A direct demonstration of proton translocation coupled to transhydrogenation in reconstituted vesicles.
Homogeneous bovine heart mitochondrial transhydrogenase was reconstituted into K+-loaded phosphatidylcholine liposomes. Transhydrogenase-catalyzed reduction of 3-acetylpyridine adenine dinucleotide by NADPH was stimulated severalfold when valinomycin was added to collapse the developing membrane potential. A rapid and extensive quenching of the fluorescence of the pH probe, 9-aminoacridine, under these conditions indicates that transhydrogenation is coupled to the acidification of the vesicle interior. A corresponding uptake of protons from the medium is demonstrated by electrode measurements that indicate 1 or less proton is translocated for each hydride ion equivalent transferred between the substrates.
D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000378:metabolism*; D010713:Phosphatidylcholines; D011188:Potassium / Q000494:pharmacology; D011510:Proteolipids; D014634:Valinomycin / Q000494:pharmacology
D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D011188:Potassium