PROTON-PUMPING N,N'-DICYCLOHEXYLCARBODIIMIDE-SENSITIVE INORGANIC PYROPHOSPHATE SYNTHASE FROM RHODOSPIRILLUM-RUBRUM - PURIFICATION, CHARACTERIZATION, AND RECONSTITUTION

A new method has been developed for the isolation of the proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive PP(i) synthase (H+-PP(i) synthase) from chromatophores of Rhodospirillum rubrum. The H+-PP(i) synthase was purified by extraction of chromatophores with a mixture of nonanoyl-N-methylglucamide and cholate, by fractionation with poly(ethylene glycol) 4000, hydroxyapatite chromatography, and affinity chromatography. The purified enzyme is homogeneous and has a specific activity of 20.4-mu-mol of PP(i) hydrolyzed min-1 mg-1 at pH 7.5 and 20-degrees-C. The hydrolytic activity of the enzyme was stimulated by addition of phospholipids and Triton X-100. Of the lipids tested, cardiolipin proved to have the maximal activating effect. Reconstitution of the H+-PP(i) synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PP(i) hydrolytic activity. The subunit composition of the purified H+-PP(i) synthase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band was obtained after silver staining with an apparent molecular weight of 56 000. The oligomeric structure of the H+-PP(i) synthase is discussed.