ELSEVIER Biochimica et BiophysicaActa 1193(1994)31-40 Btl Biochi~ic~a et Biophysica A~ta Inhibition of yeast ( 1,3)-/3-glucan synthase by phospholipase A 2 and its reaction products Yuan-Tih Ko, David J. Frost 1, Chi-Tang Ho, Richard D. Ludescher, Bruce P. Wasserman * Department of Food Science, Rutgers University, Cook College, New JerseyAgricultural Experiment Station, New Brunswick, NJ 08903-0231, USA Received 14 September 1993 Abstract Fungal (1,3)-/3-glucan synthases are sensitive to a wide range of lipophilic inhibitors and it has been proposed that enzyme activity is highly sensitive to perturbations of the membrane environment. Yeast membranes were exposed to phospholipases and various lipophilic compounds, and the resultant effects on glucan synthase activity were ascertained. Glucan synthase from Saccharomyces cerevisiae was rapidly inactivated by phospholipase A 2 (PLA2), and to a lesser extent by phospholipase C. Inactivation was time and dose-dependent and was protected against by EDTA and fatty-acid binding proteins (bovine and human serum albumins). Albumins also partially protected against inhibition by papulacandin B. PLA2 reaction products were structurally characterized and it was shown that fatty acids and lysophospholipids were the inhibitory moieties, with no novel inhibitory compounds apparent. Glucan synthase was inhibited by a range of fatty acids, monoglycerides and lysophospholipids. Inhibition by fatty acids was non-competitive, and progressive binding of [14C]oleic acid correlated with activity loss. Fluores- cence anisotropy studies using diphenylhexatriene (DPH) confirm that fatty acids increase membrane fluidity. These results are consistent with proposals suggesting that glucan synthase inhibition is due in part to non-specific detergent-like disruption of the membrane environment, in addition to direct interactions of lipophilic inhibitors with specific target sites on the enzyme complex. Key words: (1,3)-/3-Glucan synthase; Phospholipase A2; Fluorescence anisotropy; Antifungal compound; Fatty acids; Membrane perturbation I. Introduction The biosynthesis of cell wall polymers in yeast, fungi and higher plants is important for cell growth and development. In the yeast Saccharomyces cerevisiae, a predominantly (1,3)-fl-linked glucan is a major cell wall component. This glucan is synthesized by the plasma membrane-bound glycosyl transferase (1,3)-/3-glucan synthase (GS) (EC 2.4.1.34), which catalyzes the trans- fer of glucose units from UDP-Glc (uridine diphos- phate glucose) onto a growing polysaccharide chain [1-3]. Fungal GSs are thought to exist as multimeric complexes containing both integrally-bound subunits as well as a peripherally-bound GTP binding subunit * Corresponding author. Fax: + 1 (908) 9326776. 1Present address: Anti-InfectiveResearch Division, Abbott Labo- ratories, Dept. 47M, Building AP9A, One Abbott Park Rd., Abbott Park, IL 60064-3500, USA. 0005-2736/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2736(94)00079-5 which is essential for activity [4,5]. In contrast to the higher plant GSs, which are readily solubilized by either digitonin or CHAPS (3-[(3-cholamidopropyl)di- methylammonio]-l-propanesulfonate) and have been subsequently partially purified [6-11], it has not been possible to obtain detailed structural information for fungal GSs due to rapid activity losses during solubi- lization from the membrane [12-14]. Therefore it is important to understand factors which influence the stability of fungal GSs. Yeast GS is of great interest because it represents a molecular target for antifungal antibiotics to treat sys- tematic fungal infections associated with AIDS, cancer, and other diseases that result in immunocompromised states. A number of antifungal drugs have been tar- geted against GSs from various fungi including the lipopeptide echinocandin-like class of inhibitors [15- 18], aculeacin A [19,20], and the papulacandins [21-24] which are lipid-like saccharides. Each of these com-