Miao Q, Han X, Yang F
National Laboratory of Biomacromolecules, Institute of Biophysics, The Chinese Academy of Sciences, Beijing 100101, China.
Biochem J. 2001 Mar 15;354(Pt 3):681-8. doi: 10.1042/0264-6021:3540681.
The translocation of apocytochrome c (apocyt.c) across large unilamellar vesicles (LUVs) constructed from mixtures of anionic and zwitterionic phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), has been studied. It was shown that the import ratio of horse heart apocyt.c in LUVs composed of phosphatidic acid (PA) combined with PE and PC (62+/-10%) was much higher than that in LUVs made of PE and PC plus any other acidic phospholipid species (20+/-5%). This feature was shared by tuna heart and chicken heart apocyt.c. In addition, the greater efficiency of the PA/PE/PC system versus others in facilitating apocyt.c translocation was maintained using synthetic anionic phospholipids with the same acyl chains. Besides, apocyt.c induces more leakage of entrapped fluorescein sulphonate (FS) from the interior of PA/PC/PE vesicles compared with phosphatidylglycerol (PG)/PC/PE ones. By measuring the intrinsic fluorescence emission spectrum and the accessibility of the preprotein to the fluorescence quencher, acrylamide, differences could be detected in the conformational changes of apocyt.c as a consequence of its interaction with PA/PE/PC and PG/PE/PC vesicles, respectively. Particularly notable is that PE is indispensable for the PA/PE/PC system to most efficiently facilitate apocyt.c translocation across the model membranes. With the fraction of PE increasing from 0 to 30 mol%, the translocation efficiency of apocyt.c as well as its ability to induce FS efflux was significantly enhanced in PA-containing LUVs, whereas this was not observed in the case of replacement of PA by PG or phosphatidylserine. It is also interesting to note that in LUVs containing PA, dioleoyl-PE, but not dielaidoyl-PE, can exert such influences, indicative of the role of non-bilayer formation propensity. On the basis of these results it is postulated that PA might increase the bilayer-destabilizing effects of PE, and hence increase the translocation efficiency of apocyt.c and its leakage-induction ability.
研究了脱辅基细胞色素c（apocyt.c）穿过由阴离子和两性离子磷脂、磷脂酰乙醇胺（PE）和磷脂酰胆碱（PC）混合物构建的大单层囊泡（LUVs）的转运情况。结果表明，在由磷脂酸（PA）与PE和PC组成的LUVs中，马心脏脱辅基细胞色素c的导入率（62±10%）远高于由PE和PC加上任何其他酸性磷脂种类组成的LUVs（20±5%）。金枪鱼心脏和鸡心脏脱辅基细胞色素c也有此特性。此外，使用具有相同酰基链的合成阴离子磷脂时，PA/PE/PC系统在促进脱辅基细胞色素c转运方面比其他系统效率更高。此外，与磷脂酰甘油（PG）/PC/PE囊泡相比，脱辅基细胞色素c会导致PA/PC/PE囊泡内部包裹的荧光素磺酸盐（FS）泄漏更多。通过测量脱辅基细胞色素c的固有荧光发射光谱以及前体蛋白对荧光猝灭剂丙烯酰胺的可及性，分别检测到脱辅基细胞色素c与PA/PE/PC和PG/PE/PC囊泡相互作用导致的构象变化差异。特别值得注意的是，PE对于PA/PE/PC系统最有效地促进脱辅基细胞色素c穿过模型膜的转运是必不可少的。随着PE的比例从0增加到30 mol%，脱辅基细胞色素c的转运效率及其诱导FS流出的能力在含PA的LUVs中显著增强，而在用PG或磷脂酰丝氨酸替代PA的情况下未观察到这种情况。同样有趣的是，在含有PA的LUVs中，二油酰-PE而非二反油酰-PE能发挥这种影响，这表明了非双层形成倾向的作用。基于这些结果推测，PA可能会增加PE的双层去稳定作用，从而提高脱辅基细胞色素c的转运效率及其泄漏诱导能力。
