Immunochemical characterization and purification of bovine heart mitochondrial pyridine dinucleotide transhydrogenase.

Antibodies raised to homogeneous bovine heart mitochondrial pyridine dinucleotide transhydrogenase selectively immunoprecipitated the enzyme from detergent extracts of submitochondrial membranes.Antitranshydrogenase inhibited both the forward (NADH + NADP+) and reverse (NADPH + NAD') reactions catalyzed by soluble transhydrogenase maximally by 50 to 60%.Energy-linked forward transhydrogenation and reduction of NADP' by NADPH catalyzed by submitochondrial particles were inhibited to a significantly greater extent than the nonenergy-linked reactions.The following submitochondrial particle-catalyzed reactions were not inhibited by the antibody: NADH and NADPH oxidases, NADH dehydrogenase (NADH + 3-acetylpyridine adenine dinucleotide), NADPH dehydrogenase (NADPH-dichlorophenol-indophenol reductase), NADH-ferricyanide reductase, and ATP-dependent reverse electron transport between succinate and NAD'.Anti-transhydrogenase significantly inhibited NADPH -+ NAD' transhydrogenation in rat liver submitochondrial particles, but did not affect this activity from chicken liver or potato mitochondria, Escherichia coli membrane vesicles, or Rhodospirillum rubrum chromatophores.A simplified immunochemical procedure for the purification of bovine heart transhydrogenase has been developed.A transhydrogenase-free Triton extract of bovine heart submitochondrial particles, obtained by chromatography on anti-transhydrogenase immobilized on Sepharose, was used to produce antibodies to several extract proteins.These antibodies when purified and coupled to Sepharose, in conjunction with NAD affinity chromatography, provide a rapid, three-step purification of transhydrogenase with reasonable yield and the highest specific activity yet reported.The enzyme functionally incorporated into phosphatidylcholine liposomes by a cholate dialysis procedure couples the inward translocation of protons to the reduction of 3-acetylpyridine adenine dinucleotide by NADPH.Mammalian pyridine dinucleotide transhydrogenase (EC 1.6.1.1)constitutes an energy-coupling site of the inner mitochondrial membrane and functions as a proton pump according to the equation (for submitochondrial particles) (1-4) NADH + NADP' + nH', s NAD' + NADPH + nH+out