Isolation, characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump.
The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The 32Pi-ATP exchange activity of the reconstituted ATPase complex was inhibited by p-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with 14C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of 32Pi-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved: at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of 32Pi-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001436:Bacteriorhodopsins / Q000378:metabolism; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D010086:Oxidative Phosphorylation Coupling Factors / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D006180:Proton-Translocating ATPases; D013367:Submitochondrial Particles / Q000201:enzymology*
D010086:Oxidative Phosphorylation Coupling Factors; D010743:Phospholipids; D001436:Bacteriorhodopsins; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases