| Project/Area Number |
06557013
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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 | 試験 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Okayama University |
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Principal Investigator |
SEKI Shuji Okayama University Medical School ; Professor, 医学部, 教授 (50032884)
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| Co-Investigator(Kenkyū-buntansha) |
SARKER Altaf Hossan Okayama University Medical School ; Research Assistant, 医学部, 助手 (20284060)
AKIYAMA Kosuke Okayama University Medical School ; Research Assistant, 医学部, 助手 (30222540)
渡邉 晰子 (渡辺 晰子) 岡山大学, 医学部, 助手 (30093719)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1994: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Activity blotting method / Zymography / DNA repair enzyme / APEX nuclease / AP endonuclease / DNA 3' repair diesterase / DNase I / DNase I isozyme / 等電点電気泳動 / アイソザイム / 活性ブロッティング / ウエスタンブロッティング / ヒトグリオーマ細胞 / APEXヌクレアーゼ / DNA3′修復ジエステラーゼ / 活性ゲル / ゲル電気泳動 |
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| Research Abstract |
Studies on DNA modifying (repair) enzymes provide us a lot of important information for understanding not only DNA metabolism but also mutation, carcinogenesis, aging, apoptosis and so on. By this project, an activity blotting method for zymological detection of DNA-modifying enzymes in various tissue or cell extracts electrophoresed on gels has been developed. The method consists of the following six steps : (1) preparation of crude, partially purified or purified enzymes ; (2) SDS- (denatured) or native (non-denatured) polyacrylamide gel electrophoresis (PAGE) of the enzyme preparations ; (3) renaturation of proteins electrophoresed on SDS-PAGE ; (4) preparation of damaged DNA-fixed membranes ; (5) protein blotting (activity blotting) onto a damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by a repair enzyme (s) ; detection of the activity-blotted site (s) to localize the repair enzyme.
AP endonuclease and DNA 3' repair diesterase activities of APEX nuclease are semiquantitatively detected with high efficiency in tissue or cell extract by the present method. DNase I and enzymes having DNase I-like activity have also been able to detect semiquantitatively with high efficiency in various tissue or cell extracts by the present method. The activity blotting method has been applied effectively for DNA-modifying enzymes fractionated by isoelectric focussing gel electorophoresis (IEF-PAGE). Sensitive zymographical demonstration of isozyme patterns of DNase I in various tissue extracts was possible by IEF-PAGE-activity blotting.
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