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Abstract

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 P_i-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 P_i-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.