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. The³²P_i-ATP exchange activity of the reconstituted ATPase complex was inhibited byp-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with¹⁴C-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 of³²P_i-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 of³²P_i-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.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - STE-DTT buffer sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM), DTT (5 mM), pH 8.0 - F _o a membranous preparation from mitochondria conferring oligomycin (or rutamycin) sensitivity to F₁ - F₁F₆ coupling factors 1 (ATPase) and 6 - OSCP oligomycin-sensitivity-conferring protein - BSA bovine serum albumin - SDS sodium dodecyl sulfate - DTT dithiothreitol - STE buffer sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM) - TUA particles submitochondrial particles prepared by stepwise exposure of light-layer submitochondrial particles to trypsin and urea, then sonic oscillation in the presence of dilute ammonia (pH 10.4) - OG-cholate buffer glycerol (20%), Tricine (50 mM), MgSO₄ (5 mM), DTT (5mM), cholate (0.5%), octylglucoside (0.5%), pH 8.0 - p-HMB p-hydroxymercuribenzoate