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-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (Km for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.
Beauvais
A
A
Unité de Mycologie, Institut Pasteur, Paris, France.
Drake
R
R
Ng
K
K
Diaquin
M
M
Latgé
J P
JP
D000345:Affinity Labels; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000254:growth & development; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; D012995:Solubility; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; C060227:5-azidouridine 5'-diphosphoglucose; C071713:5-(3-(4-azidosalicylamide)allyluridine)- 5'-diphosphoglucose; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D014532:Uridine Diphosphate Glucose