Nonspecific inhibition of enzymes by p-bromophenacyl bromide. Inhibition of human platelet phospholipase C and modification of sulfhydryl groups.
This study demonstrates that p-bromophenacyl bromide irreversibly inhibits, in a time- and dose-dependent manner, yeast alcohol dehydrogenase, bovine pancreatic alpha-chymotrypsin, human platelet phosphatidylinositol (PI)-specific phospholipase C, in addition to the neutral-active and calcium-dependent phospholipase A2 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 A2 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 sulfhydryl 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.
D000098:Acetophenones / Q000494:pharmacology*; D001792:Blood Platelets / Q000201:enzymology*; D003545:Cysteine; D003600:Cytosol / Q000201:enzymology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D010740:Phospholipases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000097:blood
D000098:Acetophenones; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D010740:Phospholipases; D010738:Type C Phospholipases; D003545:Cysteine; C006821:4-bromophenacyl bromide