Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.
1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3. The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism; D003575:Cytochromes c1 / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases; D009206:Myocardium; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D013385:Succinate Dehydrogenase / Q000378:metabolism
D003574:Cytochrome c Group; D003580:Cytochromes; D010743:Phospholipids; D003575:Cytochromes c1; D000226:Mitochondrial ADP, ATP Translocases; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D000251:Adenosine Triphosphatases