BINDING OF CYTOCHROME-C TO LIPOSOMES AS REVEALED BY THE QUENCHING OF FLUORESCENCE FROM PYRENE-LABELED PHOSPHOLIPIDS
BINDING OF CYTOCHROME-C TO LIPOSOMES AS REVEALED BY THE QUENCHING OF FLUORESCENCE FROM PYRENE-LABELED PHOSPHOLIPIDS
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DOI：
10.1021/bi00385a006
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发表时间：
1987-06-02
期刊：
影响因子：
2.9
通讯作者：
KINNUNEN, PKJ
中科院分区：
文献类型：
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作者：
MUSTONEN, P;VIRTANEN, JA;KINNUNEN, PKJ
用共振能量转移的方法，观察了细胞色素c的血红素(Cytc)与该蛋白的结合。脂质体由蛋黄磷脂酸(PA)和蛋黄磷脂酰胆碱或二棕榈酰磷脂酰胆碱与1mol%的荧光脂质组成。通过测量荧光强度或芘的发射寿命来监测Cytc与脂质体的结合。可以再次确认膜中存在酸性磷脂以与Cytc结合的要求。当膜中磷脂酸的摩尔分数低于5%时，细胞色素c与液晶双层的结合不明显，而随着PA浓度的进一步增加，细胞色素c的结合逐渐增强，直到达到约30摩尔%的磷脂酸饱和。加入氯化钠使荧光强度和寿命恢复到细胞色素c缺失时观察到的值，从而揭示了蛋白质从膜上解离。Py标记的磷脂酸衍生物PPHPA和PPDPA比相应的磷脂酰胆碱更有效地猝灭Cytc，这显然是由于酸性头基直接参与了Cytc的结合。当使用含有5mol%磷脂酸的二棕榈酰磷脂酰胆碱(DPPC)时，在前者的相变温度以上，Cytc不与脂质体结合，而在相变温度(TM)以下发生了结合。这表明在Tm以下，PA发生相分离，从而形成带负电荷的斑块，具有足够高的表面电荷密度，为Cytc提供结合部位。因此，这种过渡诱导相在膜脂中的分离可能为Cytc型外周膜蛋白的膜结合提供了一种控制机制。这些数据还表明，膜结合的cytc对膜脂具有长程有序化作用。进一步建议将结合了cytc的膜微区填充成三角超晶格。
Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could by reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the abscence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoyphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place. This indicates that below Tm phase separation of PA occurs, thus forming negatively charged patches with high enough surface charge density to provide binding sites for cyt c. Therefore, such transition-induced phase in separation in the membrane lipids could provide a control mechanism for the membrane binding of cyt c type peripheral memebrane proteins. These data also indicate that the membrane-bound cyt c has a long-range ordering effect on the membrane lipids. The membrane microdomains with bound cyt c are further suggested to pack into a trigonal superlattice.