Characterization of inside-out oriented H(+)-ATPases in cholate-pretreated renal brush-border membrane vesicles.
Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H+ uptake as visualized by the delta pH indicator, acridine orange. The reoriented H(+)-pump is electrogenic because permeant extravesicular anions or intravesicular K+ plus valinomycin enhance H+ transport. ATP stimulates H+ uptake with an apparent Km of 93 microM. Support of H+ uptake and Pi liberation by ATP greater than GTP approximately ITP greater than UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H+ uptake (Ki, 24 microM), Mg2+ and Mn2+ support ATP-driven H+ uptake, but Ca2+, Ba2+, and Zn2+ do not, 1 mM Zn2+ inhibits MgATP-driven H+ transport completely. NEM-sensitive Pi liberation is stimulated by Mg2+ and Mg2+ and, unlike H+ uptake, also by Ca2+ suggesting Ca2(+)-dependent ATP hydrolysis unrelated to H+ transport. The inside-out oriented H(+)-pump is relatively insensitive toward oligomycin, azide, N,N'-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparent Ki, 0.77 microM), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparent Ki, 0.39 microM). Taken together, the H(+)-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of "vacuolar type" (V-type) H(+)-ATPases. Hence, V-type H(+)-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.
Simon
B J
BJ
Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
Burckhardt
G
G
D000165:Acridine Orange; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008871:Microvilli / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013552:Swine
D002793:Cholic Acids; D006859:Hydrogen; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D000165:Acridine Orange; D019826:Cholic Acid; D005033:Ethylmaleimide