| Project/Area Number |
07680690
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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 |
Functional biochemistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
FUJIWARA Kazuko the University of Tokushima, the Institute for Enzyme Research, assistant professor, 酵素科学研究センター, 助教授 (20108880)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| 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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| Keywords | lipoyltransferase / cloning / lipoic acid / lipoyl domain / glycine cleavage system / alpha-ketoacid dehydrogenase complex / ジヒドロリポイルトランスアシラーゼ |
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| Research Abstract |
Lipoic acid is a prosthetic group of H-protein of the glycine cleavage system and the acyltransferas components of the pyruvate, alpha-ketoglutarate, and branched chain alpha-ketoacid dehydrogenase complex. Previously, we purified the Lipoyl-AMP : N^<epsilon>-Iysine lipoyltransferase (lipoyltransferase) I and II which are responsible for the attachment of lipoic acid to proteins employing apoH-protein as an acceptor of lipoic acid. In this study we obtained the following results.
1) Lipoyltransferase I and II transfered a lipoyl moiety to the lipoyl domains of the acyltransferase components of the pyruvate, alpha-ketoglutarate, and branched chain alpha-keteacid dehydrogenase complex. The site-directed mutagenesis experiment suggested that the glutamic acid residue situated 3 residues to the N-terminal side of the lipoylation site is important for the lipoylation and that the glycine residue situated 11 residues C-terminal side of the lipoylation site is important for the folding of the lipoyl domains.
2) The NH_2-terminal amino acid sequences of lipoyltransferase I and II were determined up to 20 and 30 residues, respectively. Oligonucleotides were synthesized based on the sequences of Asn13-Asn18 and Trp24-Met29. One clone which hybridized with the both oligonucleotides was obtained fron independent 2.4*10^6 plaques of bovine liver lambdagt10 cDNA library. The clone includes a insert of 1.3 kbp long which is enough for a full length cDNA encoding the lipoyltransferase. Nucleotide sequence analysis is now in progress.
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