Specificity of photolabeling of beta-cell membrane proteins with an 125I-labeled glyburide analog.

The interaction between sulfonylureas and membrane proteins from a hamster insulin-secreting tumor (HIT) cell line has been examined. Four HIT cell membrane proteins were covalently linked to an 125I-labeled glyburide analog by photolabeling. Three photolabeled polypeptides of M(r) 65,000, 55,000, and 30,000 were identified as low affinity "glyburide receptors." These proteins appear to be of similar abundance, when quantitated by photolabeling, with half-maximal displacements (Ki values) by glyburide, glipizide, and tolbutamide in the low micromolar range. The glyburide analog is more tightly bound to a M(r) 140,000 protein with dissociation constants, determined by filtration binding assays and by photolabeling, of 7 and 9.0 nM, respectively. The labeled analog was displaced from the M(r) 140,000 protein by glyburide, glipizide and tolbutamide with Ki values of 3.3 nM, 103 nM, and 25 microM, respectively, as estimated by photolabeling. Optimal conditions established for visualizing the M(r) 140,000 band on autoradiograms prepared after UV cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis include irradiating the radioligand-receptor complex at 1.5 J/cm2 at 312 nm, followed by heating samples in pH 9.0 sodium dodecyl sulfate-gel sample buffer. With receptor sites partially occupied (5 nM radioligand), approximately 0.75% of the protein is photocoupled to the radioligand and visualized by autoradiography. Our results confirm that the M(r) 140,000 polypeptide contains the beta-cell high affinity glyburide binding site and show that the second generation sulfonylurea antidiabetic drugs have a selective increase in affinity for this receptor.