| Project/Area Number |
60580135
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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 | HIROSHIMA UNIVERSITY |
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Principal Investigator |
KOMINAMI Shiro Dept. Environmental Sciences, Fac. Integrated Arts and Sciences, Associate Professor, 総合科学部, 助教授 (10106776)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Liposomes / NADPH cytochrome P-450 reductase / cytochrome P- <450_(C21)> / Cytochrome P- <450_(17alpha,lyase)> / Kinetics / Adrenal steroid hormones / 副腎皮質ミクロソーム |
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| Research Abstract |
Analysis of the reaction mechanism of membrane proteins, using the adrenocortical cytochrome P-450 steroid hydroxylation system as a model system, was performed with liposomes, in which the purified cytochrome P- <450_(C21)> , cytochrome P- <450_(17alpha,lyase)> and NADPH cytochrome P-450 reductase were incorporated. The incorporation of the proteins into the liposomal memnbranes were achieved by a gradual removal of cholate from a cholate solution of phospholipids containing the proteins. The prepared proteoliposomes have the average diameter of 60 nm and 80 % of the incorported proteins exsisted at the exterior side of the unilamella vesicles. The rate of partition of the steroid substrates between the aqueous and lipid phase (liposomal membranes) and the rate of the steroid binding to P- <450_(C21)> were measured by a stopped flow method. The partition of the steroids between the two phases was accomplished within <10^(-3)> seconds. A kineric analysis of the steroid binding revealed
… More
that the substrate binding site of the P- <450_(C21)> faced lipid phase of the liposomal membranes. The study of the interaction of NADPH cytochrome P-450 reductase with liposomal membranes showed that the reductase could bind to the liposomal membranes in two different modes. The reductase in the both modes were found to be active for electron transfer to P- <450_(C21)> and well supported 21-hydroxylation reaction of P- <450_(C21)> . The reductase in the loosely bound form was mobile between vesicles and the transfer rate was controlled by the rate of the release of the reductase from the membranes. The competition for the reductase between P- <450_(C21)> and P- <450_(17alpha,lyase)> was studied using liposomes containing P- <450_(C21)> , P- <450_(17alpah,lyase)> and NADPH P-450 reductase. Theaffinity of the reductase was stronger to P- <450_(C21)> than to P- <450_(17alpha,lyase)> . This indicates the possibility that the steroidogenesis pathway in adrenocortical microsomes might be controlled by the affinity of the reductase to the P-450's in the microsomes. Less
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