Effects of boundary phospholipids on the function of membrane proteins

用时间分辨荧光仪研究了短磷脂替代天然磷脂对大鼠左心室肌浆网（SR）蛋白动态微观结构和功能的影响。将Harigaya和Schwarz（1967）方法分离的心脏SR悬浮液与含有荧光基团苯胺壬基甲基马来酰亚胺（ANM）的盐-乙酰乳状液孵育，ANM与-SH基团蛋白选择性反应（Kanaoka et al. 1973），在30℃下孵育30分钟（Suzuki et al. 1989）。然后将SR以45000xg离心，洗涤以去除多余的ANM。脂质滴定法改变SR的PL (Warren et al. 1974, Johannsson et al. 1981)；SR悬液与短酰基链的PL（两种酰基链均为单不饱和或饱和但较短；di(18:1, 16:1, 14:1或12:Q)PC）在0℃下溶解于巧克力中孵育15分钟，然后洗涤两次以去除巧克力。将SR悬浮于TES-KOH， 0.1M KCl溶液中，保存于-80℃，用二苯基-六三烯（DPH）荧光各向异性证实SR中磷脂的替代。用di(18:1)PC替代后，膜粘度降至0.78泊，用di(12:0)PC替代后，膜粘度降至0.53泊。用较短的PL代替SR的天然脂质导致Ca^<2+>依赖性atp酶活性显著降低。ANM荧光各向异性的总半衰减时间为：di(18:1)PC为30 nsec， di(14:1)PC为25 nsec， di(12:0)PC为13 nsec。这表明SR蛋白的旋转弛豫受到边界磷脂的物理性质的限制，磷脂的变化导致SR蛋白anm结合结构域的分子运动发生改变。

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.