Biosynthesis of (1 -> 3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana
2001 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 17, 4628-4638 p.Article in journal (Refereed) Published
The aim of this work was to develop a biochemical approach to study (1-->3)-beta -D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive. homotropic cooperativity of (1-->3)-beta -D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta -D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta -D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained 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 used for protein extraction. The reaction products synthesized by different detergent 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.
Place, publisher, year, edition, pages
2001. Vol. 268, no 17, 4628-4638 p.
(1 -> 3)-beta-D-glucan (callose) synthase, Arabidopsis thaliana, detergents, enzyme kinetics, polysaccharide biosynthesis, nuclear magnetic-resonance, beta-glucan synthesis, secondary cell-wall, cellulose biosynthesis, udp-glucose, (1->3)-beta-glucan synthase, cotton fiber, molecular architecture, suspension-cultures, lolium-multiflorum
IdentifiersURN: urn:nbn:se:kth:diva-20930ISI: 000170903500004OAI: oai:DiVA.org:kth-20930DiVA: diva2:339627
QC 201005252010-08-102010-08-10Bibliographically approved