CHARACTERIZATION OF THE 1,3-BETA-GLUCAN SYNTHASE OF ASPERGILLUS-FUMIGATUS

1,3-beta-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors - GTP, NaF, sucrose and EDTA - added during the extraction procedure, were essential for optimal 1,3-beta-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[P-32]UDP-glucose and 5-(125)IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a K-i of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (K-m for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.