Ruiz-Argüello M B, Goñi F M, Alonso A
Grupo Biomembranas (Unidad Asociada al C.S.I.C.), Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Biochemistry. 1998 Aug 18;37(33):11621-8. doi: 10.1021/bi980615x.
Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesicles. Phosphatidylcholine, phosphatidylethanolamine (also a substrate for the enzyme), sphingomyelin, and cholesterol have been mixed in various proportions, in binary, ternary, and quaternary mixtures. A lag period, followed by a burst of enzyme activity, has been found in all cases. The activity burst was always accompanied by an increase in turbidity of the vesicle suspension. Varying lipid compositions while keeping constant all the other parameters leads to a range of lag times extending over 2 orders of magnitude (from 0.13 to 38.0 min), and a similar variability is found in maximal enzyme rates (from 0.40 to 55.9 min-1). Meanwhile, the proportion of substrate that is hydrolyzed during the lag period remains relatively constant at 0.10% moles of total lipid, in agreement with the idea that enzyme activation is linked to vesicle aggregation through diacylglycerol-rich patches. Phosphatidylethanolamine and cholesterol enhance the enzyme activity in a dose-dependent way: they reduce the lag times and increase the maximal rates. The opposite is true of sphingomyelin. These lipids exert each its own peculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity can be to some extent predicted from its composition. Phospholipase C activity is not directly influenced by the formation of nonlamellar structures. However, the presence of lipids with a tendency to form nonlamellar phases, such as phosphatidylethanolamine or cholesterol, stimulates the enzyme even under conditions at which purely lamellar phases exist. Conversely sphingomyelin, a well-known stabilizer of the lamellar phase, inhibits the enzyme. Thus phospholipase C appears to be regulated by the overall geometry and composition of the bilayer.
蜡样芽孢杆菌的磷脂酰胆碱磷脂酶C（EC 3.1.4.3）已采用大单层囊泡形式的底物进行了测定。磷脂酰胆碱、磷脂酰乙醇胺（也是该酶的底物）、鞘磷脂和胆固醇已按不同比例混合，形成二元、三元和四元混合物。在所有情况下都发现了一个延迟期，随后是酶活性的爆发。活性爆发总是伴随着囊泡悬浮液浊度的增加。在保持所有其他参数不变的情况下改变脂质组成，会导致延迟时间范围跨越2个数量级（从0.13分钟到38.0分钟），并且在最大酶速率方面也发现了类似的变异性（从0.40分钟-1到55.9分钟-1）。同时，在延迟期内水解的底物比例在总脂质的0.10%摩尔相对恒定，这与酶激活通过富含二酰基甘油的斑块与囊泡聚集相关的观点一致。磷脂酰乙醇胺和胆固醇以剂量依赖的方式增强酶活性：它们减少延迟时间并增加最大速率。鞘磷脂则相反。这些脂质单独或组合发挥各自独特的正或负效应，因此给定混合物对酶活性的敏感性在一定程度上可以从其组成来预测。磷脂酶C活性不受非层状结构形成的直接影响。然而，即使在纯层状相存在的条件下，倾向于形成非层状相的脂质（如磷脂酰乙醇胺或胆固醇）的存在也会刺激该酶。相反，鞘磷脂是层状相的著名稳定剂，会抑制该酶。因此，磷脂酶C似乎受双层的整体几何形状和组成调节。
