Energy-linked nicotinamide nucleotide transhydrogenase. Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography.
Mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was purified by a novel procedure involving fast protein liquid chromatography and characterized with respect to molecular and catalytic properties. The method is reproducible, gives highly pure transhydrogenase as judged by silver staining, and can be modified to produce large amounts of pure transhydrogenase protein suitable for e.g. sequencing and other protein chemical studies. Transhydrogenase purified by fast protein liquid chromatography is reconstitutively active and pumps protons as indicated by an extensive quenching of 9-aminoacridine fluorescence. Under conditions which generate a proton gradient in the absence of a membrane potential the activity of reconstituted transhydrogenase is close to zero indicating a complete and proper incorporation in the membrane and a preferential regulation of the enzyme by a proton gradient rather than a membrane potential. Treatment of reconstituted transhydrogenase with N,N-dicyclohexylcarbodiimide results in an inhibition of proton pump activity without an effect on uncoupled catalytic activity, suggesting that proton translocation and catalytic activities are not obligatory linked or that this agent separates proton pumping from the catalytic activity.
D000585:Aminacrine; D000596:Amino Acids / Q000032:analysis; D000609:Aminoacridines; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004734:Energy Metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D008081:Liposomes; D008928:Mitochondria / Q000201:enzymology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*
D000596:Amino Acids; D000609:Aminoacridines; D005456:Fluorescent Dyes; D008081:Liposomes; C010847:9-amino-6-chloro-2-methoxyacridine; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000585:Aminacrine; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D006180:Proton-Translocating ATPases