PURIFICATION OF THE ENERGY-TRANSDUCING ADENOSINE-TRIPHOSPHATASE COMPLEX FROM RHODOSPIRILLUM-RUBRUM

The oligomycin- and -dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase complex extracted with Triton X-100 from the chromatophores of Rhodospi-rillum 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+-de-pendent ATP hydrolysis increased 50-fold as compared with chromatophores. The purified adenosine triphosphatase complex dissociated into a maximum of eight different po-lypeptides upon electrophoresis in the presence of sodium dodecyl sulfate. The estimated subunit molecular weights were as follows: 56 000 (a), 50000 (0), 33 000 (7), and those ranging from 17 000 to 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 as well as by a protonophorous uncoupler, carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone. © 1979, American Chemical Society. All rights reserved.