| Project/Area Number |
63571024
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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 |
Physical pharmacy
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| Research Institution | Josai University |
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Principal Investigator |
SAMEJIMA Keijiro Josai Univ., Fac. of Pharm. Sci., Professor, 薬学部, 教授 (00072413)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRAHATA Akira Josai Univ., Fac. of Pharm. Sci., Research Associate, 薬学部, 助手 (50150107)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Spermidine synthase / Spermine synthase / Active site structure / Inhibitor / Substrate specificity / Affinity adsorbent / Polyamine binding protein / Purification of enzyme / ポリアミン結合部位 / アミノプロピル基転移反応 / アミノプロピルトランスフェラ-ゼ / スペルミジン結合部位 / 拮抗阻害剤 / ポリアミンアナログ / アフィニティ-吸着体 / アミノプロピルトランスフェラーゼ / 阻害剤のデザイン |
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| Research Abstract |
1. A model of the putrescine binding site of spermidine synthase was proposed based on the study of a number of monoamine and diamine compounds as potential inhibitors and substrates of the enzyme. The active site seems to have a relatively large hydrophobic cavity adjacent to a negatively charged site, to which a protonated aminogroup of putrescine binds, with another aminogroup of putrescine being situated in the hydrophobic cavity as a free form to be aminopropylated. During these studies, several new potent inhibitors were found for spermidine synthase.
2. On the basis of the above mentioned model, another modified one was proposed for spermine synthase, and several compounds designed according to the modified model, aminopropylated derivatives of those which inhibited spermidine synthase, were found to potently inhibit spermine synthase. Also, binding site for aminopropyl moiety of spermidine seemed to be fairly narrow groove on the active site of spermine synthase.
3. In the survey of compounds which show substrate activity for spermidine synthase, two diamine compounds possessing both primary and secondary amine were found to act as aminopropyl acceptor. As it was suggested from the model and their chemical structures that their secondary amines should be aminopropylated, several experiments were carried out to confirm the enzymatic aminopropylation of secondary amines.
4. Seven different polyamine linked sepharose were prepared for affinity chromatography of spermidine and/or spermine binding proteins, and were applied for spermine synthase. Comparative studies on affinity of the enzyme to the seven columns further supported its active site model. And some of the columuns were found to be excellent for purification of the enzyme.
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