Inhibitory chemical modifications of F1-ATPase: effects on the kinetics of adenosine 5'-triphosphate synthesis and hydrolysis in reconstituted systems.
The purified, soluble F1-ATPase was modified by several covalently reacting inhibitors, either known or considered to bind to the active site bearing beta-subunit, to cause partial inhibition up to 99%. The modified enzyme was then reconstituted in the presence of OSCP (oligomycin sensitivity conferring protein) with submitochondrial particles (SMP) almost completely (greater than 99%) denuded of active F1-ATPase and was assayed for oligomycin-sensitive ATPase and oxidative phosphorylation activities. The inhibitors used were 1-fluoro-2,4-dinitrobenzene (FDNB), N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCD), quinacrine mustard (QM), 5-(dimethylamino)-naphthalene-1-sulfonyl chloride (dansyl-Cl), 5'-[p-(fluoro-sulfonyl)benzoyl]adenosine (FSBA), and N,N'-dicyclohexylcarbodiimide (DCCD). The SMP reconstituted with unmodified F1 exhibited oxidative phosphorylation and oligomycin-sensitive ATPase (in the presence of uncouplers) activities as high as 500 nmol min-1 mg-1 and 8 mumol min-1 mg-1, respectively. The systems reconstituted with F1 modified to cause various degrees of inhibition with FDNB, EEDQ, CMCD, QM, and dansyl-Cl exhibited the same degree of inhibition of oxidative phosphorylation and oligomycin-sensitive ATPase activities as the inhibition of the ATPase activity of the modified F1 before reconstitution. The systems reconstituted with FSBA-modified F1 showed the following relative degrees of inhibition: oxidative phosphorylation greater than oligomycin-sensitive ATPase of particles greater than ATPase of soluble F1. In contrast, the systems reconstituted with DCCD-modified F1 showed much greater inhibition of oligomycin-sensitive ATPase than of oxidative phosphorylation activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism; D010085:Oxidative Phosphorylation; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013367:Submitochondrial Particles / Q000201:enzymology
D000345:Affinity Labels; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004024:Dicyclohexylcarbodiimide; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000241:Adenosine