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Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

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Simon,  Bernd J.
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Burckhardt,  Gerhard
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Simon, B. J., & Burckhardt, G. (1990). Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles. Journal of Membrane Biology, 117(2), 141-151. doi:10.1007/BF01868681.


Cite as: http://hdl.handle.net/21.11116/0000-0007-E5D4-7
Abstract
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.