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 PPi synthase (H(+)-PPi synthase) from chromatophores of Rhodospirillum rubrum. The H(+)-PPi 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 mumol of PPi 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(+)-PPi synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PPi hydrolytic activity. The subunit composition of the purified H(+)-PPi 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(+)-PPi synthase is discussed.
Nyrén
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P
Institutionen för Biokemi och Biokemisk teknologi, Kungliga Tekniska Högskolan, Stockholm, Sweden.
Nore
B F
BF
Strid
A
A
D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002846:Chromatography, Affinity; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008081:Liposomes; D011092:Polyethylene Glycols; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D006882:Hydroxyapatites; D008081:Liposomes; D011092:Polyethylene Glycols; D004024:Dicyclohexylcarbodiimide; D017886:Durapatite; D011755:Pyrophosphatases; C067974:membrane-bound proton-translocating PPi synthase