In Vitro Inhibition of Stable 1,3-β-D-Glucan Synthase Activity from Neurospora

Abstract

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1 % 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, pH 7.4, containing 5mM EDTA, I mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 μM GTP, 1 mM DTT, lOmM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4°C, and a Km app of 1.66 + 0.28mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-β-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki app of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.