3. Protamine fractionation Step 4. Temperature fractionation
224 10 8
Units 2760• 4950b 3860 3350 3240
Protein (rag)
Specific activity (units/rag Yield protein) (%)
1280 620 93.5 58
2.2 3.9 6.2 35.9 74.8
100 179 140 121 157
a Activity measured on the day of preparation. Activity measured on the following day. The specific activity of the crude extract increased about 2-fold upon standing overnight. with ammonium sulfate. The purified enzyme is stored at 4 ° as a suspension in ammonium sulfate. Samples of the enzyme m a y be prepared for assay by the centrifugation of desired aliquots of the ammonium sulfate suspension followed by removal of ammonium sulfate and solution of the pellet in appropriate buffers. Properties
Stability. The enzyme is stable for several months when stored at 4 ° at a suspension in half-saturated ammonium sulfate solution. The activity of the dissolved enzyme decreases slowly over a period of several days at room temperature in S u c r o s e - T r i s - E D T A , 4 m M ATP. The enzyme is stable for about 1 day in S u c r o s e - T r i s - E D T A and in buffers which do not contain sucrose provided A T P is present. Aqueous solutions of the A T P a s e are stable for at least 5 days when stored at --70 ° if frozen quickly in liquid nitrogen and thawed rapidly at 300. 7 Cold Lability. ATPase activity and coupling factor activity of the purified enzyme are cold labile in aqueous buffers, la,2 The rate of cold inactivation is greater at low protein concentrations (the half-time at 0 ° is about 7 minutes at 0.05 m g / m l and about 21 minutes at 3 m g / m l ) , is accelerated by anions (the order of effectiveness of anions in accelerating cold inactivation is I- > NO3- > Br- > C1- > S O ( - ) and is more labile at 0 ° (half-time about 7 minutes) than at 10 ° (half-time about 30 minutes). 7 The enzyme is stabilized in the cold by factors derived from beef heart mitochondria such as a heat stable dialyzable inhibitor, 1~ a heat stable protein inhibitor, 1G mitochondrial phospholipid, 1~ and Fo, an insoluble protein containing phospholipid which confers sensitivity to E. Racker, Federation Proc. 21, 54 (1962). le M. E. Pullman and G. Monroy, J. Biol. Chem. 238, 3762 (1963).
526
COUPLING FACTORS
[83]
oligomycin on the ATPase. 17 The enzyme also is stabilized in the cold by ethylene glycol, methanol, glycerol, and ethanolJ The cold-inactivated enzyme may be partially reactivated by rewarming at concentrations greater than 0.1 mg/ml. Physical Properties. At 25 ° in 20 mM P~, pH 7.2, 2 mM EDTA, 4 mM ATP, 0.1 M KCI, ~0.w is 12.9 °, the molecular weight determined by equilibrium ultracentrifugation is 284,000, the intrinsic viscosity is 3.2 ml/g, and the partial specific volume is 0.74 ml/gJ When incubated in the cold, the enzyme dissociates forming an equilibrium mixture of 3.4 S, 9.2 S, and 11.9 S components. Rewarming of the cold-treated enzyme under conditions which restore enzymatic activity results in the formation of a single sedimenting species with a sedimentation coefficient identical with that of the native enzyme. The ATPase dissociates at 25 ° in 1% sodium dodecyl sulfate to form a single sedimenting species with an S2o,w of 2.6 S and approximate molecular weight of 29,000. Chemical Analysis. The minimum molecular weight based on the half-cystine content is 26,100. TM Tryptophan, galactosamine, glucosamine, and fatty acidsTM were not detected. Sulfhydryl group analyses indicate 10 or 11 SH per mole. There is no evidence for disulfide bridges in the moleculeJ Other Praperties. The specificity of the ATPase, activators, and inhibitors of the enzyme and the pH optima have been described2 ,7 E. Racker, Biochem. Biophya. Res. Commun. 10, 435 (1963). The amino acid analyses were kindly performed by Dr. S. Moore. Fatty acid analyses were carried out by Dr. S. Fleischer and Dr. I. Mossbach.
[83] P r e p a r a t i o n of 3 H - A c e t y l - A T P a s e ( C o u p l i n g F a c t o r 1)
By Yasuo ICCGAWA1 General larinciple ATPase TM containing 5-6 moles of 3H-acetyl groups per mole of enzyme is prepared by reaction with SH-acetie anhydride. The labeled preparations appear to retain all the properties of the native enzyme and are useful in studies of the binding of the soluble ATPase to insoluble mitochondrial fractions. 1See footnote 1, page 505. ,I See this volume [82].