Resolution of enzymes catalyzing energy-linked transhydrogenation. IV. Reconstitution of adenosine triphosphate-driven transhydrogenation in depleted chromatophores of Rhodospirillum rubrum by the transhydrogenase factor and a soluble oligomycin-insensitive Mg ++ -adenosine triphosphatase.

The catalysis of the energy-linked transhydrogenation reaction of Rhodospirillum rubrum chromatophores is dependent upon a soluble protein, the transhydrogenase factor. This factor can be detached from the chromatophore membrane by the relatively mild procedures of extensive washing, passage of chromatophores through Sephadex G-ZOO, or sedimentation of chromatophores through a dense sucrose solution. Fractionation of the crude factor preparation on DEAE-Sephadex yields a highly purified protein which is resolved on polyacrylamide gels into two bands of apparent equal concentration. The transhydrogenase factor has no noticeable flavin absorption, is heat-inactivated, trypsinsensitive, and has a molecular weight of about 70,000. It contains sulfhydryl groups which are essential for its activity. A soluble, oligomycin-insensitive Mg++ dependent ATPase protein isolated from R. rubrum chromatophores has been successfully used together with the transhydrogenase factor in reconstitution experiments with chromatophore particles resolved with respect to these two factors.