INHIBITION OF HUMAN PHOSPHOLIPASE-C BY AUROTHIOMALATE AND (TRIETHYLPHOSPHINE) GOLD COMPLEXES

The effect of several gold complexes on the activity of phospholipase C from human blood platelets was studied in vitro. Aurothiomalate and auranofin - 2 agents used for the chrysotherapy of rheumatoid arthritis-, gold chloride, (triethylphosphine)gold chloride, and 5 newly synthesized (triethylphosphine)gold complexes dose-dependently inhibited the enzyme with IC50 values between 0.8-mu-mol/l ((triethylphosphine)gold chloride) and over 100-mu-mol/l (auranofin). None of the compounds affected phospholipase A2 from human polymorphonuclear leukocytes at concentrations up to 100-mu-mol/l. Inhibition of phospholipase C by (triethylphosphine)gold chloride, aurothiomalate, and compound 3 was not significantly different at substrate concentrations of 20 and 100-mu-mol/l phosphatidylinositol, suggesting that these gold complexes do not inhibit phospholipase C by competing with the substrate. After confirming the Ca2+ dependence of the human platelet phospholipase C by demonstrating inhibition by the Ca2+ chelators EDTA and EGTA - but no inhibition by the Zn2+ chelator 1,10-phenanthroline - the inhibition of the enzyme by (triethylphosphine)gold chloride, aurothiomalate, and compound 3 was studied at 3 different concentrations of Ca2+ in the range of 0.2 to 2 mmol/l. Inhibition by (triethylphosphine)gold chloride was not affected by changes of Ca2+, whereas inhibition by aurothiomalate and compound 3 was markedly suppressed by increasing the Ca2+ concentration. This indicates that inhibition of phospholipase C by the latter 2 compounds could be due to competition with Ca2+ within the active site of the enzyme. Mercuric chloride and 4-chloromercuribenzoic acid were found to be potent inhibitors of phospholipase C (IC50 0.6 and 1.2-mu-mol/l, respectively), whereas the alkylating agents N-ethyl-maleimide and iodoacetamide, and the thiol compounds 2-mercaptoethanol and 1,4-dithio-D,L-threitol (DTT) had no significant effect. However, when the above 2 mercurials or the gold complexes (triethylphosphine)gold chloride and aurothiomalate were preincubated with the enzyme in the presence of an excess of 2-mercaptoethanol, inhibition was completely prevented. These results suggest that gold complexes and mercurials inhibit human platelet phospholipase C by blockade of an essential sulfhydryl group of the enzyme.