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Journal Article

Effect of cholate on H+-ATPase and other proteins of dog renal brush-border membrane

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

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Citation

Noël, J., Fleser, A., Bellemare, F., Thiéry, G., Laprade, R., Burckhardt, G., et al. (1993). Effect of cholate on H+-ATPase and other proteins of dog renal brush-border membrane. Biochemistry and Cell Biology-Biochimie et Biologie Cellulaire, 71(7-8), 390-400. doi:10.1139/o93-057.


Cite as: https://hdl.handle.net/21.11116/0000-0008-28C7-B
Abstract
A short treatment of dog renal brush-border membrane vesicles (BBMV) with sodium cholate, followed by dialysis of the detergent, reorients the polarity of H+-ATPase in the membrane and exposes its ATP binding sites to the extravesicular space, as previously shown with pig BBMV. In cholate-pretreated vesicles, the H+-ATPase remains fully active, but is inserted under the reversed polarity in sealed vesicles. A large spontaneous N-ethylmaleimide-sensitive ATPase activity is thus observed, as well as a steep intravesicular acidification upon external ATP addition, two findings absent in native vesicles. The ability of nitrate plus ATP to dissociate the hydrolytic subunits ot the proton pump in cholate-pretreated vesicles, but not in native vesicles, demonstrates that most of the ATP binding subunits are accessible to ATP following cholate treatment. The sensitivity of the cytoplasmic domain of the H+-ATP activity to trypsin also confirms the reorientation of the enzyme in cholate-pretreated vesicles. The H+-ATPase and alkaline phosphatase remain largely associated with the membranes after the treatment with cholate, but gamma-glutamyltranspeptidase, aminopeptidase N, and neutral endopeptidase are largely solubilized. Upon dialysis of cholate, all these enzymes are in part reinserted in the membrane according to their original polarity. The reorientation process is however specific for the H+-ATPase. Cholate treatment does not increase the formation of inside-out vesicles. Thus the treatment with cholate really reorients the polarity of the H+-ATPase in vesicles and allows for study of the proton pumping capacity of vacuolar H+-ATPase of proximal tubules.