Basáñez G, Nieva J L, Goñi F M, Alonso A
Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Biochemistry. 1996 Dec 3;35(48):15183-7. doi: 10.1021/bi9616561.
When phospholipase C is added to a suspension of large unilamellar vesicles of egg phosphatidylcholine, maximal rates of hydrolysis occur only after a latency period. No lag period is seen when the substrate is in the form of small (sonicated) vesicles, or of short-chain phosphatidylcholine monomers. For a given vesicle concentration, the lag time may vary as a function of Ca2+, enzyme concentration, or temperature, but activation occurs at a fixed molar fraction of diacylglycerol produced. Lag times decrease gradually with vesicle size, and also with the amount of diacylglycerol present in the bilayers when it is mixed with phospholipid prior to enzyme addition. Parallel recordings of enzyme activity and suspension turbidity reveal that in all cases the latency period ends concomitantly with the start of a process of vesicle aggregation. Both the lag time and the amount of diacylglycerol formed before activation decrease with vesicle concentration, suggesting that enzyme activation is somehow related to vesicle aggregation. The latency period of phospholipase C may be explained in terms of a hypothesis according to which (a) full enzyme activity requires the presence of membrane surface irregularities or defects, (b) the diacylglycerol generated in the lag phase produces some kind of phase separation, with the formation of diacylglycerol-rich "patches" or domains, (c) vesicles aggregate through contacts between those patches, and (d) aggregation causes (and/or increases, and/or stabilizes) the surface inhomogeneities that allow fast enzyme activity. These data and suggestions may be relevant to the process of model membrane fusion promoted by phospholipase C.
当将磷脂酶C添加到卵磷脂酰胆碱的大单层囊泡悬浮液中时，只有在一段延迟期后才会出现最大水解速率。当底物为小（超声处理的）囊泡或短链磷脂酰胆碱单体形式时，未见延迟期。对于给定的囊泡浓度，延迟时间可能随Ca2+、酶浓度或温度而变化，但激活发生在产生的二酰基甘油的固定摩尔分数时。延迟时间随着囊泡大小逐渐减小，并且在添加酶之前与双层中存在的二酰基甘油量混合时也会减小。酶活性和悬浮液浊度的平行记录表明，在所有情况下，延迟期都与囊泡聚集过程的开始同时结束。延迟时间和激活前形成的二酰基甘油量均随囊泡浓度降低，这表明酶激活在某种程度上与囊泡聚集有关。磷脂酶C的延迟期可以根据以下假设来解释：（a）完整的酶活性需要膜表面存在不规则性或缺陷；（b）在延迟期产生的二酰基甘油产生某种相分离，形成富含二酰基甘油的“斑块”或结构域；（c）囊泡通过这些斑块之间的接触聚集；（d）聚集导致（和/或增加和/或稳定）允许快速酶活性的表面不均匀性。这些数据和建议可能与磷脂酶C促进的模型膜融合过程相关。
