Poveda José A, Encinar José A, Fernández Asia M, Mateo C Reyes, Ferragut José A, González-Ros José M
Centro de Biología Molecular y Celular, Universidad Miguel Hernández, 03206 Elche (Alicante), Spain.
Biochemistry. 2002 Oct 8;41(40):12253-62. doi: 10.1021/bi0200099.
Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.
从电鳐中提取的纯化乙酰胆碱受体（AcChR）已以低（约1:3500）和高（约1:560）的蛋白质与磷脂摩尔比，重构到含有卵磷脂酰胆碱、胆固醇和不同的二肉豆蔻酰磷脂（二肉豆蔻酰磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰甘油和磷脂酸）的囊泡中，作为探针，通过差示扫描量热法、红外光谱和荧光光谱来探究该蛋白质对磷脂组织的影响。所有实验结果表明，即使在较低的蛋白质与磷脂摩尔比下，AcChR蛋白质的存在也会引导磷脂酸探针的单阴离子磷酰形式发生横向相分离，导致形成特定的富含磷脂酸的脂质结构域，这些结构域与主体脂质分离，其范围（囊泡中总磷脂酸群体中隔离到该结构域中的磷脂酸）取决于蛋白质。此外，利用蛋白质色氨酸残基作为能量供体以及荧光探针反式-帕里南酸或二苯基己三烯作为受体的荧光能量转移，证实AcChR包含在该结构域中。在相同条件下，其他二肉豆蔻酰磷脂探针（磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰甘油）无法模拟用磷脂酸探针观察到的蛋白质诱导的结构域形成，导致重构囊泡中所有脂质成分理想混合。同样，在没有蛋白质的情况下，所有磷脂探针，包括磷脂酸，都表现出理想混合行为。由于磷脂酸和胆固醇与重构的AcChR的功能调节有关，因此有人提出这种特定的调节作用可能由相关脂质类别的结构域隔离介导。
