Identification of antitumor sulfonylurea binding proteins of HeLa plasma membranes.

Plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [3H]LY181984 with high affinity (Kd of about 25 nM). The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance protein of the total plasma membrane proteins. The binding proteins appeared to contain one or more thiols in the binding site as high affinity binding of [3H]LY181984 was reduced by treatment with the covalent thiol blocking reagent, N-ethylmaleimide (NEM), or by oxidation with dilute hydrogen peroxide but was protected by glutathione or dithiothreitol. Elimination of binding of [3H]LY181984 by NEM was prevented by excess unlabeled LY181984 (an active sulfonylurea) but less so by excess LY181985 (an inactive sulfonylurea). The binding proteins were specifically labeled with thiol reagents following reaction of unprotected thiols with unlabeled thiol reagents. Binding proteins at ca. 34 kDa were labeled. Plasma membrane proteins after solubilization with SDS under strongly reducing conditions still bound sulfonylurea. [3H]LY181984 binding to plasma membrane proteins resolved on SDS-PAGE correlated as well with proteins in the 30-40 kDa range.