ATP-dependent proton transport by isolated brain clathrin-coated vesicles. Role of clathrin and other determinants of acidification.
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 chromatograpy 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 (Cl- greater than Br- much greater than NO3- much greater than gluconate, SO2-(4), HPO2-(4), mannitol; Km for Cl- congruent to 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 less than 10 microM), dicyclohexylcarbodiimide (DCCD) (IC50 congruent to 10 microM), chlorpromazine (IC50 congruent to 15 microM), and oligomycin (IC50 congruent to 3 microM). In contrast to N-ethylmaleimide, chlorpromazine rapidly dissipated preformed pH gradients. Valinomycin stimulated H+ transport in the presence of potassium salts (gluconate much greater than NO3- greater than 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 greater than potassium gluconate greater than KCl) 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Van Dyke
R W
RW
Scharschmidt
B F
BF
Steer
C J
CJ
D000165:Acridine Orange / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000648:ultrastructure*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D011992:Endosomes / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology
D002712:Chlorides; D002966:Clathrin; D014634:Valinomycin; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D010042:Ouabain; D006180:Proton-Translocating ATPases; D000165:Acridine Orange; D002746:Chlorpromazine