Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel.
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.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002540:Cerebral Cortex / Q000648:ultrastructure; D005905:Glyburide / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D002352:Carrier Proteins; D015221:Potassium Channels; D000255:Adenosine Triphosphate; D005905:Glyburide