We have systematically investigated certain characteristics of the ATP-dependent proton transport mechanism of bovine brain clathrin-coated vesicles. H+transport specific activity was shown by column chromatography to co-purify with coated vesicles, however, the clathrin coat is not required for vesicle acidification as H+transport was not altered by prior removal of the clathrin coat. Acidification of the vesicle interior, measured by fluorescence quenching of acridine orange, displayed considerable anion selectively (CI-> Br-≫NO3-≫ gluconate, SO42-, HPO42-, mannitol; Kmfor Cl-≈- 15 mM), but was relatively insensitive to cation replacement as long as Cl-was present. Acidification was unaffected by ouabain or vanadate but was inhibited by N-ethylmaleimide (IC50< 10 μM), dicyclohexylcarbodiimide (DCCD) (lC50≈- 10 μM), chlorpromazine (lC50≈- 15 μM), and oligomycin (lC50≈- 3 μM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+transport in the presence of potassium salts (gluconate ≫ NO3-> Cl-), and the membrane-potential-sensitive dye Oxonol V demonstrated an ATP-dependent interior-positive vesicle membrane potential which was greater in the absence of permeant anions (mannitol > potassium gluconate > KCI) and was abolished by N-ethylmaleimide, protonophores or detergent. Total vesicle-associated ouabain-insensitive ATPase activity was inhibited 64% by 1 mM N-ethylmaleimide, and correlated poorly with H+transport, however N-ethylmaleimide-sensitive ATPase activity correlated well with proton transport (r = 0.95) in the presence of various Cl-salts and KNO3-Finally, vesicles prepared from bovine brain synaptic membranes exhibited H+transport activity similar to that of the coated vesicles. Collectively these findings indicate that: (1) the H+transport mechanism of bovine brain clathrin-coated vesicles is not dependent upon the clathrin coat, and closely resembles that of rat liver clathrin-coated vesicles, (2) the H+transport mechanism is intrinsically electrogenic and pH and electrical gradients established by the proton transport mechanism vary inversely in the presence of permeable or impermeable anions, (3) at concentrations above 3 μM, oligomycin inhibits vesicle acidification, (4) chlorpromazine inhibits coated vesicle acidification, at least in part, by increasing proton conductance, and (5) other smooth membranes from bovine brain, including synaptic membranes, exhibit a similar H+transport mechanism. © 1985.
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