Proton-translocating ATPase from bovine kidney medulla: partial purification and reconstitution.
The proton-translocating ATPase that is responsible both for urinary and vacuolar acidification was partially purified from bovine kidney medulla microsomes. ATPase activity was purified to a maximum specific activity of 1.7 mumol.min-1.mg prot-1 and was inhibited completely by N-ethylmaleimide. The relative molecular weight (Mr) of the intact protein estimated by high-pressure size-exclusion liquid chromatography was 586,000. Nondenaturing gels of the isolated enzyme revealed two protein bands at MrS of 551,000 and 523,000. Sodium dodecyl sulfate-gel electrophoresis of the isolated H+-ATPase revealed component subunits at MrS of 70,000, 56,000, 45,000, 42,000, 38,000, 31,000, 15,000, 14,000, and 12,000. The properties of the isolated H+-ATPase and of microsomal ATP-dependent proton transport correlated closely. The isolated H+-ATPase was reconstituted into phospholipid liposomes and demonstrated N-ethylmaleimide-inhibitable ATP-dependent potential generation, consistent with electrogenic proton transport. In overall structure, the enzyme appears to be a new type of H+-ATPase with several features of the F0F1 class of ion-translocating ATPases but is immunologically and structurally different from the mitochondrial F1-ATPase.
D000818:Animals; D002417:Cattle; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007679:Kidney Medulla / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D014617:Vacuoles / Q000201:enzymology
D002793:Cholic Acids; D006180:Proton-Translocating ATPases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate