Lampe P D, Pusey M L, Wei G J, Nelsestuen G L
J Biol Chem. 1984 Aug 10;259(15):9959-64.
The electron microscopic and hydrodynamic properties of factor V and factor Va-vesicle complexes were determined. Images of negatively stained factor V bound to vesicles showed the protein as a relatively large globular domain (9.5 nm diameter) connected to the membrane through a narrow protein region 0.5-3 nm in length. This connecting region was not always visible and was measured as the distance between the globular region and the apparent vesicle edge. Factor V protein alone usually appeared as two connected globular regions of 10.2 and 6.5 nm diameter. The two-domain protein structure appeared consistent with both the image of factor V alone and bound to the membrane. Factor V had no biological activity in a phospholipid-free prothrombinase assay system used. The proteolytically activated form of factor V generated by digestion with thrombin (factor Va) was at least 30,000 times more active. The electron microscopic images of factor Va-vesicle complexes showed a smaller protein that was more closely associated with the vesicle surface than was factor V. The light chain (Mr about 80,000) component of factor Va also bound to the surface of the vesicles and appeared to be largely external to the membrane. Protein-induced hydrodynamic radius changes for the factor V-vesicle and factor Va-vesicle complexes were 12.8 and 6.3 nm, respectively. The images observed in the electron microscope were used to calculate protein-induced radius changes. Comparison of these values with the experimentally determined hydrodynamic radius changes showed approximate agreement for factor Va-membrane complexes. However, the images of factor V-vesicle complexes suggested smaller hydrodynamic radius changes than were actually observed.
测定了因子V和因子Va-囊泡复合物的电子显微镜和流体动力学特性。负染的与囊泡结合的因子V图像显示，该蛋白为一个相对较大的球状结构域（直径9.5 nm），通过一个长度为0.5 - 3 nm的狭窄蛋白区域与膜相连。这个连接区域并非总是可见，其测量值为球状区域与明显的囊泡边缘之间的距离。单独的因子V蛋白通常呈现为两个直径分别为10.2和6.5 nm的相连球状区域。这种双结构域蛋白结构与单独的因子V以及与膜结合的因子V的图像均相符。在无磷脂的凝血酶原酶测定系统中，因子V没有生物活性。经凝血酶消化产生的因子V的蛋白水解激活形式（因子Va）的活性至少高30000倍。因子Va-囊泡复合物的电子显微镜图像显示，有一个比因子V与囊泡表面结合更紧密的较小蛋白。因子Va的轻链（分子量约80000）成分也与囊泡表面结合，且似乎大部分位于膜外部。因子V-囊泡和因子Va-囊泡复合物的蛋白诱导流体动力学半径变化分别为12.8和6.3 nm。利用电子显微镜观察到的图像来计算蛋白诱导的半径变化。将这些值与实验测定的流体动力学半径变化进行比较，结果显示因子Va-膜复合物的结果大致相符。然而，因子V-囊泡复合物的图像显示的流体动力学半径变化比实际观察到的要小。
