Purification of the energy-transducing adenosine triphosphatase complex from Rhodospirillum rubrum

Abstract

The oligomycin- and N,N''-dicyclohexylcarbodiimide-sensitive ATPase complex extracted with Triton X-100 from the chromatophores of R. rubrum was extensively purified. The purification procedure included (diethylamino)ethylcellulose chromatography and glycerol gradient centrifugation. The specific activity of Mg2+-dependent ATP hydrolysis in the purified preparation increased about 11-fold, while that of Ca2+-dependent ATP hydrolysis increased 50-fold as compared with chromatophores. The purified ATPase complex dissociated into a maximum of 8 different polypeptides upon electrophoresis in the presence of sodium dodecyl sulfate. The estimated subunit MW were as follows: 56,000 (.alpha.), 50,000 (.beta.), 33,000 (.gamma.) and those ranging from 17,000-9400 for the remaining smaller subunits. The purified preparation was incorporated into phospholipid vesicles by using the freeze-thaw technique. The reconstituted vesicles catalyzed [32P]ATP exchange, which was almost completely inhibited by both oligomycin and N,N''-dicyclohexylcarbodiimide and by a protonophorous uncoupler, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone.