Abstract
The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The32Pi-ATP exchange activity of the reconstituted ATPase complex was inhibited byp-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with14C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of32Pi-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved; at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of32Pi-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.
Similar content being viewed by others
Abbreviations
- DCCD:
-
N,N′-dicyclohexylcarbodiimide
- STE-DTT buffer:
-
sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM), DTT (5 mM), pH 8.0
- F o :
-
a membranous preparation from mitochondria conferring oligomycin (or rutamycin) sensitivity to F1
- F1F6 :
-
coupling factors 1 (ATPase) and 6
- OSCP:
-
oligomycin-sensitivity-conferring protein
- BSA:
-
bovine serum albumin
- SDS:
-
sodium dodecyl sulfate
- DTT:
-
dithiothreitol
- STE buffer:
-
sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM)
- TUA particles:
-
submitochondrial particles prepared by stepwise exposure of light-layer submitochondrial particles to trypsin and urea, then sonic oscillation in the presence of dilute ammonia (pH 10.4)
- OG-cholate buffer:
-
glycerol (20%), Tricine (50 mM), MgSO4 (5 mM), DTT (5mM), cholate (0.5%), octylglucoside (0.5%), pH 8.0
- p-HMB:
-
p-hydroxymercuribenzoate
References
Alfonzo, M., and Racker, E. (1979).Can. J. Biochem. 57 1351–1358.
Ames, B. N., and Dubin, D. T. (1960).J. Biol. Chem. 235 769–775.
Banerjee, R., Epstein, M., Kandrach, M., Zimniak, P., and Racker, E. (1979).Membr. Biochem. 2 283–296.
Becker, B. M., and Cassim, J. Y. (1975).Prep. Biochem. 5(2), 161–178.
Bensadoun, A., Weinstein, D. (1976).Anal. Biochem. 70 241–250.
Bulos, B., and Racker, E. (1968).J. Biol. Chem. 243 3891–3900.
Carroll, R., and Racker, E. (1977).J. Biol. Chem. 252 6981–6990.
Criddle, R. S., Packer, L., and Shieh, P. (1977).Proc. Natl. Acad. Sci. USA 74 4306–4310.
Dunn, S. D., and Futai, M. (1980).J. Biol. Chem. 255 113–118.
Futai, M., and Kanazawa, H. (1980).Curr. Top. Bioenerg. 10 181–215.
Gómez-Puyou, A., Gómez-Puyou, M. T., and Ernster, L. (1979).Biochim. Biophys. Acta 547 252–257.
Green, D. E., Lester, R. L., and Ziegler, D. M. (1957).Biochim. Biophys. Acta 23 516–524.
Horstman, L. L., and Racker, E. (1970).J. Biol. Chem. 245 1336–1344.
Kagawa, Y. (1978).Biochim. Biophys. Acta 505 45–93.
Kagawa, Y., Kandrach, A., and Racker, E. (1973).J. Biol. Chem. 248 676–684.
Kagawa, Y., and Racker, E. (1966).J. Biol. Chem. 241 2467–2474.
Kagawa, Y., and Racker, E. (1971).J. Biol. Chem. 246 5477–5487.
Kanner, B. I., Serrano, R., Kandrach, M. A., and Racker, E. (1976).Biochem. Biophys. Res. Commun. 69 1050–1056.
Kasahara, M., and Hinkle, P. C. (1976).Proc. Natl. Acad. Sci. USA 73 396–400.
Lanyi, J. K., and MacDonald, R. E. (1979). InMethods in Enzymology S. Fleischer and L. Packer, eds., Academic Press, New York, Vol. 56, pp. 398–407.
Lindberg, O., and Ernster, L. (1956). InMethods of Biochemical Analysis Interscience, New York, Vol. 3, p. I.
Lohmann, K., and Jendrassik, L. (1926).Biochem. Z. 178 419–426.
Ludwig, B., and Schatz, G. (1980).Proc. Natl. Acad. Sci. USA 77 196–200.
MacLennan, D. H., and Tzagoloff, A. (1968).Biochemistry 7 1603–1610.
McCarty, R. E. (1979).Annu. Rev. Plant Phys. 30 79–104.
Nelson, N. (1976).Biochim. Biophys. Acta. 456 314–338.
Nelson, N., Eytan, E., Notsani, B., Sigrist, H., Sigrist-Nelson, K., and Gitler, C. (1977).Proc. Natl. Acad. Sci. USA 74 2375–2378.
Nelson, N., Nelson, H., and Racker, E. (1972).J. Biol. Chem. 247 7657–7662.
Oesterhelt, D., and Stoeckenius, W. (1974). InMethods in Enzymology S. Fleischer and L. Packer, eds. Academic Press, New York, Vol. 31, pp. 667–686.
Pick, U., and Racker, E. (1979).J. Biol. Chem. 254 2793–2799.
Pullman, M. E., and Monroy, G. C. (1963).J. Biol. Chem. 238 3762–3769.
Racker, E. (1962).Proc. Natl. Acad. Sci. USA 48 1659–1663.
Racker, E. (1979).Acc. Chem. Res. 12 338–344.
Racker, E., and Stoeckenius, W. (1974).J. Biol. Chem. 249 662–663.
Racker, E., Violand, B., O'Neal, S., Alfonzo, M., and Telford, J. (1979).Arch. Biochem. Biophys. 198 470–477.
Ragan, C. I., and Racker, E. (1973).J. Biol. Chem. 248 2563–2569.
Schatz, G., and Mason, T. L. (1974).Ann. Rev. Biochem. 43 51–87.
Schneider, D. L., Kagawa, Y., and Racker, E. (1972).J. Biol. Chem. 247 4074–4079.
Senior, A. E. (1971).Bioenergetics 2 141–150.
Sone, N., Ohyama, T., and Kagawa, Y. (1979).FEBS Lett. 106 39–42.
Swank, R. T., and Munkres, K. D. (1971).Anal. Biochem. 39 462–477.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Alfonzo, M., Kandrach, M.A. & Racker, E. Isolation, characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump. J Bioenerg Biomembr 13, 375–391 (1981). https://doi.org/10.1007/BF00743211
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00743211