Catalytic hydrolysis and synthesis of adenosine 5'-triphosphate by stereoisomers of covalently labeled F1-adenosinetriphosphatase and reconstituted submitochondrial particles.
Bovine heart F1-adenosinetriphosphatase (F1) was labeled specifically and precisely with 7-chloro-4-nitro-2,1,3-[14C]benzoxadiazole ([14C]NBD-Cl). The stereospecifically labeled F1 (O-beta'-[14C]-NBD-F1) was partially reactivated by LiCl treatment, which could cause rearrangement of the beta subunits to form O-beta', beta''-[14C]NBD-F1. Both labeled enzymes were used to combine with F1-deficient submitochondrial particles (ASU) to form the reconstituted particles O-beta'-NBD-F1-ASU and O-beta', beta''-NBD-F1-ASU, respectively. A comparison of the observed steady-state rates of catalytic ATP hydrolysis and oxidative phosphorylation by these specifically labeled submitochondrial particles (SMP) with those of the unlabeled control samples suggests that oxidative phosphorylation involves more active sites of F1 than catalytic ATP hydrolysis. A comparison of the observed ATPase activity of uncoupled labeled SMP and the activity for ATP-driven reverse electron transport in coupled labeled SMP with the corresponding values of the unlabeled control samples shows that the observed fractional inhibition ATP hydrolysis is the same for both the coupled SMP and uncoupled SMP and is determined only by the state of stereospecific labeling of F1. The effect of preincubation under simulated oxidative phosphorylation conditions on the ATPase activity of the unperturbed, specifically NBD-labeled submitochondrial particles was also examined. The data show that respiration-generated proton flux does not cause the beta subunits in bovine heart proton-ATPase to continue switching places with each other during oxidative phosphorylation. Samples of NBD-F1 with specific labels on its nonhydrolytic beta'' subunits but none on its hydrolytic beta' subunit were prepared by a three-cycle process.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010085:Oxidative Phosphorylation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013237:Stereoisomerism; D013367:Submitochondrial Particles / Q000201:enzymology*
D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases