Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.): Properties of the membrane-bound glucan synthases

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP-[U-14C]glucose, the main components of which were identified as (1,3)(1,4)-β- and (1,3)-β-D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)-β-glucans, (1,3)-β-glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)-β-glucan synthesis or (1,3)-β-glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4)- and (1,3)-β-glucan synthesis reactions even when both occurred together. The synthesis of both β-glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)-β-glucan synthesis and (1,3)-β-glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)-β-glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)-β-glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)-β-Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment. The Km with respect to UDP-glucose was 1.5 mM for (1,3)-β-glucan synthesis and approximately 1 mM for (1,3)(1,4)-β-glucan synthesis. The (1,3)(1,4)-β-glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)-β-glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.