Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies.

摘要: Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), enriched 25-fold over homogenate, abolished pretreatment with protonophores or a nonionic detergent, exhibited pH optimum 7.5, inhibited N-ethylmaleimide (NEM) (IC50 approximately 5 microM) N,N'-dicyclohexylcarbodiimide microM), resistant to inhibition vanadate, ouabain, oligomycin. Acidification no specific cation requirement; however, maximal rates acidification depended upon Cl- (Km 20 mM). Other anions less effective supporting (Cl- Br- gluconate, NO-3, SO2-4, mannitol), indeed NO-3 even 150 mM Cl-. The transport mechanism appeared be electrogenic based on: (a) enhancement valinomycin K (b) quenching bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, membrane potential-sensitive anionic dye. Furthermore, magnitude electrical gradients generated vary inversely absence Finally, MVB ATPase activity that ouabain oligomycin, but 32.3% 1 NEM, 33.7% 200 microM dicyclohexylcarbodiimide, 18.7% KNO3. In isolated MVB, therefore, NEM-sensitive may represent enzymatic equivalent pump. These studies identify characterize an process liver which shares many properties pump described clathrin-coated vesicles, endosomes, lysosomes, Golgi, endoplasmic reticulum other tissues. differed somewhat vesicles response suggesting these two might differ.