| Project/Area Number |
62580139
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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 |
物質生物化学
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| Research Institution | Tokyo Metropolitan Institute of Gerontology |
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Principal Investigator |
IWASHITA Yoshiko Tokyo Metropolitan Institute of Gerontology, 生化学部, 主任研究員 (50111498)
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| Co-Investigator(Kenkyū-buntansha) |
IWASHITA Shintaro Mitsubishi-Kasei Institute of Life Sciences, 細胞生理, 主任研究員 (90092147)
SAITO Megumi Tokyo Metropolitan Institute of Gerontology, 生化学部, 研究員 (90132901)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Cholesterol / Cell Membrane / Thiol-Activated Cytolysins / theta-Toxin / Erythrocytes / Limited Broteolysis / Reductive Methylation / 膜流動性 / θ-毒素 |
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| Research Abstract |
1. A hemolysin, theta-toxin, produced by Clostridium perfringens has one thiol group which is essential for its hemolytic activity. Modification of the residue with thiol-blocking agents reduced the binding affinity of the toxin to sheep erythrocytes to 1/100 that of intact toxin, resulting in a failure of binding. Thus the failure of hemolysis is primarily ascribed to a decreased affinity of the socin for erythrocytes.
2. The primary receptor for theta-toxin is the membrane cholesterol on the cell surface. The effect of theta-toxin on membrane fluidity was tested in hepatocyte preparations. Incubation of hepatocytes for 5 min with 600 or 2000 units/ml of theta-toxin resulted in a significant increase (32 and 46%, respectively) of D__- as compared to the controls of the same age group when measured by fluorescence recovery after photobleaching technique. Various tests like heat denaturation or cholesterol saturation of theta-toxin before its application revealed that the increase of D__- is a specific toxin effect due most probably to the reaction of theta-toxin with cholesterol.
3. A nicked theta-toxin (Ctheta), obtained by limited proteolysis with subtilisin carlsberg, causes almost no hemolysis while it retains nearly intact cholesterol binding site below 20゜C. Using Ctheta as a probe due to its high affinity for membrane cholesterol without causing any obvious membrane changes, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes. Ctheta binds only to cholesterol among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes but not to the protein components derived from them. These results strongly suggest that cholesterol is an essential component of the both high- and low-affinity sites, and also imply that the modes of existence of cholesterol in the red cell membrane are heterogeneous.
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