| Project/Area Number |
60060005
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Research Institution | Kumamoto University |
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Principal Investigator |
MORINO Yoshimasa Kumamoto University Medical School, 医学部, 教授 (30028352)
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| Co-Investigator(Kenkyū-buntansha) |
KAGAMIYAMA Hiroyuki Osaka Medical College, 教授 (80028555)
SHIMADA Kazunori Kumamoto University Medical School, 医学部, 教授 (40037354)
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| Project Period (FY) |
1985 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥230,000,000 (Direct Cost: ¥230,000,000)
Fiscal Year 1988: ¥28,000,000 (Direct Cost: ¥28,000,000)
Fiscal Year 1987: ¥30,000,000 (Direct Cost: ¥30,000,000)
Fiscal Year 1986: ¥70,000,000 (Direct Cost: ¥70,000,000)
Fiscal Year 1985: ¥102,000,000 (Direct Cost: ¥102,000,000)
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| Keywords | Aspartate aminotransferase / Malate dehydrogenase / Malate-aspartate shuttle / Enzyme structure-function / Site-directed mutagenesis / Isozyme genes / 酵素の機構と機能相関 / リンゴ酸アスパラギン酸シヤトル / アイソザイム遺伝子の比較 / 酵素前駆体のミトコンドリヤ移入シグナル / アイソザイムの発現調節機構 / 酵素の構造機能相関 / アスパラギン酸・リンゴ酸シャトル / 哺乳動物酵素の酵母による発現 / リンゴ酸脱水素酵素 / トランスアミナーゼ / 酵素の構造と機能 / 酵素の発現調節機構 |
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| Research Abstract |
Cytosolic and mitochondrial isozymes of both aspartate aminotransferese(AspAT) and malate dehydrogenase(MDH) play pivotal roles in operating the malate-aspartate shuttle that links the cytosoilcally generated NADH to the mitochondrial oxidative phosphorylation in most cells of higher organisms. The purpose of the present research project is two-fold. One is to inquire into the molecular basis of the catalytic competence of AspAT and the mode of cellualar organization of the mitochondrial isozyme in particular. The technique of oligonuclectide-directed in vitro mutagenesis is comployed to examine the functional roles of critical amino acid residues involved in the formation of the coenzyme binding domain, substrate anchoring site, structural component assisting catalysis such as intramolecular proton transfer, interdomain interaction/subunit assembly, and the signal sequence requisite to mitochondrial localization. About 50 mutant enzymes were analyzed for the state of bound coenzyme, k
… More
inetic properties and thermodynamic aspects. These results are interpreted in terms of X-ray data to elicit the structure-function relationships of AspAT catalyzed reaction on molecular basis.
The other is to elucidate the regulatory mechanism for a coordinate gene expression of these metabolically related isozymes. The approach includes the isolation and structural comparison of the genomic DNAs for mouse cytosolic and mitochondrial isozymes of both AspAT and MDH and the identification of the structural components, particularly in their 5' regulatory regions, involved in the regulatory expression of these isozymes on the cellular and animal levels with combined use of various metabolic modulators. Comparison of nucleotide sequences in the 5' flanking regions of these isoenzyme genes revealed several characteristic features: absence of conventional 5' transcriptional regulatory sequences such as TATA and CAAT boxed, presence of G+C rich sequences. Moreover, there are several highly conserved regions in the 5' flanking sequences between mitochondrial and cytosolic AspATs, between mitochondrial AspAT and MDH, and, between cytosolic AspAT and MDH. the use of dna transfection technique permitted identification of a sequence with promotor activity in the 5' flanking region of each gene. Less
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