ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein.

摘要: The exposure to trypsinolysis of subunits F1F0-ATPase and its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment submitochondrial particles with guanidine hydrochloride removed the F1-ATPase oligomycin-sensitivity conferral protein (OSCP), exposed sites that were occluded intact complex. These identified by purifying isolated complexes before after proteolysis vesicles, characterizing them N-terminal sequencing electrospray-ionization mass spectrometry. In stripped subunit F6 was completely digested away either trypsin or chymotrypsin. Trypsin also cleaved b, first at bond arginine-166-glutamine-167, then consecutive linkages, lysine-120-arginine-121 arginine-121-histidine-122. Chymotrypsin-sensitive observed adjacent methionines 164 165. amino acids 1-3 d, minor cleavage d between 24 25, g 5 6, acid 40 e. other remained protected proteolysis. membrane-bound F1F0-ATPase, N-terminus accessible trypsin, e more susceptible than F0. Otherwise OSCP protected. Subunits alpha beta same their regions as purified F1-ATPase. trypsinized incapable binding presence OSCP. experiments vitro re-assembly described elsewehere, guided results experiments, helped establish a central role for b formation stalk connecting F1 domains