Tissue Slice and Particulate β-Glucan Synthetase Activities from Pisum Epicotyls

Abstract

beta-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg(2+) optimum, apparent K(m)) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various beta-glucanases all show that both beta-1,3 and beta-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. beta-1,4 Linkages predominate at low substrate (5 mum) concentration. Kinetic data indicate that the capacity to synthesize beta-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.