Influence of proteinase inhibitors and substrates on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding capacity of the rat hepatic Ah receptor.

These studies investigated the effects of various serine proteinase inhibitors and substrates on the TCDD-binding capacity of the rat hepatic Ah receptor. TCDD binding to the Ah receptor was inhibited by serine proteinase inhibitors phenylmethylsulfonyl fluoride (PMSF), tosyl-lysine chloromethyl ketone (TosLysCH2Cl), tosylamide-phenylethyl chloromethyl ketone (TosPheCH2Cl) and substrates tosyl-L-arginine methyl ester (TosArgOMe) and D-tryptophan methyl ester (TrpOMe). The order of potency was TosPheCH2Cl greater than TosLysCH2Cl much greater than PMSF approximately equal to TosArgOMe approximately equal to TrpOMe. Reactivity of the chloromethyl ketones with sulfhydryl groups was suggested by their steep inhibition curves above the concentration of nonprotein sulfhydryl groups, and the partial mitigation of inhibition by 1 mM dithiothreitol. Inhibition by these reagents was irreversible, while that by TosArgOMe and TrpOMe was completely reversible by gel filtration. The mechanism of inhibition by TosArgOMe and TrpOMe was formally competitive, with inhibition constants similar to those reported in steroid hormone receptor systems. Neither inhibitors nor substrates displaced previously bound TCDD.