1989 Fiscal Year Final Research Report Summary
Effects of boundary phospholipids on the function of membrane proteins
| 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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| Keywords | Membrane protein / Sarcoplasmic reticulum / Ca^<2+>-ATPase / Boundary phospholipids / Phospholipid titration / Dynamic microstructure / Time-resolved fluorometer / Fluorescence anisotropy |
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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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Research Products
(13 results)
