(1,3)BETA-GLUCAN SYNTHASE ACTIVITY OF NEUROSPORA-CRASSA - OF A SUBSTRATE-BINDING PROTEIN

(1,3)beta-Glucan synthase activity from the filamentous Ascomycete Neu rospora crassa was purified 1300-fold to a specific activity of 14000 nmol glucose incorporated/min per mg protein. Hyphae were disrupted an d crude membrane fractions obtained by high-speed centrifugation. Memb rane fractions were extracted with Tergitol NP-40 and a second high-sp eed particulate fraction was obtained. Enzyme activity was solubilized with holamidopropyl)dimethylammonio)-1-propanesulfonate and octyl-bet a-D-glucoside from Tergitol-extracted membrane preparations. Solubiliz ed enzyme activity was purified by product entrapment and recovered by low-speed centrifugation through a layer of sucrose. SDS-PAGE analysi s revealed 2 proteins of 165 and 100 kDa as likely candidates for subu nits of the (1,3)beta-glucan synthase enzyme complex. 5-Azido-[P-32]UD P-glucose was photo-crosslinked to UDP-glucose-binding proteins in eac h fraction of the purification procedure. Autoradiograms of SDS-PAGE g els revealed a single protein of 165 kDa enriching with enzyme activit y and labeling with the substrate analog. Photoincorporation of 5-azid o-[P-32]UDP-glucose by the 165 kDa protein was competed by 0.25 mM UDP -glucose (80%) and TDP-glucose (65%) while ADP-glucose (27%), CDP-gluc ose (36%), and GDP-glucose (8%) where less effective. These results we re similar to in vitro inhibition of enzyme activity by the same compo unds. These data strongly suggest that the 165 kDa protein is a substr ate-binding subunit of (1,3)beta-glucan synthase.