Isolation and polypeptide composition of l,3‐ß‐glucan synthase from plasma membranes of Brassica oleracea
ABSTRACT The l,3-ß-glucan synthase (callose synthase, EC 18.104.22.168) was solubilized from cauliflower (Brassica oleracea L.) plasma membranes with digitonin, and partially purified by ion exchange chromatography and gel filtration [fast protein liquid chromatography (FPLC)] using 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) in the elution buffers. These initial steps were necessary to obtain specific precipitation of the enzyme during product entrapment, the final purification step. Five polypeptides of 32, 35, 57, 65 and 66 kDa were highly enriched in the final preparation and are thus likely components of the callose synthase complex. The purified enzyme was activated by Ca2+, spermine and cellobiose in the same way as the enzyme in situ, indicating that no essential subunits were missing. The polyglucan produced by the purified enzyme contained mainly 1,3-linked glucose.
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ABSTRACT: The aim of this work was to optimize the conditions for in vitro synthesis of (13)--D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca2+, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca2+, 8 mM Mg2+, and 20 mM cellobiose. The in vitro-glucans were identified by hydrolysis of radioactive products, using specific enzymes. 13C-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (13)--D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.Cellulose 01/2004; 11(3):313-327. · 3.03 Impact Factor
Article: Formulations used in nutrition01/2005;
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ABSTRACT: Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 22.214.171.124) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca2+ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca2+, and in the presence of 2 mM Ca2+ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Planta 08/1993; 191(4):470-481. · 3.38 Impact Factor