Clague M J, Schoch C, Zech L, Blumenthal R
Section of Membrane Structure and Function, LMMB, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1990 Feb 6;29(5):1303-8. doi: 10.1021/bi00457a028.
To identify the initial stages of membrane fusion induced by vesicular stomatitis virus, we performed stopped-flow kinetic measurement with fluorescently labeled virus attached to human erythrocyte ghosts that contained symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically using an assay based on mixing of the lipid fluorophore octadecylrhodamine. At 37 degrees C and pH values near the threshold for fusion, a lag phase of 2 s was observed. The lag time decreased steeply as the pH decreased, while the initial rate of fusion showed the reverse functional dependence on pH. The observed rapid fluorescence changes resulted from fusion of virus bound to the target, and the time lags were not due to association-dissociation reactions between virus and target. For a given pH value, the temperature dependence of the lag time was similar to that of the initial rate of fusion. The results were fitted to a multistate model similar to that resulting from ion channel gating kinetics. The model allows testing of hypotheses concerning the role of cooperativity and conformational changes in viral spike glycoprotein-mediated membrane fusion.
为了确定水泡性口炎病毒诱导的膜融合的初始阶段，我们对附着在含有对称双层磷脂分布的人红细胞血影上的荧光标记病毒进行了停流动力学测量。使用基于脂质荧光团十八烷基罗丹明混合的测定法，通过荧光分光光度法监测融合。在37℃和接近融合阈值的pH值下，观察到2秒的延迟期。随着pH值降低，延迟时间急剧减少，而融合的初始速率则显示出对pH的反向功能依赖性。观察到的快速荧光变化是由于病毒与靶标的融合引起的，时间延迟并非由于病毒与靶标之间的缔合 - 解离反应。对于给定的pH值，延迟时间的温度依赖性与融合初始速率的温度依赖性相似。结果拟合到一个类似于离子通道门控动力学产生的多状态模型。该模型允许测试关于协同性和构象变化在病毒刺突糖蛋白介导的膜融合中的作用的假设。
