Partial purification and immunological characterization of 1,3-?-glucan synthase from suspension cells of Glycine max

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Abstract
The plasma-membrane-localized 1,3-β-glucan synthase (EC 2.4.1.34) from suspension cultures of Glycine max (L.) Merr. was greatly enriched by a three-step purification procedure. Starting with a microsomal preparation, a six- to eightfold enrichment of the enzyme was achieved by isolating plasma-membrane vesicles in a polyethyleneglycol/dextran two-phase system. The enzyme was solubilized with the nonionic detergent digitonin and further purified 12-fold by successive centrifugations on two linear sucrose density gradients. The most purified enzyme preparation showed enrichment in a 31-kilodalton (kDa) polypeptide and was used to raise polyspecific antibodies which precipitated 1,3-β-glucan synthase activity. These antibodies were purified by affinity chromatography against immobilized membrane protein fractions of lower molecular weight which were devoid of 1,3-β-glucan synthase activity. The purified antibodies specifically labelled a single polypeptide of 31 kDa in the 1,3-β-glucan-synthase-containing heavy fractions of the first sucrose gradient indicating that this polypeptide represents part of the active enzyme complex.