PARTIAL-PURIFICATION OF A GTP-INSENSITIVE (1-]3)-BETA-GLUCAN SYNTHASEFROM PHYTOPHTHORA-SOJAE

A (1 --> 3)-beta-glucan synthase activity was identified in cell membr ane preparations from the oomycete Phytophthora sojae, a soybean patho gen, The activity could be solubilized using the zwitterionic detergen t CHAPS at relatively low concentrations (3 mg/ml), High salt concentr ations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increas e of calculated V-max values (2.5 vs. 0.4 nkat/mg protein) but only mi nor alteration of the K-m (10.6 vs. 10.7 mM), Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular wei ght (1 --> 3)-beta-glucan. Glucan synthase activity in both membrane a nd solubilized preparations was not activated by GTP or divalent catio ns as reported for other fungal or plant glucan synthases, The activit y was inhibited, as expected, in a competitive manner by UDP with a K- i of 2.9 mM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment, This procedure resulted in the selective enrichment of a protein band with apparent M-r 108 000 in SDS-PAGE which was not visible in any of the steps prec eding product entrapment. The glucan pellets from product entrapment c ontained up to 3% of the initial enzyme activity present in the fracti on used for the procedure. (C) 1998 Federation of European Biochemical Societies.