2004 Fiscal Year Final Research Report Summary
Analyses of Physiological Functions of Protein-Modifying Enzyme Trabsglutaminase
| Project/Area Number |
15580077
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Kyoto Institute of Technology |
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Principal Investigator |
IKURA Koji Kyoto Institute of Technology, Faculty of textile Science, Professor, 繊維学部, 教授 (00101246)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Protein-modifying enzyme / Transglutaminase / Betaine-homocysteine methyltransferase / Homocysteine metabolism |
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| Research Abstract |
In order to elucidate physiological functions of protein-modifying enzyme transglutaminase at the molecular level, analyses of its protein substrates and interacting proteins were done, and ectopic gene expressing experiments using Drosophila as a model system were designed.
1.It was conmirmed that betaine-homocysteine S-methyltransferase(BHMT) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutaminase. Primary amines were incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. Glutamine residues of BHMT which are reactive for transglutaminase reaction were located in the region near the carboxyl terminal of BHMT subunit, BHMT activity was regulated repressively by the transglutaminase modification, specially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.
2.The experiments using immuno-affinity gel beads with monoclonal antibody to guinea pig liver transglutaminase suggested that a 55 kDa protein is a candidate of an interacting protein of tissue-type transglutaminase. Yeast two-hybrid experiments using Sos/Ras-relay system were done to detect proteins interacting with tissue-type transglutaminase in mouse liver. The results suggested that various proteins including enzymes and proteinase inhibitor can be candidates of interacting proteins. We also prepared materials to apply the two-hybrid experiments on human brain.
3.A search on the data-base of Drosophila gene (Fly Base) indicated that Drosophila has one transglutaminase gene and two splicing variants are transcribed from the gene. To make the GAL4-UAS expression system of transglutaminase in Drosophila, we are now subcloning the cDNA of Drosophila transglutaminase into pUASP vector.
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