Resolution of Enzymes Catalyzing Energy-linked Transhydrogenation

Abstract 1. The energy-linked transhydrogenase system of Rhodospirillum rubrum chromatophores has been resolved into a water-soluble protein component, the transhydrogenase factor (TH1) and an insoluble membrane component. TH1 was removed from the chromatophore membrane by washing with large volumes of buffer and concentrated by ammonium sulfate precipitation. 2. Addition of TH1 to chromatophores depleted of TH1 (Ct-particles) reconstituted energy-linked transhydrogenation to rates observed with unresolved chromatophores. However, TH1 did not stimulate transhydrogenation appreciably in unresolved chromatophores. 3. The formation of a stable complex between TH1 and Ct-particles required the presence of low concentrations of NADP+ or NADPH; half-maximal binding occurred at 0.4 µm and 0.2 µm, respectively. 4. NADPH at concentrations in excess of 10 µm inhibited TH1 binding to Ct-particles, whereas excess NADP+ did not inhibit binding of TH1. NADH did not stimulate TH1 binding, but totally inhibited that supported by NADP+ or NADPH. High concentrations of NADP+ were unable to reverse the inhibition of TH1 binding observed in the presence of reduced pyridine nucleotides. Dissociation of TH1 from chromatophores was stimulated, whereas the binding of TH1 to Ct-particles was inhibited in the presence of a complete mixture of the components of the transhydrogenase assay medium. NADP+ stabilized the TH1-membrane complex during washing of the chromatophores. These findings suggest that dissociation of TH1 from chromatophores during washing is a result of the removal of membrane-bound pyridine nucleotides. Further indicated is that the TH1-membrane complex is reversibly dissociated during the process of active transhydrogenation.