Biosynthesis of (1 -> 3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana

The aim of this work was to develop a biochemical approach to study (1-->3) -beta -D-glucan (callose) biosynthesis using suspension cultures of Arabido psis thaliana. Optimal conditions for in vitro synthesis of callose corresp onded to an assay mixture containing 50 mM Mops buffer, pH6.8, 1 mM UDP-glu cose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and ad dition of Mg2+ to the reaction mixture did not favour cellulose biosynthesi s. Enzyme kinetics suggested the existence of positive. homotropic cooperat ivity of (1-->3)-beta -D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeri c complex containing several catalytic subunits. Detergents belonging to di fferent families were tested for their ability to extract and preserve memb rane-bound (1-->3)-beta -D-glucan synthase activity. Cryo-transmission elec tron microscopy experiments showed that n-octyl-beta -D-glucopyranoside all owed the production of micelle-like structures, whereas vesicles were obtai ned with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3) -beta -D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of them in vitro products are influenced by the detergent us ed for protein extraction. The reaction products synthesized by different d etergent extracts were characterized by infrared spectroscopy, methylation analysis, C-13-N-MR spectroscopy, electron microscopy and X-ray diffraction . These products were identified as linear (1-->3)-beta -D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.