Resolution of Enzymes Catalyzing Energy-linked Transhydrogenation

Abstract 1. A highly purified transhydrogenase factor (TH1) has been isolated from Rhodospirillum rubrum chromatophores by washing the membranes first with buffer containing 10 µm NADP+ under conditions in which TH1 remains bound, then with buffer lacking NADP+ resulting in the dissociation of TH1 from the membrane. 2. Purified TH1 stimulated the energy-linked reduction of NADP+ by NADH, the nonenergy-linked reduction of AcPyAD+ by NADPH, and the reduction of AcPyAD+ by NADH in the presence of chromatophores resolved with respect to these activities. TH1 did not catalyze these reactions independently of these resolved chromatophores. 3. The energy-linked reduction of NADP+ by NADH has been demonstrated to occur by the transfer of hydrogen without prior exchange to the medium from the 4A (nicotinamide) locus of NADH to the 4B (nicotinamide) locus of NADPH. This stereospecificity is identical with that found for the analogous mitochondrial reaction. 4. It is concluded that TH1 functions in the transfer of hydrogen between NAD(H) and NADP(H) in both the energy- and nonenergy-linked transhydrogenase reactions.