Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Jun;83(12):4233–4237. doi: 10.1073/pnas.83.12.4233

Steady-state ATP synthesis by bacteriorhodopsin and chloroplast coupling factor co-reconstituted into asolectin vesicles.

J Krupinski, G G Hammes
PMCID: PMC323706  PMID: 2872676

Abstract

A method was developed for the co-reconstitution of bacteriorhodopsin and chloroplast coupling factor in asolectin vesicles. First, bacteriorhodopsin was reconstituted from a mixture of octyl glucoside, asolectin, and protein in the presence of ethylenediaminetetraacetic acid by passage through a Sephadex G-50 centrifuge column. Then, the purified coupling factor was reconstituted from a mixture of sodium cholate, bacteriorhodopsin vesicles, and coupling factor in the presence of Mg2+ by passage through the centrifuge column. Sucrose density-gradient centrifugation indicated a band of vesicles with slightly different positions in the gradient for maximum vesicle concentration, bacteriorhodopsin vesicle concentration, ATP synthesis, and ATP hydrolysis. The rate of light-driven ATP synthesis reaches a limiting value as the concentration of bacteriorhodopsin and the light intensity are increased. A steady-state rate of ATP synthesis of 1 mumol per mg of coupling factor X min-1 has been achieved. Apparently this rate is limited by the heterogeneity within the vesicle population and by the ability of bacteriorhodopsin to form a sufficiently large pH gradient.

Full text

PDF
4235

Images in this article

4235Image
on p.4235

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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. 1981 Dec;13(5-6):375–391. doi: 10.1007/BF00743211. [DOI] [PubMed] [Google Scholar]
  2. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  3. Bokranz M., Mörschel E., Kröger A. Phosphorylation and phosphate-ATP exchange catalyzed by the ATP synthase isolated from Wolinella succinogenes. Biochim Biophys Acta. 1985 Dec 16;810(3):332–339. doi: 10.1016/0005-2728(85)90218-x. [DOI] [PubMed] [Google Scholar]
  4. Dewey T. G., Hammes G. G. Steady state kinetics of ATP synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor. J Biol Chem. 1981 Sep 10;256(17):8941–8946. [PubMed] [Google Scholar]
  5. Hauska G., Samoray D., Orlich G., Nelson N. Reconstitution of photosynthetic energy conservation. II. Photophosphorylation in liposomes containing photosystem-I reaction center and chloroplast coupling-factor complex. Eur J Biochem. 1980 Oct;111(2):535–543. doi: 10.1111/j.1432-1033.1980.tb04969.x. [DOI] [PubMed] [Google Scholar]
  6. Huang K. S., Bayley H., Khorana H. G. Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid. Proc Natl Acad Sci U S A. 1980 Jan;77(1):323–327. doi: 10.1073/pnas.77.1.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Krupinski J., Hammes G. G. Phase-lifetime spectrophotometry of deoxycholate-purified bacteriorhodopsin reconstituted into asolectin vesicles. Biochemistry. 1985 Nov 19;24(24):6963–6972. doi: 10.1021/bi00345a032. [DOI] [PubMed] [Google Scholar]
  8. Lozier R. H., Niederberger W., Bogomolni R. A., Hwang S., Stoeckenius W. Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane. Biochim Biophys Acta. 1976 Sep 13;440(3):545–556. doi: 10.1016/0005-2728(76)90041-4. [DOI] [PubMed] [Google Scholar]
  9. Oren R., Weiss S., Garty H., Caplan S. R., Gromet-Elhanan Z. ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin. Arch Biochem Biophys. 1980 Dec;205(2):503–509. doi: 10.1016/0003-9861(80)90133-2. [DOI] [PubMed] [Google Scholar]
  10. Penefsky H. S. A centrifuged-column procedure for the measurement of ligand binding by beef heart F1. Methods Enzymol. 1979;56:527–530. doi: 10.1016/0076-6879(79)56050-9. [DOI] [PubMed] [Google Scholar]
  11. Pick U., Racker E. Purification and reconstitution of the N,N'-dicyclohexylcarbodiimide-sensitive ATPase complex from spinach chloroplasts. J Biol Chem. 1979 Apr 25;254(8):2793–2799. [PubMed] [Google Scholar]
  12. Racker E., Stoeckenius W. Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation. J Biol Chem. 1974 Jan 25;249(2):662–663. [PubMed] [Google Scholar]
  13. Ryrie I. J., Critchley C., Tillberg J. E. Structure and energy-linked activities in reconstituted bacteriorhodopsin--yeast ATPase proteoliposomes. Arch Biochem Biophys. 1979 Nov;198(1):182–194. doi: 10.1016/0003-9861(79)90409-0. [DOI] [PubMed] [Google Scholar]
  14. Sone N., Takeuchi Y., Yoshida M., Ohno K. Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin. J Biochem. 1977 Dec;82(6):1751–1758. doi: 10.1093/oxfordjournals.jbchem.a131873. [DOI] [PubMed] [Google Scholar]
  15. Stoeckenius W., Bogomolni R. A. Bacteriorhodopsin and related pigments of halobacteria. Annu Rev Biochem. 1982;51:587–616. doi: 10.1146/annurev.bi.51.070182.003103. [DOI] [PubMed] [Google Scholar]
  16. Takabe T., Hammes G. G. pH dependence of adenosine 5'-triphosphate synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor. Biochemistry. 1981 Nov 24;20(24):6859–6864. doi: 10.1021/bi00527a018. [DOI] [PubMed] [Google Scholar]
  17. Winget G. D., Kanner N., Racker E. Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin. Biochim Biophys Acta. 1977 Jun 9;460(3):490–499. doi: 10.1016/0005-2728(77)90087-1. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES