The sulfonylurea-inhibited NADH oxidase activity of HeLa cell plasma membranes has properties of a protein disulfide-thiol oxidoreductase with protein disulfide-thiol interchange activity

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsi ve oxidation of NADH. The NADH oxidation takes place in an argon or nitroge n atmosphere and in samples purged of oxygen. Direct assay of protein thiol s by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's rea gent), suggests that protein disulfides may be the natural electron accepte rs for NADH oxidation by the plasma membrane vesicles. In the presence of N ADH, protein disulfides of the membranes were reduced with a concomitant st oichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC50 of ca. 30 nM. LY181984, with an EC50 of 30 nM, also inhibited a protein disulfide-thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxi dation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide-thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols invo lved was demonstrated using detergents and the impermeant thiol reagent p-c hloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a p hysiological role of the protein in NADH oxidation. Rather, it may carry ou t some other role more closely related to a function in growth, such as pro tein disulfide-thiol interchange coupled to cell enlargement.