Effects of boundary phospholipids on the function of membrane proteins

边界磷脂对膜蛋白功能的影响

• 批准号: 62480100
• 负责人: KOYAMA Tomiyasu
• 金额: $ 3.9万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62480100/
• 关键词: 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

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.

用时间分辨荧光法研究了短磷脂（PL）替代天然磷脂（PL）对大鼠左心室肌浆网（SR）蛋白质动态结构和功能的影响。将通过Harigaya和施瓦茨（1967）的方法分离的分离的心脏SR的悬浮液与含有荧光团苯胺基萘基马来酰亚胺（ANM）的盐水-丙酮乳液在30 ℃下孵育30分钟（Suzuki等1989），所述荧光团选择性地与蛋白质的-SH基团反应（Kanaoka等1973）。然后将SR以45000 xg离心并洗涤以除去过量的ANM。通过脂质滴定法改变SR的PL（Warren等，1974，Johannsson等，1981）; SR悬浮液与溶解在胆酸盐中的短酰基链的PL（两个酰基链都是单不饱和的或饱和的但短的;二（18：1，16：1，14：1或12：Q）PC）在0 ℃孵育15分钟，然后洗涤两次以除去胆酸盐。将SR悬浮在TES-KOH、0.1M KCl溶液中并储存在-80 ℃。SR中磷脂的置换用二苯基己三烯（DPH）荧光各向异性证实。通过用二（18：1）PC替换，膜粘度降低至0.78泊，通过用二（12：0）PC替换，膜粘度降低至0.53泊。用短PL取代SR的天然脂质导致Ca^<2+>依赖性ATP酶活性显著降低。ANM荧光各向异性的总体半衰减时间对于di（18：1）PC为30 nsec，对于di（14：1）PC为25 nsec，并且对于di（12：0）PC为13 nsec。这一结果表明，SR蛋白的旋转松弛是有限的边界磷脂的物理性质和磷脂的变化引起的改变，在SR蛋白的ANM结合域的分子运动。

项目成果

Araiso T & Koyama T: "A picosecond fluorescence study of the cone angle of the wobbling motion of rod-like fluorophore in lipid bilayers" Chemistry and Physics of Lipids. 50. 105-108 (1989)

荒矶T

Koyama T,Zhu M-Y,Araiso T,& Kinjo M: "Effects of phospholipid bilayer on function and dynamic microstructure of membrane proteins in rat cardiac sarcoplasmic reticulum"

小山T,朱明宇,荒矶T,

Koyama,T;Araiso,T.;Nitta,J.: Biorheology. 24. 311-317 (1987)

小山，T；荒矶，T.；新田，J.：生物流变学。

Tajima M, Araiso T, Koyama T et al.: "Membrane viscosity of lymphocytes and influence of phytohemagglutinin." Biorheology vol. 26, 45-54 (1989).

Tajima M、Araiso T、Koyama T 等人：“淋巴细胞的膜粘度和植物血凝素的影响。”

Koyama T, Zhu M-Y, Araiso T, Kinjo M: "Microdynamics of outer and inner membranes of mitochondria from bullfrog myocardium."

Koyama T、Zhu M-Y、Araiso T、Kinjo M：“牛蛙心肌线粒体外膜和内膜的微动力学。”