| Project/Area Number |
06660118
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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 |
応用微生物学・応用生物化学
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| Research Institution | Kyoto Prefectural University |
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Principal Investigator |
ICHIHARA Kenichi Kyoto Prefectural University, Department of Agriculture, Associate Professor, 農学部, 助教授 (50046512)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Kunisuke Kyoto Prefectural University, Department of Agriculture, Professor, 農学部, 教授 (90027194)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Acyl-CoA Synthetase / Oil Synthesis / Oil Plants / Fatty Acid Activation Enzyme / 可溶化 / トリアミルグリセロール |
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| Research Abstract |
A membrane-bound acyl-CoA synthetase was purified from maturing seeds of safflower (Carthomus tinctorius L.). The enzyme was first solubilized with a detergent from microsomal membranes. The solubilized enzyme was purified 400-fold by ion-exchange chromatography and affinity chromatography. According to SDS-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was 76,000. The amino end of the protein was blocked. When the enzyme was solubilized, the fatty acid specificity was changed. This indicates that changes in protein structure affected the recognition of the enzyme for fatty acyl structures. A purified enzyme preparation that was partially inactivated was re-activated by the addition of phospholipid liposomes, indicating that phospholipids are required to maintain the structure of the enzyme protein and the enzymatic activity.
cDNA libraries were prepared from maturing safflower and rice seeds. PCR was carried out with the cDNA libraries as templates. Probes used were oligonuleotides whose sequences were conserved in animal and bacterial acy1-CoA synthetases. PCR gave a 1,150bp nucleotide for rice and a 1,230 bp nucleotide for safflower. Highly conservative sequences were detected in the amino acid sequences deduced from the above nucleotides, . These conservative peptide regions may be substrate-binding sites or the active site of the enzyme. The acy1-CoA synthetase was partially similar to firefly luciferase in amino acid sequence, which indicates that these two defferent enzyme proteins originated from an identical protein molecule in past.
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