| Project/Area Number |
62480100
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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 |
General physiology
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| Research Institution | Hokkaido University |
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Principal Investigator |
KOYAMA Tomiyasu Physiology Section, Research Institute of Applied Electricity, Hokkaido University. Professor, 応用電気研究所, 教授 (50001681)
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| Co-Investigator(Kenkyū-buntansha) |
KANAZAWA Tooru Department of Physiology, Asahikawa Medical College. Professor, 医学部, 教授 (80028141)
KINJO Masataka Physiology Section, Research Institute of Applied Electricity, Hokkaido Universi, 応用電気研究所, 助手 (70177971)
ARAISO Tunehisa Physiology Section, Research Institute of Applied Electricity, Hokkaido Universi, 応用電気研究所, 助教授 (30151145)
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| Project Period (FY) |
1987 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1987: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Membrane protein / Sarcoplasmic reticulum / Ca^<2+>-ATPase / Boundary phospholipids / Phospholipid titration / Dynamic microstructure / Time-resolved fluorometer / Fluorescence anisotropy / Ca^<2+>ATPase / 時間分解蛍光計 / 微視的粘性 / 燐脂質分子搖動角 / 心筋ミトコンドリア外膜 / 内膜 / 境界燐脂質層 / 低秩序燐脂質層 / 蛍光異方性 / サブナノ秒高速蛍光計 / 時間分解異方性曲線 / 相分離 / 燐脂質分子揺動角 |
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| Research Abstract |
The effects of replacement of native phospholipids (PL) with shorter PL on the dynamic microstructure and function of protein of sarcoplasmic reticulum (SR) isolated from rat left ventricles were studied with a timeresolved fluorometer. The suspension of isolated cardiac SR isolated by the method of Harigaya and Schwarz (1967) was incubated with saline-aceton emulsion containing the fluorophore, anilinonaphthylmaleimide (ANM), which selectively reacts with -SH group of proteins (Kanaoka et al. 1973), for 30 minutes at 30 C (Suzuki et al. 1989). SR was then centrifuged at 45000xg and washed to eliminate excess ANM. PL of SR were changed by lipid titration method (Warren et al. 1974, Johannsson et al. 1981); SR suspension was incubated with PL of short acyl chains (both acyl chains are monounsaturated or saturated but short; di(18:1, 16:1, 14:1 or 12:Q)PC) dissolved in cholate for 15 minutes at 0 C, then washed twice to eliminate cholate. SR was suspended in TES-KOH, 0.1M KCl solution and stored at -80 C. Replacement of phosholipids in SR was confirmed with diphenyl-hexatriene (DPH) fluorescence anisotropy. The membrane viscosity was reduced to 0.78 poise by replacement with di(18:1)PC and to 0.53 poise by replacement with di(12:0)PC. The replacement of native lipids of SR with shorter PL caused a remarkable decrease in Ca^<2+>-depending ATPase activity. The overall half decay time of ANM fluorescence anisotropy was 30 nsec with di(18:1)PC, 25 nsec with di(14:1)PC and 13 nsec with di(12:0)PC. This result suggests that the rotational relaxation of SR protein is limited by the physical properties of boundary phospholipids and that changes in phospholipids causes alterations in the molecular motion of ANM-binding domain of SR protein.
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