title: Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.
abstract: Kidney cortex microsomes enriched in Golgi markers and probably also containing endosomes were isolated by cell fractionation and found to contain a proton-translocating ATPase that was inhibited by N-ethylmaleimide (NEM). This NEM-sensitive ATPase was solubilized with n-octyl glucoside and purified using anion-exchange sievorptive chromatography on sequential DEAE-Sephadex and QAE-Sephadex columns followed by a final hydroxyapatite HPLC column. The purified enzyme, with a specific activity of 4.4 mumol.mg-1.min-1 was completely inhibited by NEM. Addition of asolectin and removal of the detergent by dialysis resulted in reconstitution of NEM-sensitive electrogenic proton transport. This vacuolar ATPase is composed of five polypeptides with apparent molecular masses of 68, 58, 40, 37, and 16 kDa.
authors.affiliation: Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
authors.forename: G P
authors.initials: GP
authors.lastname: Young
authors.forename: J Z
authors.initials: JZ
authors.lastname: Qiao
authors.forename: Q
authors.initials: Q
authors.lastname: Al-Awqati
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000201:enzymology / Q000648:ultrastructure*; D007425:Intracellular Membranes / Q000201:enzymology*; D007672:Kidney Cortex / Q000648:ultrastructure; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000032:analysis*
chemical_list: D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide