| Project/Area Number |
08457055
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Wakayama Medical College |
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Principal Investigator |
NISHIKIMI Morimitsu Wakayama Medical College, Medicine, Professor, 医学部, 教授 (20022816)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUI LEE In Sook Wakayama Medical College, Medicine, Research associate, 医学部, 助手 (50209475)
SHIRAISHI Noriyuki Wakayama Medical College, Medicine, Lecturer, 医学部, 講師 (30133169)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Copper-binding protein / S100b protein / Hemolysis / Reactive oxygen species / Antioxidant / Escherichia coli / 銅結合性タンパク質 / キレート / アスコルビン酸 |
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| Research Abstract |
This study was aimed at elucidation of the importance of copper-binding protein in suppression of copper-induced cellular oxidative damage. We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation of L-ascorbate and identified it as S100b protein, an EF-hand calcuim binding protein, by sequencing its proteolytis peptides. Copper binding studies showed that this protein has four copper binding sites per dimeric protein molecule with a dissociation constant of 0.46 muM and that in the presence of L-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes indused by CuCl_2. We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be extressed as a fusion protein with glutathione S-transferase.The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl_2 plus H_2O_2 as compared with the control cells expressing glutathions S-transferase alone. A study by gel chromatography and affinity chromatography for S100beta fusion protein showed that the polymeric form of the fusion protein expressed in the cells mainly bound the copper that had been incorporated into the cells. These results indicate S100b protein does suppress oxidative cell damage by sequestering copper ions.
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