Does β-Glucan Synthesis Need a Primer?

Maclachlan, Gordon

doi:10.1007/978-1-4684-1116-4_16

Cited by 18 publications

(10 citation statements)

References 30 publications

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“…The very low apparent Ka for Ca2" for these enzymes in the presence of physiological levels of Mg2" supports the notion that small variations in levels of free intracellular Ca2" probably form a key regulatory mechanism for callose synthesis in vivo (16 (20) were not incorporated into product, suggesting that these activators do not serve as primers; rather, the kinetic studies described here suggest that they serve as allosteric effectors.…”

Section: Discussionsupporting

confidence: 47%

UDP-Glucose: (1→3)-β-Glucan Synthases from Mung Bean and Cotton

Hayashi¹,

Read²,

Bussell³

et al. 1987

Plant Physiol.

102

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A re-examination of the kinetic properties of UDP-glucose: (1-.3)-i% glucan (callose) synthases from mung bean seedlings ( Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca2" and millimolar concentrations of j-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V. of the enzyme, and by lowering the apparent K,. for UDPglucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg2" markedly enhances the affinity of the mung bean enzyme for Ca2" but not for fglucoside; with saturating Ca2', Mg2' only slightly stimulates further production of glucan. However, the presence of Mg2' during synthesis, or NaBH. treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca24, #-glucoside-activated cotton fiber enzyme, with or without Mg2', is very similar in size to callose isolated from cotton fibers, but is a linear (1-.3)-#-glucan lacking the small amount of branches at C0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg2' in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg2' on the conformation of the enzyme. Rate-zonal centrifuption of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.Essentially all higher plants contain a UDP-glucose: (1-+3)-f3-glucan (callose) synthase. This enzyme is largely found on the plasma membrane, and in most cases is latent and only becomes activated by perturbed conditions which lead to some loss of membrane permeability (5, 16). Some years ago, Ray (26) named this enzyme Glucan Synthetase II, and assayed it at high concentrations of UDP-Glc in the absence of divalent cations, although others have reported that activity can be enhanced by Mg2' (6),

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Section: Discussionsupporting

confidence: 47%

UDP-Glucose: (1→3)-β-Glucan Synthases from Mung Bean and Cotton

Hayashi¹,

Read²,

Bussell³

et al. 1987

Plant Physiol.

102

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“…In addition, our kinetic studies did not show activation of the allosteric type in the presence of either of these disaccharides (data not shown). It has also been suggested that sugars such as cellobiose may mimic an endogenous primer [43]. However, the use of radioactive cellobiose and laminaribiotol showed that these activators are not incorporated into the GS product [27].…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of (1→3)‐β‐d‐glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana

Him

Pélosi

Chanzy

et al. 2001

European Journal of Biochemistry

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The aim of this work was to develop a biochemical approach to study (1 !3)-b-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, pH 6.8, 1 mM UDP-glucose, 8 mM Ca 21 and 20 mM cellobiose. The enzyme was Ca 21 -dependent, and addition of Mg 21 to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive homotropic cooperativity of (1 !3)-b-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)-b-D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-b-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)-b-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 the 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, 13 C-NMR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1 !3)-b-D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.

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“…Both celluloses assembled in vitro were labeled with cellobiohydrolase Igold complex, and the electron diffraction patterns of both products from SE, and SE, revealed cellulose I and cellulose II, respectively. Contamination of native cellulose was ruled out by extensive evidente from autoradiography of the ethanol-insoluble and aceticl nitric acid-insoluble materials, including three different controls.To understand the mechanism of cellulose biosynthesis, many attempts to synthesize cellulose in vitro have been made with cell-free systems from different sources (Franz and Heininger, 1981;Robinson and Quader, 1981;Carpita, 1982;Maclachan, 1982;Blaschek et al, 1983;Delmer, 1987;Brown, 1989aBrown, , 1989bDhugga and Ray, 1991;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model system, and sufficiently high rates of synthesis of P-1,4-glucan from UDP-Glc were achieved (Aloni et al…”

mentioning

confidence: 99%

“…To understand the mechanism of cellulose biosynthesis, many attempts to synthesize cellulose in vitro have been made with cell-free systems from different sources (Franz and Heininger, 1981;Robinson and Quader, 1981;Carpita, 1982;Maclachan, 1982;Blaschek et al, 1983;Delmer, 1987;Brown, 1989aBrown, , 1989bDhugga and Ray, 1991;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model system, and sufficiently high rates of synthesis of P-1,4-glucan from UDP-Glc were achieved (Aloni et al…”

mentioning

confidence: 99%

[beta]-Glucan Synthesis in the Cotton Fiber (IV. In Vitro Assembly of the Cellulose I Allomorph)

Kudlicka

Brown

et al. 1995

Plant Physiol.

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In vitro assembly of cellulose from plasma membrane extracts of the cotton (Gossypium hirsutum) fiber was enriched by a combination of 3 4 N-morpholino)propanesulfonic acid extraction buffer and two independent digitonin solubilization steps consisting of 0.05% digitonin (SE,) followed by 1 % digitonin (SE,). Clucan synthase activity assays revealed that, although the SE, fraction possessed higher activity, only 8.6% of the in vitro product survived aceticl nitric acid treatment. On the other hand, the SE, fraction was less active, but 32.1 % of the total glucan in vitro product was resistant to acetichitric acid. In vitro products synthesized from the SE, fraction contained P-1,3-glucan and fibrillar cellulose I, whereas the SE, fraction produced P-1,3-glucan and cellulose II. Both celluloses assembled in vitro were labeled with cellobiohydrolase Igold complex, and the electron diffraction patterns of both products from SE, and SE, revealed cellulose I and cellulose II, respectively. Contamination of native cellulose was ruled out by extensive evidente from autoradiography of the ethanol-insoluble and aceticl nitric acid-insoluble materials, including three different controls.To understand the mechanism of cellulose biosynthesis, many attempts to synthesize cellulose in vitro have been made with cell-free systems from different sources (Franz and Heininger, 1981;Robinson and Quader, 1981;Carpita, 1982;Maclachan, 1982;Blaschek et al., 1983;Delmer, 1987;Brown, 1989aBrown, , 1989bDhugga and Ray, 1991;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model system, and sufficiently high rates of synthesis of P-1,4-glucan from UDP-Glc were achieved (Aloni et al

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Does β-Glucan Synthesis Need a Primer?

Cited by 18 publications

References 30 publications

UDP-Glucose: (1→3)-β-Glucan Synthases from Mung Bean and Cotton

UDP-Glucose: (1→3)-β-Glucan Synthases from Mung Bean and Cotton

Biosynthesis of (1→3)‐β‐d‐glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana

[beta]-Glucan Synthesis in the Cotton Fiber (IV. In Vitro Assembly of the Cellulose I Allomorph)

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