| Project/Area Number |
60580132
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Kyoto University |
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Principal Investigator |
KATSURA Izui Instructor, Faculty of Science, 理学部, 助手 (20025414)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Phosphoenolpyruvate carboxylase / Escherichia coli / Anacystis nidulans / Zea mays / allosteric enzyme / chimeric gene / 遺伝子操作 |
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| Research Abstract |
1. The genes for phosphoenolpyruvate carboxylase (PEPC) were cloned from Escherichia coli, Anacystis nidulans and Zea mays, and were sequenced.
2. Comparison of the three deduced amino acid sequences of PEPCs revealed that the extent of homology was 30-40% and the homology was about 10% higher in the C-terminal half than in the N-terminal half. Since the allosteric regulatory properties of three enzymes were quite different from each other, the former was presumed to be involved in the catalytic function and the latter in the regulatory function.
3. The enzyme which was encoded by the A. nidulans gene and synthesized in E. coli cells was purified to homogeneity. The determination of the amino acid composition and the N-terminal amino acid sequence verified the sequence deduced from DNA base sequence.
4. Differential chemical modification of E. coli enzyme in the presence and absence of PEP analog suggested the essential cysteine residue to be Cys-568.
5. Deletion introduced at the Stu <I> and Sac <II> sites of the E. coli ppc gene suggested that the region around these sites (corresponding to 344 and 670th amino acid residue, respectively) are not directly involved in the catalytic and regulatory functions. However, two clones with larger deletion gave enzymes with altered allosteric properties.
6. Chimeric genes were constructed between the E. coli and the A. nidulans genes. It was suggested that the N-terminal half of the E. coli gene is able to confer its original allosteric properties to the chimeric enzyme.
7. Chimeric genes were also constructed between the A. nidulans and the Z. mays genes. Recombination was performed at one of the Sac <II> sites where both enzyme have homologous amino acid sequence. To our regret, in the E. coli cells transformed by this chimeric plasmid neither enzyme activity nor immunoreactive protein was detected.
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