| Project/Area Number |
63480390
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Ophthalmology
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| Research Institution | Tohoku University |
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Principal Investigator |
ISHIGURO Sei-ichi Tohoku University School of Medicine Assistant Professor, 医学部, 構師 (20111271)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAI Makoto Tohoku University School of Medicine Professor, 医学部, 教授 (90004720)
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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 |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | Bovine Retima / Rod Outer Segments / Retinol Dehydrogenase / Purification / Stereospecificity / Immunohisto chemisrty / Enzyme-Linked Immunosorbent Assay / Retinal Degeneration / ビタミンA / 酵素免疫組織化学 / 膜酵素 / 単クロ-ン抗体 / レチノール脱水素酵素 |
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| Research Abstract |
We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, Concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. SDS polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all- trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds.
In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37 KDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1 : 140.
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