Purification and properties of the coupling-factor ATPases F1 from Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides.
The coupling-factor ATPases from photosynthetically grown Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides were purified by the same procedure to homogeneity. Gel chromatography on Sephacryl S-300 Superfine shortened the process of purification and improved its yield. Solubilization of the ATPase from both bacteria was found to be dependent on a specific sonication treatment of the cell suspensions, indicating a very weakly bound F1-ATPase in R. palustris. Depleted chromatophores could be restored in photophosphorylation and membrane-bound ATPase activities by adding the solubilized ATPase protein. The purified enzymes did not show a markedly trypsin-stimulated or dithiothreitol-stimulated activity. Isoelectric focusing and chromatofocusing revealed isoelectric points of 5.0 for both F1-ATPases. The molecular weights were determined by gel chromatography plus high-performance liquid chromatography. Hence, we calculated a molecular weight of 350000 for both F1-ATPases. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed five subunits for both enzymes. Kinetic parameters, regarding substrate specificity, the effect of divalent cations, Km and Ki values for the membrane-bound and solubilized ATPases were determined.
D001426:Bacterial Proteins / Q000302:isolation & purification; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D012241:Rhodopseudomonas / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008565:Membrane Proteins; D006180:Proton-Translocating ATPases