Purification and Properties of a Coupling Factor (Ca²⁺‐Dependent Adenosine Triphosphatase) from <i>Rhodospirillum rubrum</i>

Abstract

A coupling factor has been purified from an acetone powder extract of Rhodospirillum rubrum chromatophores. This restores to decoupled chromatophores (extracted with respect to coupling factor) both photophosphorylation and Mg 2+ ‐dependent ATPase activities. The coupling factor had been partially purified by gel filtration through a Sepharose 6B column, and further purified by ultra‐filtration and sucrose‐density‐gradient centrifugation. At each stage of purification, the coupling factor activity was coincident with a Ca 2+ ‐dependent ATPase activity, increasing in total activity during the purification. Mg 2+ , Mn 2+ , Co 2+ or Zn 2+ could not substitute for Ca 2+ in activation of this soluble ATPase, but these four metals strongly inhibited the activity of the Ca 2+ ‐dependent ATPase. By incubation with decoupled chromatophores, the soluble Ca 2+ ‐dependent ATPase was rebound to the membrane in the presence of either Mg 2+ or Ca 2+ . This binding resulted in a partial masking of the Ca 2+ ‐dependent ATPase activity of the soluble enzyme. The purified ATPase has a K m value of 1.2 mM for ATP. The K i values for inhibition of the soluble Ca 2+ ‐dependent ATPase by Mg 2+ (170 μM), and by ADP (42 μM) are in close agreement with the corresponding K m values for Mg 2+ ‐dependent ATPase and photophosphorylation, respectively, as measured with chromatophores. The membrane‐bound Mg 2+ ‐ or Ca 2+ ‐dependent ATPase activities were inhibited by oligomycin. Azide and parahydroxymercuribenzoate inhibited both the soluble and the membrane‐bound ATPase activities. Treatment with phospholipase A inhibited both the membrane‐bound ATPase, but had no effect on the soluble Ca 2+ ‐dependent ATPase. The sedimentation constant at 1 mg protein/ml of the extensively purified coupling factor is s 20, w = 13.1 × 10 −13 s, and the approximate molecular weight 350000.