CHARACTERIZATION AND CILOFUNGIN INHIBITION OF SOLUBILIZED ASPERGILLUS-FUMIGATUS (1,3)-BETA-D-GLUCAN SYNTHASE

(1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is t he target of antifungal drugs of the lipopeptide class. Aspergillus fi lmigatus (1,3)-beta-D glucan synthase was prepared and its activity wa s measured by incorporation of [C-14]glucose from UDP-[U-C-14]glucose into an insoluble polymer in the presence of cx-amylase. Solubilizatio n of the (1,3)-beta-D-glucan synthase was attempted with several deter gents, and the maximum percent solubilization was obtained with a poly oxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations we re extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this gl ucan synthase was done by three methods. The first was enzymatic end p roduct degradation by alpha-amylase (no degradation) and beta-glucanas e (85 to 95% degradation). The second was gas chromatography-mass spec troscopy analysis of the partially methylated alditol acetate derivati ves prepared from total carbohydrate polymers present in the sample. T his method identified the presence of (1,3)- and (1,2)-glucosidic link ages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of t he newly synthesized, radioactive polymers from the contaminating carb ohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with bet a-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan s ynthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-r esponse experiments,vith this inhibitor showed that the solubilized en zyme was maximally inhibited at a cilofungin concentration of 1.25 mu g/ml and showed < 5% inhibition at 0.02 mu g/ml. The apparent K-m (K-m app) for the solubilized glucan synthase was 400 +/- 80 mu M, and the apparent K-i (K-i app) for cilofungin was 0.19 +/- 0.03 mu M. Inhibit ion of A. fumigatus (1,3)-beta-D-glucan synthase with cilofungin was n oncompetitive, as it was for the Candida albicans (1,3)-beta-D-glucan synthase.