IN-VITRO SYNTHESIS OF A MICROFIBRILLAR (1-]3)-BETA-GLUCAN BY A RYEGRASS (LOLIUM-MULTIFLORUM) ENDOSPERM (1-]3)-BETA-GLUCAN SYNTHASE ENRICHED BY PRODUCT ENTRAPMENT

A simple, rapid procedure has been developed to purify (1-->3)-beta-glucan synthase (UDP-Glc:(1-->3)-beta-glucan 3-beta-D-glucosyl transferase (EC 2.4.1.34)) over 400-fold from membrane preparations of Italian ryegrass (Lolium multiflorum) and 60-fold from CHAPS-extracted membranes. When a CHAPS-extract of the membranes is treated with 8 mM CaCl2, proteinaceous material is precipitated. Although less than 10% of CHAPS-solubilized protein is removed in this step, the total activity recovered in the supernatant increases fourfold. Thus, CaCl2 precipitation appears to be important in removing inhibitors of the (l-->3)-beta-glucan synthase. In the presence of 1 mM UDP-glucose, the supernatant after CaCl2 treatment produces a high molecular weight, insoluble product that entraps a (1-->3)-beta-glucan synthase of high specific activity. The product-entrapped enzyme preparation contains six major polypeptides, and comparison of the SDS-PAGE pattern of this fraction with the polypeptide profile of an immunoprecipitated (1-->3)beta-glucan synthase preparation suggests that polypeptides at 30-31 and 55-58 kDa are the most likely candidates for participation in (1-->3)-beta-glucan synthesis. When the reaction is performed on a larger scale, milligram quantities of product can be seen precipitating from the reaction mixture within 1 h of substrate addition. This product has been characterized by methylation analysis, H-1- and C-13-nmr spectroscopy, X-ray diffraction, electron microscopy, size exclusion chromatography, UV-induced fluorescence in the presence of the (1-->3)-beta-glucan-specific fluorochrome from aniline blue, and enzymic hydrolysis with a specific (1-->3)-beta-glucanase. These physical, chemical and enzymic analyses clearly demonstrate that the product is a microfibrillar (1-->3)-beta-glucan with a degree of polymerization of about 1500.