Effects of boundary phospholipids on the function of membrane proteins

用时间分辨荧光仪研究了天然磷脂(PL)替代天然磷脂(PL)对大鼠左心室肌浆网(SR)蛋白质动态微结构和功能的影响。用Harigaya和Schwarz(1967)方法分离的心脏SR悬液与含有荧光团苯胺萘马来酰亚胺(ANM)的盐丙酮乳剂孵育，ANM选择性地与-SH蛋白质基团反应(Kanaoka等人)。1973)，在30℃下持续30分钟(Suzuki et al.(1989年)。然后将SR在45000xg的温度下离心并洗涤以去除多余的ANM。用脂质滴定法改变SR的PL(Warren等人)。1974年，Johannsson等人提出。(1981)；SR悬浮液与短酰基链(两个酰基链都是单不饱和或饱和但短的；二(18：1，16：1，14：1或12：Q)PC)的PL在0℃下溶解在胆酸盐中15min，然后洗涤两次以除去胆酸盐。SR悬浮在TES-KOH，0.1M KCl溶液中，在-80℃下保存，用二苯基己三烯(DPH)荧光各向异性证实SR中磷脂的取代。用di(18：1)PC置换膜粘度降至0.78泊，用di(12：0)PC置换膜粘度降至0.53泊。用较短的PL取代SR中的天然脂类，可显著降低Ca~(2+)-依赖的ATPase活性。测得的ANM荧光各向异性半衰期分别为：di(18：1)pc为30nsec，di(14：1)pc为25nsec，di(12：0)pc为13nsec。这一结果表明，SR蛋白的旋转弛豫受边界磷脂的物理性质的限制，并且磷脂的变化导致SR蛋白ANM结合域的分子运动发生变化。

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.