Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement Access content directly
Journal Articles Proceedings of the National Academy of Sciences of the United States of America Year : 1988

Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel

Abstract

Sulfonylurea and particularly glibenclamide are potent blockers of ATP-regulated K+ channels in insulin-secreting cells. A very good correlation exists between binding of sulfonylurea to brain and insulinoma cell membranes. The [3H]glibenclamide-binding component from pig brain microsomes was solubilized with digitonin with a complete retention of its properties of interaction with glibenclamide and other sulfonylureas. A four-step purification was achieved that used (i) hydroxylapatite chromatography, (ii and iii) affinity chromatographies on ADP-agarose and wheat germ agglutinin-agarose columns, and (iv) a final chromatographic step on a mixture of AMP-agarose/GMP-agarose/hydroxylapatite. This procedure led to a 2500-fold purification. NaDodSO4/polyacrylamide gel electrophoresis of the purified material in reducing and nonreducing conditions showed that the sulfonylurea-binding component is made of a single major polypeptide chain of Mr 150,000 +/- 10,000. Direct photoaffinity labeling of the receptor with [3H]glibenclamide at different steps of the purification also showed that radioactivity was specifically incorporated into a polypeptide of Mr 150,000 +/- 5000, thus confirming the subunit structure indicated by the purification.

Dates and versions

hal-02725025 , version 1 (02-06-2020)

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Henri Bernardi, Michel Fosset, Michel Lazdunski. Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel. Proceedings of the National Academy of Sciences of the United States of America, 1988, 85 (24), pp.9816-9820. ⟨10.1073/pnas.85.24.9816⟩. ⟨hal-02725025⟩

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