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. 1996 Aug 15;318(Pt 1):351–357. doi: 10.1042/bj3180351

ATP synthase from bovine heart mitochondria: reconstitution into unilamellar phospholipid vesicles of the pure enzyme in a functional state.

G Groth 1, J E Walker 1
PMCID: PMC1217628  PMID: 8761492

Abstract

A highly purified and monodisperse preparation of proton-translocating F1F0-ATPase from bovine heart mitochondria is an assembly of 16 unlike polypeptides. This preparation has been reconstituted in the presence of various detergents into unilamellar phospholipid vesicles. Incorporation of the enzyme into vesicles increases the ATP hydrolase activity of the enzyme by 10-20-fold, depending on the detergent, and the highest activities of ATP hydrolysis, 70 units/mg, were obtained by reconstitution from dodecylmaltoside or CHAPS. This activity is mostly sensitive to inhibitors that act on the F0 membrane sector of the complex. From the quenching of the pH-sensitive probe, 9-amino-6-chloro-2-methoxyacridine, it was shown that the reconstituted enzyme was able to form a transmembrane proton gradient in an ATP-dependent manner. By co-reconstitution of the enzyme with bacteriorhodopsin, it was demonstrated that in the presence of a light-induced proton gradient the enzyme can synthesize ATP from ADP and phosphate. Therefore, the characteristic biological functions of the F1F0-ATPase in mitochondria have been demonstrated with the purified enzyme. Thus, in terms of both its physical and biochemical properties, the purified enzyme fulfils important pre-requisites for formation of two- and three-dimensional crystals.

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Selected References

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  1. Abrahams J. P., Leslie A. G., Lutter R., Walker J. E. Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 1994 Aug 25;370(6491):621–628. doi: 10.1038/370621a0. [DOI] [PubMed] [Google Scholar]
  2. Aggeler R., Haughton M. A., Capaldi R. A. Disulfide bond formation between the COOH-terminal domain of the beta subunits and the gamma and epsilon subunits of the Escherichia coli F1-ATPase. Structural implications and functional consequences. J Biol Chem. 1995 Apr 21;270(16):9185–9191. doi: 10.1074/jbc.270.16.9185. [DOI] [PubMed] [Google Scholar]
  3. Almog S., Litman B. J., Wimley W., Cohen J., Wachtel E. J., Barenholz Y., Ben-Shaul A., Lichtenberg D. States of aggregation and phase transformations in mixtures of phosphatidylcholine and octyl glucoside. Biochemistry. 1990 May 15;29(19):4582–4592. doi: 10.1021/bi00471a012. [DOI] [PubMed] [Google Scholar]
  4. Ansorge W. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J Biochem Biophys Methods. 1985 May;11(1):13–20. doi: 10.1016/0165-022x(85)90037-5. [DOI] [PubMed] [Google Scholar]
  5. Berden J. A., Henneke M. A. The uncoupler-binding protein in the proton-pumping ATPase from beef-heart mitochondria. FEBS Lett. 1981 Apr 20;126(2):211–214. doi: 10.1016/0014-5793(81)80244-x. [DOI] [PubMed] [Google Scholar]
  6. Boyer P. D. The binding change mechanism for ATP synthase--some probabilities and possibilities. Biochim Biophys Acta. 1993 Jan 8;1140(3):215–250. doi: 10.1016/0005-2728(93)90063-l. [DOI] [PubMed] [Google Scholar]
  7. Buchanan S. K., Walker J. E. Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria. Biochem J. 1996 Aug 15;318(Pt 1):343–349. doi: 10.1042/bj3180343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang T., Penefsky H. S. Aurovertin, a fluorescent probe of conformational change in beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1973 Apr 25;248(8):2746–2754. [PubMed] [Google Scholar]
  9. Collinson I. R., Runswick M. J., Buchanan S. K., Fearnley I. M., Skehel J. M., van Raaij M. J., Griffiths D. E., Walker J. E. Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase. Biochemistry. 1994 Jun 28;33(25):7971–7978. doi: 10.1021/bi00191a026. [DOI] [PubMed] [Google Scholar]
  10. Deisinger B., Nawroth T., Zwicker K., Matuschka S., John G., Zimmer G., Freisleben H. J. Purification of ATP synthase from beef heart mitochondria (F0F1) and co-reconstitution with monomeric bacteriorhodopsin into liposomes capable of light-driven ATP synthesis. Eur J Biochem. 1993 Dec 1;218(2):377–383. doi: 10.1111/j.1432-1033.1993.tb18387.x. [DOI] [PubMed] [Google Scholar]
  11. Dreyfus G., Célis H., Ramírez J. Isolation of the mitochondrial F1-F0 adenosine triphosphatase by Sepharose-hexylammonium chromatography: properties and reconstitution in liposomes. Anal Biochem. 1984 Oct;142(1):215–220. doi: 10.1016/0003-2697(84)90541-4. [DOI] [PubMed] [Google Scholar]
  12. Dreyfus G. Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes. J Biol Chem. 1985 Oct 5;260(22):12112–12117. [PubMed] [Google Scholar]
  13. Duncan T. M., Bulygin V. V., Zhou Y., Hutcheon M. L., Cross R. L. Rotation of subunits during catalysis by Escherichia coli F1-ATPase. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10964–10968. doi: 10.1073/pnas.92.24.10964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galante Y. M., Wong S. Y., Hatefi Y. Composition of complex V of the mitochondrial oxidative phosphorylation system. J Biol Chem. 1979 Dec 25;254(24):12372–12378. [PubMed] [Google Scholar]
  15. Gräber P., Schlodder E., Witt H. T. Conformational change of the chloroplast ATPase induced by a transmembrane electric field and its correlation to phosphorylation. Biochim Biophys Acta. 1977 Sep 14;461(3):426–440. doi: 10.1016/0005-2728(77)90231-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hatefi Y., Stiggall D. L., Galante Y., Hanstein W. G. Mitochondrial ATP-Pi exchange complex. Biochem Biophys Res Commun. 1974 Nov 6;61(1):313–321. doi: 10.1016/0006-291x(74)90568-3. [DOI] [PubMed] [Google Scholar]
  17. Jagendorf A. T., Uribe E. ATP formation caused by acid-base transition of spinach chloroplasts. Proc Natl Acad Sci U S A. 1966 Jan;55(1):170–177. doi: 10.1073/pnas.55.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joshi S., Hughes J. B., Shaikh F., Sanadi D. R. On the role of coupling factor B in the mitochondrial Pi-ATP exchange reaction. J Biol Chem. 1979 Oct 25;254(20):10145–10152. [PubMed] [Google Scholar]
  19. Junge W., Rumberg B., Schröder H. The necessity of an electric potential difference and its use for photophosphorylation in short flash groups. Eur J Biochem. 1970 Jul;14(3):575–581. doi: 10.1111/j.1432-1033.1970.tb00326.x. [DOI] [PubMed] [Google Scholar]
  20. Kagawa Y. Incorporation of purple membrane into vesicles capable of light-stimulated ATP synthesis. Methods Enzymol. 1979;55:777–780. doi: 10.1016/0076-6879(79)55085-x. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lichtenberg D., Robson R. J., Dennis E. A. Solubilization of phospholipids by detergents. Structural and kinetic aspects. Biochim Biophys Acta. 1983 May 24;737(2):285–304. doi: 10.1016/0304-4157(83)90004-7. [DOI] [PubMed] [Google Scholar]
  23. McEnery M. W., Buhle E. L., Jr, Aebi U., Pedersen P. L. Proton ATPase of rat liver mitochondria. Preparation and visualization of a functional complex using the novel zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. J Biol Chem. 1984 Apr 10;259(7):4642–4651. [PubMed] [Google Scholar]
  24. Penin F., Godinot C., Comte J., Gautheron D. C. Vesicular preparation of a highly coupled ATPase-ATP synthase complex from pig heart mitochondria. Biochim Biophys Acta. 1982 Feb 17;679(2):198–209. doi: 10.1016/0005-2728(82)90291-2. [DOI] [PubMed] [Google Scholar]
  25. Racker E. Reconstitution of membrane processes. Methods Enzymol. 1979;55:699–711. doi: 10.1016/0076-6879(79)55078-2. [DOI] [PubMed] [Google Scholar]
  26. Richard P., Rigaud J. L., Gräber P. Reconstitution of CF0F1 into liposomes using a new reconstitution procedure. Eur J Biochem. 1990 Nov 13;193(3):921–925. doi: 10.1111/j.1432-1033.1990.tb19418.x. [DOI] [PubMed] [Google Scholar]
  27. Rigaud J. L., Bluzat A., Buschlen S. Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation. Biochem Biophys Res Commun. 1983 Mar 16;111(2):373–382. doi: 10.1016/0006-291x(83)90316-9. [DOI] [PubMed] [Google Scholar]
  28. Rigaud J. L., Paternostre M. T., Bluzat A. Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 2. Incorporation of the light-driven proton pump bacteriorhodopsin. Biochemistry. 1988 Apr 19;27(8):2677–2688. doi: 10.1021/bi00408a007. [DOI] [PubMed] [Google Scholar]
  29. SUGINO Y., MIYOSHI Y. THE SPECIFIC PRECIPITATION OF ORTHOPHOSPHATE AND SOME BIOCHEMICAL APPLICATIONS. J Biol Chem. 1964 Jul;239:2360–2364. [PubMed] [Google Scholar]
  30. Schuldiner S., Rottenberg H., Avron M. Determination of pH in chloroplasts. 2. Fluorescent amines as a probe for the determination of pH in chloroplasts. Eur J Biochem. 1972 Jan 31;25(1):64–70. doi: 10.1111/j.1432-1033.1972.tb01667.x. [DOI] [PubMed] [Google Scholar]
  31. Serrano R., Kanner B. I., Racker E. Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria. J Biol Chem. 1976 Apr 25;251(8):2453–2461. [PubMed] [Google Scholar]
  32. Stiggall D. L., Galante Y. M., Hatefi Y. Preparation and properties of an ATP-Pi exchange complex (complex V) from bovine heart mitochondria. J Biol Chem. 1978 Feb 10;253(3):956–964. [PubMed] [Google Scholar]
  33. Strotmann H., Bickel S., Huchzermeyer B. Energy-dependent release of adenine nucleotides tightly bound to chloroplast coupling factor CF1. FEBS Lett. 1976 Jan 15;61(2):194–198. doi: 10.1016/0014-5793(76)81036-8. [DOI] [PubMed] [Google Scholar]
  34. Turina P., Rumberg B., Melandri B. A., Gräber P. Activation of the H(+)-ATP synthase in the photosynthetic bacterium Rhodobacter capsulatus. J Biol Chem. 1992 Jun 5;267(16):11057–11063. [PubMed] [Google Scholar]

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