DIFFERENTIAL INTERACTION OF GLIMEPIRIDE AND GLIBENCLAMIDE WITH THE BETA-CELL SULFONYLUREA RECEPTOR .2. PHOTOAFFINITY-LABELING OF A 65 KDA PROTEIN BY [H-3] GLIMEPIRIDE

Glimepiride is a novel sulfonylurea for the treatment of type II-diabe tic patients exhibiting different receptor binding kinetics to beta-ce ll membranes with 8-9-fold higher k(off) rate and 2.5-3-fold higher k( on) rate compared to glibenclamide (see accompanying paper (Muller, G. et al. (1994) Biochim. Biophys. Acta 1191, 267-277)). To elucidate th e molecular basis for this differential behaviour of glimepiride and g libenclamide, direct photoaffinity labeling studies using beta-cell tu mor membranes were performed. [H-3]Glimepiride was specifically incorp orated into a membrane polypeptide of M(r) = 65000 under conditions, w hich led to predominant labeling of a 140 kDa protein by [H-3]glibencl amide (Kramer, W. et al. (1988) FEBS lett. 229, 355-359). Labeling of the 140 kDa protein by [H-3]glibenclamide was inhibited by unlabeled g limepiride and, vice versa, glibenclamide inhibited labeling of the 65 kDa protein by [H-3]glimepiride. The 65 kDa protein was also specific ally photolabeled by the sulfonylurea [I-125]35623, whereas an 4-azido benzoyl derivative of glibenclamide, N-3[H-3]33055, exclusively labele d a 33 kDa protein. Competitive Scatchard analysis of [H-3]glimepiride -binding and [H-3]glibenclamide-binding to RNm5F cell membranes using glibenclamide and glimepiride, respectively, as heterologous displacin g compounds yielded non-linear plots. These findings may be explained by cooperative interactions between the 140 and 65 kDa sulfonylurea-bi nding proteins. The possibility that sulfonylureas of different struct ure have different access to the 140 and 65 kDa receptor proteins due to the beta-cell membrane barrier was investigated by photoaffinity la beling of solubilized beta-cell membrane proteins. Interestingly, solu bilization of beta-cell tumor membranes led to a shift of specific [H- 3]glibenclamide binding from the 140 kDa to the 65 kDa binding protein , exclusively, and to an increased labeling of the 65 kDa protein by [ 3H]glimepiride. The labeling of a unique protein is in agreement with similar K-d values measured for both sulfonylureas upon solubilization of beta-cell tumor and RINm5F cell membranes (see accompanying paper) . Furthermore, competitive Scatchard plots of [H-3]glimepiride binding to solubilized RINm5F cell membrane proteins in the presence of glibe nclamide and vice versa approximate linearity suggesting loss of coope rativity between the 140 kDa glibenclamide-binding and 65 kDa glimepir ide-binding proteins upon solubilization. The physiological significan ce of the differential interaction of glimepiride and glibenclamide wi th different binding proteins was also substantiated by photoaffinity labeling of RINm5F cells leading to labeling of a 140 kDa protein by [ H-3]glibenclamide and of a 65 kDa protein by [H-3]glimepiride. In conc lusion, this report presents the first evidence that different sulfony lurea drugs bind to different components of the sulfonylurea receptor complex which are characterized by accessibility for the drugs.