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]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM 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 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-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.

The Genetic Basis of Cellular Morphogenesis in the Filamentous FungusNeurospora crassa

“…Following the filtration enrichment step, the remaining conidia and small germlings were suspended in Vogels minimal agar medium containing 0.05% sucrose and 2% sorbose, plated, and incubated at 25°C to allow survivors of the mutagenesis and filtration to form colonies. (2% sorbose restricts hyphal extension, leading to the formation of compact colonies [Davis and de Serres, 1970;Taft et al, 1991]. )…”

Fungal ß(1,3)-D-glucan synthesis

“…Direct measurement of chitin or mannan biosynthetic activity in the presence of aculeacin A revealed complete lack of inhibition at concentrations that inhibited b (1,3)-glucan synthase activity by as much as 70% [35]. The enzymes used in these experiments were derived from organisms, including Geotrichum lactis [36], C. albicans [37], S. cerevisiae [31], N. crassa [38], H. anomola, Cryptococcus laurentii and Schizophyllum commune [39]. Three themes emerged from these early studies: First, inhibition of enzymatic activity was not competitive with the UDP-glucose substrate; second, reactions were inhibited to 70 or 80% of control, but not to completion; and third, susceptibility to inhibition is conserved across several fungal genera.…”

Characterization of Rho GTPase GAP/GEF modules in the ascomycete Neurospora crassa

“…Chemicals and culture media components, as well as all supplements in stress tests, were used from AppliChem GmbH, Carl Roth GmbH & Co. KG, Invitrogen GmbH, Merck KGaA, Roche Diagnostics GmbH and Sigma-Aldrich Chemie GmbH. To restrict radial growth on VMM supplement plates, sucrose was exchanged by 2% L-sorbose, 0.05% glucose and 0.05% fructose (Mishra & Tatum, 1972, Taft et al, 1991. 1mg/ml Congo Red, 5% NaCl, 0.3µM Benomyl, 0.01% SDS, 5mg/ml Lysing enzyme, 0.5µM…”