Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1.
A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1). The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed. The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M. (1985) J. Biol. Chem. 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores. The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R. rubrum Fo-F1 complex. The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin. In contrast, chromatophores reconstituted with the native R. rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin. These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes. They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.
D000255:Adenosine Triphosphate / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D006096:Gramicidin / Q000494:pharmacology; D006868:Hydrolysis; D046911:Macromolecular Substances; D010766:Phosphorylation; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D046911:Macromolecular Substances; D006096:Gramicidin; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases