1997 Fiscal Year Final Research Report Summary
Studies on steric structure of substrate binding site of rat spermidine synthase
| Project/Area Number |
07672324
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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 |
Physical pharmacy
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| Research Institution | Josai University |
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Principal Investigator |
SAMEJIMA Keijiro Josai University, Fac.Pharm.Sci.Prof., 薬学部, 教授 (00072413)
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| Co-Investigator(Kenkyū-buntansha) |
NIITSU Masaru Josai University, Fac.Pharm.Sci.Docent, 薬学部, 講師 (00077976)
SHIRAHATA Akira Josai University, Fac.Pharm.Sci, Asso.Prof., 薬学部, 助教授 (50150107)
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| Project Period (FY) |
1995 – 1997
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| Keywords | spermidine synthase / amino acid sequence / active site structure / MALDI-TOF-MS / SH-reagent / two functional reagent / decarboxylated S-adenosylmethionine |
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| Research Abstract |
1.Primary structure of rat spermidine synthase : It was interesting, using insufficient amount of rat spermidine synthase for Edman method, how to get a satisfactory information on its amino acid sequence making reference to the known sequence of mouse or human enzyme deduced from DNA.The strategy was the limited proteolysis of the rat enzyme with lysylendopeptidase and arginylendopeptidase, and specific cleavage at cystein residue with chemical reagents, followed by separation of the resulting peptides by semimicro HPLC and measurement of their mass by MALDI-TOF-MS.At least the three specific cleavage methods were necessary to assure the reliability of sequence. The results showed that the sequence of rat enzyme was principally the same as that of mouse enzyme except for Pro 11 corresponding to human enzyme, and the N terminal was acetylated. 2.Probable presence of free SH group (s) at the active site : The rat enzyme was not labeled with commercially available fluorescent SH-reagent, IAEDANS,in the presence of decarboxylated S-edenosymethionine (deAdoMet), whereas was labeled in the absence of deAdoMet, suggesting the presence of SH group around the binding site of deAdoMet. After analysis of the labeled enzyme, 4 to 6 cystein residues of known lacation were found to be labeled with IAEDANS.3.Basic research to survey amino acid residues spacially close to protein SH group : Bifunctional (SH reactive and photoaffinity labeling) reagent, AIAB,was prepared, and evaluated its usefulness for SH-protein.
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