Barley Proteome Analysis, Starch Degrading Eznymes and Proteinaceous Inhibitors

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概要

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• Proteomes of barley seeds were described by 2-D gel electrophoresis and spots selected for proteinidentification by mass spectrometry and database searches. Proteins were categorised according to temporalappearance during seed development and maturation. Fragments of β-amylases appeared transiently at midgrain filling and during germination. The α-amylase/trypsin inhibitors increased during grain filling andtypical housekeeping enzymes were present throughout the period. Germination altered the proteome and dissection of micromalted seeds enabled localization of selected proteins to specific tissues. The embryo was particularly rich in soluble proteins. Barley α-amylase/subtilisin inhibitor (BASI) and its target α-amylaseisozyme 2 occurred in endosperm and aleurone. Immunoblotting showed how the α-amylase changed duringgermination. Mutational analysis in a-amylase and BASI was guided by the three-dimensional structures.Mutants along the 10 binding subsites in barley a-amylase 1 addressed roles of individual residues in thepreference for starch over oligosaccharide substrates and vice versa, action patterns on oligosaccharides, andmultiple attack on amylose. Although the wild-type enzyme was proficient in transglycosylation, mutants ofthe catalytic nucleophile did not act as glycosynthase. C-terminal fusion of a starch binding domain from Aspergillus niger glucoamylase to barley α-amylase isozyme 1 enhanced degradation of starch granules. Recent heterologous expression of BASI allowed mutation of residues critical in enzyme binding as monitored by activity inhibition and surface plasmon resonance assays. A fully hydrated Ca<SUP>2+</SUP> at the protein interface secured contact between BASI and the catalytic residues in the α-amylase.

• 日本応用糖質科学会の論文
• 2003-07-14

著者

• Nielsen Peter

  Department Of Chemistry Carlsberg Laboratory

• Bak-jensen Kristian

  Department Of Chemistry Carlsberg Laboratory

• MORI Haruhide

  Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University

• Finnie Christine

  Department Of Chemistry Carlsberg Laboratory

• Nohr Jane

  Institute Of Food Research Norwich Research Park

• Juge Nathalie

  Institute Of Food Research Norwich Research Park

• Ostergaard Ole

  Department Of Chemistry Carlsberg Laboratory

• Kramhoft Birte

  Department Of Chemistry Carlsberg Laboratory

• Bonsager Birgit

  Department Of Chemistry Carlsberg Laboratory

• Svensson Birte

  Department Of Chemistr Carlsberg Laboratory

• Mori Haruhide

  Department Of Biochemistry And Molecular Biology University Of Southern Denmark

関連論文

• Molecular Cloning of Isomaltotrio-dextranase Gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and Its Expression in Escherichia coli
• A Catalytic Amino Acid and Primary Structure of Active Site in Aspergillus niger α-Glucosidase
• Molecular Cloning and Nucleotide Sequences of cDNA and Gene Encoding endo-Inulinase from Penicillium purpurogenum
• Barley Proteome Analysis, Starch Degrading Eznymes and Proteinaceous Inhibitors
• Impact on Substrate Specificity of Mutational Subsite Isozyme Mimicry in Barley α-Amylase
• Improving the Bioavailability of PNA-Peptide Conjugates
• Studies on Structure,Function,and Protein Engineering of Starch-Degrading Enzymes (平成10年度日本応用糖質科学会学会賞受賞講演)
• Low Dose-Ethanol Modulates Toxic Effect of Iron-Overloading in the Liver
• Starch-hydrolyzing Enzymes in Germinating Kidney Bean
• Barley Proteome Analysis, Starch Degrading Eznymes and Proteinaceous Inhibitors
• Impact on Substrate Specificity of Mutational Subsite Isozyme Mimicry in Barley α-Amylase

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