SEPARATION AND PARTIAL PEPTIDE CHARACTERIZATION OF BETA-1-3 GLUCAN SYNTHASE FROM SAPROLEGNIA

The membrane-localized 1,3-beta-glucan synthase (EC 2.4.1.34; UDPglucose: 1,3-beta-D-glucan 3-beta-glucosyltransferase) from Saprolegnia monoica was greatly enriched by a two-step purification procedure. Starting with a microsomal preparation, the enzyme was solubilized with a cholamidopropyl-dimethyl-ammonio-propanesulfonate (Chaps)/octylglucoside mixture and further purified by ultracentrifugation on a linear glycerol density gradient. The most purified enzyme preparation showed enrichment in 34, 48 and 50 kDa polypeptides. The true involvement of these three bands in the 1,3-beta-glucan synthase structure was demonstrated by three experimental procedures. After enzyme purification, entrapment gave selective synthase sedimentation with its reaction product and subsequent SDS-PAGE electrophoresis showed the presence of these three polypeptides. Lectin binding on solubilized preparation provoked a shift in the density gradient of the synthase activity correlating with the distribution of these three bands. Western analysis also showed that the doublet 48, 50 kDa was glycosylated. Finally, the three bands were used to raise polyspecific antibodies which precipitated 1,3-beta-glucan synthase activity.