Biosynthesis of (1,3)(1,4)-beta-glucan and (1,3)-beta-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)-beta- and (1,3)-beta-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)-beta-glucans, (1,3)-beta-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)-beta-glucan synthesis or (1,3)-beta-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)-beta-glucan synthesis reactions even when both occurred together. The synthesis of both beta-glucans was optimal at 20 degrees C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)-beta-glucan synthesis and (1,3)-beta-glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)-beta-glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)-beta-glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)-beta-Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Becker
M
M
Department of Biological and Molecular Sciences, University of Stirling, Scotland, UK.
Vincent
C
C
Reid
J S
JS
D002236:Carbohydrate Conformation; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D005936:Glucans / Q000096:biosynthesis* / Q000737:chemistry; D005964:Glucosyltransferases / Q000378:metabolism; D001467:Hordeum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013696:Temperature; D047071:beta-Glucans
D002413:Cations, Divalent; D005936:Glucans; D047071:beta-Glucans; C067858:beta-glucan, (1-3)(1-4)-; C033363:beta-1,3-glucan; D005964:Glucosyltransferases; C040232:glucan synthase