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[50] Preparation of coupling factor 6 (F6)
398
ATP
SYNTHESIS AND REGULATION
[50] Preparation
of Coupling
Factor
[50]
6 ( F 6)
By EFRAIM RACKER
Assay M e t h o d Principle. Coupling factor 6 (F6) stimulates the a*P~-ATP exchange in submitochondrial particles which have been exposed to silicotungstate (STA) 1 and to which the other coupling factors have been added. Reagents Buffer A: 250 mM sucrose, 0.25 mM E D T A , and 10 mM Tris-SO4 (pH 8.0) Coupling factor 2 (Fz; factor B), ca. 1 mg/ml Other reagents as for assay of rutamycin-sensitive ATPase (RSA) (see Article [35], this volume) Preparation o f S T A Particles. In a final volume of 11 ml, 220 mg of A particles 2 are added dropwise with stirring to a cold solution containing in final concentration 0.15 M sucrose, 1% STA, p H 5.5, and 30 mM T r i s T E S , p H 7.5 (pH measured at 22°). The mixture is kept at 4 ° for 6 min and then diluted with 10 volumes of cold 0.25 M sucrose. After centriguation for 20 min at 105,000 g in a Spinco 40 rotor, the supernatant is discarded. The pellet is resuspended in 0.25 M s u c r o s e - 5 mM dithiothreitol and centrifuged at 105,000 g for 20 min. The pellet is again suspended in 0.25 M sucrose-5 mM dithiothreitol at a protein concentration of 15-20 mg/ml and stored in small aliquots in liquid nitrogen. Under these conditions, the particles are stable for several weeks but deteriorate within a few hours at 4°. Procedure Protein fractions of F6 are incubated for 20 min at 30° with STA particles 1 (200 /zg) together with 120 /zg o f F1, 20 /zg of oligomycinsensitivity-conferring protein, 25/zg of Fz, a and 4 mg of defatted bovine serum albumin in a final volume of 0.5 ml of buffer A. The reaction is started by addition of an equal volume of a mixture containing 40 mM KPi (with about 10 r cpm of a~P0, 500 mM sucrose, 20 mM MgSO4, 20 mM Na-ATP, and 40 mM Tris-SO4 (pH 8.0). After 10 min incubation at 1 E. Racker, L. L. Horstman, D. Kling, and J. M. Fessenden-Raden, J. Biol. Chem. 244, 6668 (1969). J. M. Fessenden and E. Racker, this series, Vol. 10 [35]. a E. Racker, J. M. Fessenden-Raden, M. A. Kandrach, K. W. Lam, and D. R. Sanadi, Biochem. Biophys. Res. Commun. 41, 1474-1479(1970). METHODS IN ENZYMOLOGY, VOL. LV
Copyright © 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
[51]
MITOCHONDRIALATPase INHIBITOR
399
30° the reaction is terminated and assayed as described for the assay of RSA (!Article [35], this volume). P r e p a r a t i o n of F6 The RSA complex (38-45 P fraction) is prepared as d e s c r i b e d ? The preparation of about 25-30 mg protein per milliliter is heated in a test tube for 4 rain at 75 ° in a heating block with occasional shaking. The denatured proteins are removed by centrifugation at 4 ° for l0 rain at 12,000 g. The supernatant (S,) is removed with a Pasteur pipette, and 2,8 ml of ice-cold 95% ethanol is added per milliliter of $1 (final ethanol concentration 70%, v/v). The mixture is agitated for 20-30 sec on a Vortex mixer and centrifuged as above. The pellet, which contains mitochondrial ATPase inhibitor, is resuspended in buffer A at 0.5-1.0 mg protein per milliliter. The supernatant, which contains F0, is taken to dryness by lyophilization. The residue is taken up in 0.5 ml of buffer A for each milliliter of starting material, and insoluble material is discarded after centrifugation as described above. In sodium dodecyl sulfate-urea acrylamide gels this preparation appears as a single protein band corresponding to a molecular weight of 8000. A contaminating yellow pigment can be removed by dialysis against several changes of l0 mM Tris-sulfate (pH 8.0) for several days. A small amount of phospholipid can be removed by lyophilization and extraction with large volumes (200 ml for each l0 mg of protein) of chloroform-methanol (2: 1). The bulk of the solvent can be removed after centrifugation by aspiration, but since the pellet is loose, residual solvent is removed under a stream of nitrogen. If desirable, the preparation can be again dialyzed. 4 E. Racker, this volume Article [35].
[51] Mitochondrial By
LARS
ERNSTER,
ATPase Inhibitor: Applications
CHRISTINE
CARLSSON,
Properties
TORILL
and
HUNDAL, and
KERSTIN NORDENBRAND Properties Mitochondrial ATPase inhibitor was discovered in 1963 by Pullman and Monroy, 1 and its preparation and properties were reviewed by the 1 M. E. Pullman and G. C. Monroy, J. METHODS IN ENZYMOLOGY, VOL. LV
Biol. Chem.
238, 3762 (1963). Copyright © 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
ATP
SYNTHESIS AND REGULATION
[50] Preparation
of Coupling
Factor
[50]
6 ( F 6)
By EFRAIM RACKER
Assay M e t h o d Principle. Coupling factor 6 (F6) stimulates the a*P~-ATP exchange in submitochondrial particles which have been exposed to silicotungstate (STA) 1 and to which the other coupling factors have been added. Reagents Buffer A: 250 mM sucrose, 0.25 mM E D T A , and 10 mM Tris-SO4 (pH 8.0) Coupling factor 2 (Fz; factor B), ca. 1 mg/ml Other reagents as for assay of rutamycin-sensitive ATPase (RSA) (see Article [35], this volume) Preparation o f S T A Particles. In a final volume of 11 ml, 220 mg of A particles 2 are added dropwise with stirring to a cold solution containing in final concentration 0.15 M sucrose, 1% STA, p H 5.5, and 30 mM T r i s T E S , p H 7.5 (pH measured at 22°). The mixture is kept at 4 ° for 6 min and then diluted with 10 volumes of cold 0.25 M sucrose. After centriguation for 20 min at 105,000 g in a Spinco 40 rotor, the supernatant is discarded. The pellet is resuspended in 0.25 M s u c r o s e - 5 mM dithiothreitol and centrifuged at 105,000 g for 20 min. The pellet is again suspended in 0.25 M sucrose-5 mM dithiothreitol at a protein concentration of 15-20 mg/ml and stored in small aliquots in liquid nitrogen. Under these conditions, the particles are stable for several weeks but deteriorate within a few hours at 4°. Procedure Protein fractions of F6 are incubated for 20 min at 30° with STA particles 1 (200 /zg) together with 120 /zg o f F1, 20 /zg of oligomycinsensitivity-conferring protein, 25/zg of Fz, a and 4 mg of defatted bovine serum albumin in a final volume of 0.5 ml of buffer A. The reaction is started by addition of an equal volume of a mixture containing 40 mM KPi (with about 10 r cpm of a~P0, 500 mM sucrose, 20 mM MgSO4, 20 mM Na-ATP, and 40 mM Tris-SO4 (pH 8.0). After 10 min incubation at 1 E. Racker, L. L. Horstman, D. Kling, and J. M. Fessenden-Raden, J. Biol. Chem. 244, 6668 (1969). J. M. Fessenden and E. Racker, this series, Vol. 10 [35]. a E. Racker, J. M. Fessenden-Raden, M. A. Kandrach, K. W. Lam, and D. R. Sanadi, Biochem. Biophys. Res. Commun. 41, 1474-1479(1970). METHODS IN ENZYMOLOGY, VOL. LV
Copyright © 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8
[51]
MITOCHONDRIALATPase INHIBITOR
399
30° the reaction is terminated and assayed as described for the assay of RSA (!Article [35], this volume). P r e p a r a t i o n of F6 The RSA complex (38-45 P fraction) is prepared as d e s c r i b e d ? The preparation of about 25-30 mg protein per milliliter is heated in a test tube for 4 rain at 75 ° in a heating block with occasional shaking. The denatured proteins are removed by centrifugation at 4 ° for l0 rain at 12,000 g. The supernatant (S,) is removed with a Pasteur pipette, and 2,8 ml of ice-cold 95% ethanol is added per milliliter of $1 (final ethanol concentration 70%, v/v). The mixture is agitated for 20-30 sec on a Vortex mixer and centrifuged as above. The pellet, which contains mitochondrial ATPase inhibitor, is resuspended in buffer A at 0.5-1.0 mg protein per milliliter. The supernatant, which contains F0, is taken to dryness by lyophilization. The residue is taken up in 0.5 ml of buffer A for each milliliter of starting material, and insoluble material is discarded after centrifugation as described above. In sodium dodecyl sulfate-urea acrylamide gels this preparation appears as a single protein band corresponding to a molecular weight of 8000. A contaminating yellow pigment can be removed by dialysis against several changes of l0 mM Tris-sulfate (pH 8.0) for several days. A small amount of phospholipid can be removed by lyophilization and extraction with large volumes (200 ml for each l0 mg of protein) of chloroform-methanol (2: 1). The bulk of the solvent can be removed after centrifugation by aspiration, but since the pellet is loose, residual solvent is removed under a stream of nitrogen. If desirable, the preparation can be again dialyzed. 4 E. Racker, this volume Article [35].
[51] Mitochondrial By
LARS
ERNSTER,
ATPase Inhibitor: Applications
CHRISTINE
CARLSSON,
Properties
TORILL
and
HUNDAL, and
KERSTIN NORDENBRAND Properties Mitochondrial ATPase inhibitor was discovered in 1963 by Pullman and Monroy, 1 and its preparation and properties were reviewed by the 1 M. E. Pullman and G. C. Monroy, J. METHODS IN ENZYMOLOGY, VOL. LV
Biol. Chem.
238, 3762 (1963). Copyright © 1979 by Academic Press Inc. All rights of reproduction in any form reserved. ISBN 0-12-181955-8