Nonspecific inhibition of enzymes by p-bromophenacyl bromide inhibition of human platelet phospholipase C and modification of sulfhydryl groups

Abstract

This study demonstrates that β-bromophenacyl bromide irreversibly inhibits, in a time- and dose-dependent manner, yeast alcohol dehydrogenase, bovine pancreatic α-chymotrypsin, human platelet phosphatidylinositol (PI)-specific phospholipase C, in addition to the neutral-active and calcium-dependent phospholipase A 2 of human platelets. The PI-specific phospholipase C has maximal activity at pH 5,5 is calcium-dependent, and is strongly inhibited by sulfhydryl reagents 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and methylmethane thiosulfonate. Increasing concentrations of DTNB produced concomitant inhibition of phospholipase C activity and titration of sulfhydryl groups. In contrast, human platelet phospholipase A 2 activity was unaffected by concentrations of DTNB that titrated sulfhydryl groups, and completely inhibited PI-specific phospholipase C activity. Treatment of cysteine with p-bromophenacyl bromide resulted in modification of the amino acid as demonstrated by paper chromatography, and loss of titratable sulhydryl groups. These data show that p-bromophenacyl bromide inhibits a wide spectrum of enzymatic activities including PI-specific phospholipase C. This reagent modifies amino acid residues other than active-site histidines and therefore has a broader reactivity than previously considered. Thus, it should not be used as a selective inhibitor of enzymes in crude cellular experiments.