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A Brush Border Membrane-Bound H+ -ATPase from the Dog Proximal Tubule

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

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Citation

Noël, J., Laprade, R., Burckhardt, G., Gougoux, A., & Vinay, P. (1992). A Brush Border Membrane-Bound H+ -ATPase from the Dog Proximal Tubule. Cellular Physiology and Biochemistry, 2(1), 18-36. doi:10.1159/000154623.


Cite as: http://hdl.handle.net/21.11116/0000-0008-511E-C
Abstract
We have characterized the H+-ATPase activity (ATP hydrolysis and proton transport) of brush border membranes (BBMs) isolated from the dog kidney cortex. In solubilized BBMs, two thirds of total ATPase activity is insensitive to 10–3 M ouabain and 10–5M oligomycin, but sensitive to 10–6M NN’-dicyclohexylcarbodiimide (DCCD), N-ethyl-maleimide (NEM) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. The NEM- and DCCD-sensitive ATPase activity is not affected by 2–20 mM potassium, 10–3M omeprazole or 10–3M vanadate. A comparable enrichment was observed in solubilized BBMs for this ATPase activity and BBM enzyme markers. The NEM-sensitive ATPase activity of intact BBMs is increased 6.6-fold upon solubilization with 0.1 % deoxycholate, indicating the localization of most of the ATP-binding sites inside the native vesicles. Addition of ATP to BBM vesicles resulted in a large intravesicular acidification (acridine orange) only in vesicles pretreated with cholate which reverses the polarity of the H+ pump. Both proton transport and H+-ATPase activity were optimal around pH 7.0–7.5, and presented a comparable sensitivity to inhibitors. In intact dog cortical tubules, the large ouabain-insensitive but oligomycin-sensitive respiration suggests that this pump may represent a major energy-consuming process. In contrast, rabbit cortical tubules present a lower H+-ATPase activity and specific respiration. We therefore propose that an ATP-driven H+ transport may contribute significantly to H+ secretion in dog proximal tubules.