| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
1.Cross-linking of the recombinant E.coli T-protein (ET) and H-protein (EH) of the glysine cleavage system with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), a zero-length cross-linker of NH_2 and COOH,produced covalently bound product consisted of one moleculef eachof ET and EH.HPLC mapping of its lysylpeptides showed not only intermolecular cross-linking between ET and EH,but also intramolecular cross-linking in ET.The latter cross-linking was EH-dependent but not-dependent folate. The ET with 7 amino acid deletion in N-terminus (ETDELTA7) which showed a remarkably reduced affinity for EH compared with wild-type ET also formed a cross-linked product with EH.However the intramolecular cross-linking in ETDELTA7 was not observed. These results suggest the contribution of the N-terminal region of ET to the functional conformation.
2. ^<14>C-labeled methylenetetrahydropteroyltetraglutamate (CH_2-H_4PteGlu_4), a physiological folate substrate of T-protein, was enzymatically synthes
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ized from methylenetetrahydrofolate and ^<14>C-glutamic acid, and subjected to cross-linking with ET using EDC.The product also consisted of one molecule each of ET and CH_2-H_4PteGlu_4. HPLC mapping and amino acid sequence of its lysylpeptides revealed that three lysine residues, Lys-78, Lys-81 and Lys-352 were involved in cross-linking with polyglutamate tail of CH_2-H_4PteGlu_4.
3. The Lys-352, which is conserved in T-proteins from seven different species so far determined, was replaced by glutamate, glutamine, and arginine by site-directed mutagenesis. The mutants were overexpressed, purified, and characterized. All of them showed similar specific activity and Km values for CH_2-H_4PteGlu_4 to that of wild-type ET.The mutations of the lysine residue at 78 and 81 are now in progress.
4. Overexpression of normal and mutant human T-proteins with the point mutations identified in nonketotic hyperglycinemia patients are also underway to elucidate the relationship between the structure and the function of T-protein. Less
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