INHIBITION OF YEAST (1,3)-BETA-GLUCAN SYNTHASE BY PHOSPHOLIPASE A(2) AND ITS REACTION-PRODUCTS

Fungal (1,3)-beta-glucan synthases are sensitive to a wide range of li pophilic inhibitors and it has been proposed that enzyme activity is h ighly sensitive to perturbations of the membrane environment. Yeast me mbranes were exposed to phospholipases and various lipophilic compound s, and the resultant effects on glucan synthase activity were ascertai ned. Glucan synthase from Saccharomyces cerevisiae was rapidly inactiv ated by phospholipase A(2) (PLA(2)), and to a lesser extent by phospho lipase C. Inactivation was time and dose-dependent and was protected a gainst by EDTA and fatty-acid binding proteins (bovine and human serum albumins). Albumins also partially protected against inhibition by pa pulacandin B. PLA(2) reaction products were structurally characterized and it was shown that fatty acids and lysophospholipids were the inhi bitory moieties, with no novel inhibitory compounds apparent. Glucan s ynthase was inhibited by a range of fatty acids, monoglycerides and ly sophospholipids. Inhibition by fatty acids was non-competitive, and pr ogressive binding of [C-14]oleic acid correlated with activity loss. F luorescence anisotropy studies using diphenylhexatriene (DPH) confirm that fatty acids increase membrane fluidity. These results are consist ent with proposals suggesting that glucan synthase inhibition is due i n part to non-specific detergent-like disruption of the membrane envir onment, in addition to direct interactions of lipophilic inhibitors wi th specific target sites on the enzyme complex.