Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.): Properties of the membrane-bound glucan synthases

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP-[U-14C]glucose, the main components of which were identified as (1,3)(1,4)-β- and (1,3)-β-D-glucans....

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Veröffentlicht in:Planta 1995-01, Vol.195 (3), p.331-338
Hauptverfasser: Becker, Martin, Vincent, Christine, Reid, J.S. Grant
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description Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP-[U-14C]glucose, the main components of which were identified as (1,3)(1,4)-β- and (1,3)-β-D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)-β-glucans, (1,3)-β-glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)-β-glucan synthesis or (1,3)-β-glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4)- and (1,3)-β-glucan synthesis reactions even when both occurred together. The synthesis of both β-glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)-β-glucan synthesis and (1,3)-β-glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)-β-glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)-β-glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)-β-Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment. The Km with respect to UDP-glucose was 1.5 mM for (1,3)-β-glucan synthesis and approximately 1 mM for (1,3)(1,4)-β-glucan synthesis. The (1,3)(1,4)-β-glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)-β-glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.
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Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)-β-glucan synthesis or (1,3)-β-glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4)- and (1,3)-β-glucan synthesis reactions even when both occurred together. The synthesis of both β-glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)-β-glucan synthesis and (1,3)-β-glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)-β-glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)-β-glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)-β-Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment. The Km with respect to UDP-glucose was 1.5 mM for (1,3)-β-glucan synthesis and approximately 1 mM for (1,3)(1,4)-β-glucan synthesis. The (1,3)(1,4)-β-glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)-β-glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.</description><identifier>ISSN: 0032-0935</identifier><identifier>EISSN: 1432-2048</identifier><identifier>DOI: 10.1007/BF00202589</identifier><identifier>PMID: 7766040</identifier><identifier>CODEN: PLANAB</identifier><language>eng</language><publisher>Berlin: Springer-Verlag</publisher><subject>Agronomy. 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Grant</creatorcontrib><title>Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.): Properties of the membrane-bound glucan synthases</title><title>Planta</title><addtitle>Planta</addtitle><description>Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP-[U-14C]glucose, the main components of which were identified as (1,3)(1,4)-β- and (1,3)-β-D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)-β-glucans, (1,3)-β-glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)-β-glucan synthesis or (1,3)-β-glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4)- and (1,3)-β-glucan synthesis reactions even when both occurred together. The synthesis of both β-glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)-β-glucan synthesis and (1,3)-β-glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)-β-glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)-β-glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)-β-Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment. The Km with respect to UDP-glucose was 1.5 mM for (1,3)-β-glucan synthesis and approximately 1 mM for (1,3)(1,4)-β-glucan synthesis. The (1,3)(1,4)-β-glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)-β-glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.</description><subject>Agronomy. Soil science and plant productions</subject><subject>Barley</subject><subject>beta-Glucans</subject><subject>Biological and medical sciences</subject><subject>Biosynthesis</subject><subject>Biotechnology</subject><subject>Carbohydrate Conformation</subject><subject>Cations, Divalent</subject><subject>Cell Membrane - enzymology</subject><subject>Cell walls</subject><subject>Coleoptiles</subject><subject>Economic plant physiology</subject><subject>Endosperm</subject><subject>Enzymes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glucans</subject><subject>Glucans - biosynthesis</subject><subject>Glucans - chemistry</subject><subject>Glucosyltransferases - metabolism</subject><subject>Hordeum - metabolism</subject><subject>Hydrogen-Ion Concentration</subject><subject>Kinetics</subject><subject>Metabolism</subject><subject>Metabolism. Physicochemical requirements</subject><subject>Nitrogen metabolism and other ones (excepting carbon metabolism)</subject><subject>Nutrition. Photosynthesis. Respiration. Metabolism</subject><subject>Plant physiology and development</subject><subject>Plants</subject><subject>Polymers</subject><subject>Product labeling</subject><subject>Temperature</subject><issn>0032-0935</issn><issn>1432-2048</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkMtKxDAUhoMoOo5u3AtZiDhg9eTSpnXniDcY0IWuSyY90Q69jEkrzCv4OD6Iz2TQMrpJwvl-vnNyCDlgcMYA1Pn0BoADj9Nsg4yYFDziINNNMgIIb8hEvEN2vV8ABKjUNtlWKklAwoh8TMvWr5ruFX3paWvpCTsVk3DISfT1Gb1UvdEN1U3xC_7VyobOtatwRU_uWldgX9P3vnrRDunsbHJBH127RNeV-KMNDWiN9dzpBqN52wfh4Pnprj36PbJldeVxf7jH5Pnm-unqLpo93N5fXc6iBY9VF8lYJjy2xiaSc0ikllYXMjMKEJPCFJhyyWzK0BomMGZgkYPSoZQya7QQY3L861269q1H3-V16Q1WVRit7X2uFAcmJAvBwyHYz2ss8qUra-1W-bC8wI8Grr3RlQ1_M6Vfx4TIwrazP83Cd61bYy5EKnjg3w8Fhck</recordid><startdate>19950101</startdate><enddate>19950101</enddate><creator>Becker, Martin</creator><creator>Vincent, Christine</creator><creator>Reid, J.S. Grant</creator><general>Springer-Verlag</general><general>Springer</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>19950101</creationdate><title>Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.): Properties of the membrane-bound glucan synthases</title><author>Becker, Martin ; Vincent, Christine ; Reid, J.S. Grant</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-j257t-454625fcf6422064a4fad49c70ee6dcde8241f81efc13e510fe207a1f881fca33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>Agronomy. Soil science and plant productions</topic><topic>Barley</topic><topic>beta-Glucans</topic><topic>Biological and medical sciences</topic><topic>Biosynthesis</topic><topic>Biotechnology</topic><topic>Carbohydrate Conformation</topic><topic>Cations, Divalent</topic><topic>Cell Membrane - enzymology</topic><topic>Cell walls</topic><topic>Coleoptiles</topic><topic>Economic plant physiology</topic><topic>Endosperm</topic><topic>Enzymes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glucans</topic><topic>Glucans - biosynthesis</topic><topic>Glucans - chemistry</topic><topic>Glucosyltransferases - metabolism</topic><topic>Hordeum - metabolism</topic><topic>Hydrogen-Ion Concentration</topic><topic>Kinetics</topic><topic>Metabolism</topic><topic>Metabolism. Physicochemical requirements</topic><topic>Nitrogen metabolism and other ones (excepting carbon metabolism)</topic><topic>Nutrition. Photosynthesis. Respiration. Metabolism</topic><topic>Plant physiology and development</topic><topic>Plants</topic><topic>Polymers</topic><topic>Product labeling</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Becker, Martin</creatorcontrib><creatorcontrib>Vincent, Christine</creatorcontrib><creatorcontrib>Reid, J.S. Grant</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>Planta</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Becker, Martin</au><au>Vincent, Christine</au><au>Reid, J.S. Grant</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.): Properties of the membrane-bound glucan synthases</atitle><jtitle>Planta</jtitle><addtitle>Planta</addtitle><date>1995-01-01</date><risdate>1995</risdate><volume>195</volume><issue>3</issue><spage>331</spage><epage>338</epage><pages>331-338</pages><issn>0032-0935</issn><eissn>1432-2048</eissn><coden>PLANAB</coden><abstract>Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP-[U-14C]glucose, the main components of which were identified as (1,3)(1,4)-β- and (1,3)-β-D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)-β-glucans, (1,3)-β-glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)-β-glucan synthesis or (1,3)-β-glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4)- and (1,3)-β-glucan synthesis reactions even when both occurred together. The synthesis of both β-glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)-β-glucan synthesis and (1,3)-β-glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)-β-glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)-β-glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)-β-Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment. The Km with respect to UDP-glucose was 1.5 mM for (1,3)-β-glucan synthesis and approximately 1 mM for (1,3)(1,4)-β-glucan synthesis. The (1,3)(1,4)-β-glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)-β-glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.</abstract><cop>Berlin</cop><pub>Springer-Verlag</pub><pmid>7766040</pmid><doi>10.1007/BF00202589</doi><tpages>8</tpages></addata></record>
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subjects Agronomy. Soil science and plant productions
Barley
beta-Glucans
Biological and medical sciences
Biosynthesis
Biotechnology
Carbohydrate Conformation
Cations, Divalent
Cell Membrane - enzymology
Cell walls
Coleoptiles
Economic plant physiology
Endosperm
Enzymes
Fundamental and applied biological sciences. Psychology
Glucans
Glucans - biosynthesis
Glucans - chemistry
Glucosyltransferases - metabolism
Hordeum - metabolism
Hydrogen-Ion Concentration
Kinetics
Metabolism
Metabolism. Physicochemical requirements
Nitrogen metabolism and other ones (excepting carbon metabolism)
Nutrition. Photosynthesis. Respiration. Metabolism
Plant physiology and development
Plants
Polymers
Product labeling
Temperature
title Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.): Properties of the membrane-bound glucan synthases
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