Proton ATPase in rat renal cortical endocytotic vesicles.
To relate ATPase activity to the ATP-driven H+-pump in rat renal endocytotic vesicles we applied an in vitro coupled optical test and a Pi-liberation assay. Endocytotic vesicles contain an ouabain-, vanadate- and oligomycin-insensitive ATPase. The ionophores for K+ and H+, valinomycin and carbonylcyanide p-chloro-methoxyphenylhydrazone (CCCP), respectively, stimulated ATPase activity, indicating its relation to the electrogenic H+-pump. This conclusion is supported by a similar distribution on a Percoll gradient of ATP-driven H+ uptake into endosomes and ionophore-stimulated ATPase activity. Coupled optical and Pi-liberation assays were then used to characterize the H+-ATPase with respect to the requirement for pH, nucleotides, anions, and mono- and divalent cations. The H+-ATPase activity was decreased by widely used blockers: N-ethylmaleimide (NEM), dicyclohexylcarbodiimide (DCCD) and diethylstilbestrol (DES). Different sensitivities to these blockers proved that alkaline phosphatase and H+-ATPase are separate entities. To investigate whether the NEM-, DCCD- and DES-sensitive ATPase activity is confined to intact endocytotic vesicles, cellular membranes from rat kidney cortex were separated on a Percoll density gradient. Surprisingly, endocytotic vesicles contain only a small fraction of the total NEM-, DCCD- and DES-sensitive ATPase activity. The majority of the blocker-sensitive ATPases belongs to membranes of as yet undefined cellular origin.
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000838:Anions; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002412:Cations; D002712:Chlorides / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004054:Diethylstilbestrol / Q000494:pharmacology; D004705:Endocytosis; D000431:Ethanol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006096:Gramicidin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000648:ultrastructure*; D009711:Nucleotides / Q000378:metabolism; D009940:Organoids / Q000187:drug effects / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry; D013379:Substrate Specificity; D014634:Valinomycin / Q000494:pharmacology
D000838:Anions; D002412:Cations; D002712:Chlorides; D009711:Nucleotides; D000643:Ammonium Chloride; D006096:Gramicidin; D014634:Valinomycin; D000431:Ethanol; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D004054:Diethylstilbestrol; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide