Photoaffinity Labeling of the Cerebral Sulfonylurea Receptor Using a Novel Radioiodinated Azidoglibenclamide Analogue

In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145–150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [ 3 H]glibenclamide or a 125 I‐labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4‐azido‐5‐iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high‐affinity binding of this novel probe to the particulate ( K D = 0.13 n M ) and solubilized ( K D = 0.56 n M ) sulfonylurea receptor from cerebral cortex. The novel probe showed >100‐fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175‐kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a K D value of 0.38 n M . Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed K D values for these sulfonylureas of 0.06 n M , 1.6 n M , and 1.2 µ M , respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175‐kDa protein represents the cerebral sulfonylurea receptor.