Megli F M, Selvaggi M, Liemann S, Quagliariello E, Huber R
Centro di Studio sui Mitocondri e Metabolismo Energetico, Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Italy.
Biochemistry. 1998 Jul 21;37(29):10540-6. doi: 10.1021/bi9801255.
The fluidity of the hydrophobic interior of phospholipid vesicles after calcium-dependent binding of human annexin V (AVH) was studied using EPR spectroscopy. Vesicles (SUVs) composed of PC or PE and an acidic phospholipid (alternatively PS, PA, or CL) were probed at different bilayer depths by either phosphatidylcholine, or the accompanying acidic phospholipid, bearing a spin label probe at position C-5, C-12, or C-16 of the sn-2 acyl chain. Alternatively, the vesicle surface was probed with a polar head spin labeled PE (PESL). The EPR spectra of annexin-bound bilayer domain(s) were obtained by computer spectral subtraction. The order parameter values (S) from the resulting difference spectra revealed that the bilayer hydrophobic interior has a greatly altered fluidity gradient, with an increased rigidity up to the C-12 position. Thereafter, the rigidification progressively vanished. The effect is not linked to the phospholipid class, since all the acidic phospholipid spectra, as well as phosphatidylcholine, shared the same sensitivity to the bound protein. The observed membrane rigidification appears to parallel the "crystallizing" tendency of vesicle-bound annexin V, but may not be involved in the calcium channeling activity of this protein.
利用电子顺磁共振波谱法研究了人膜联蛋白V（AVH）钙依赖性结合后磷脂囊泡疏水内部的流动性。由PC或PE与酸性磷脂（分别为PS、PA或CL）组成的小单层囊泡（SUVs），通过在sn-2酰基链的C-5、C-12或C-16位置带有自旋标记探针的磷脂酰胆碱或伴随的酸性磷脂，在不同的双层深度进行探测。或者，用极性头部自旋标记的PE（PESL）探测囊泡表面。通过计算机光谱减法获得膜联蛋白结合的双层结构域的电子顺磁共振谱。所得差异谱的序参量值（S）表明，双层疏水内部的流动性梯度发生了很大变化，直至C-12位置刚性增加。此后，刚性化逐渐消失。这种效应与磷脂类别无关，因为所有酸性磷脂谱以及磷脂酰胆碱对结合蛋白具有相同的敏感性。观察到的膜刚性化似乎与囊泡结合的膜联蛋白V的“结晶”趋势平行，但可能不参与该蛋白的钙通道活性。
