Moreau H, Pieroni G, Jolivet-Reynaud C, Alouf J E, Verger R
Centre de Biochimie et Biologie Moléculaire du CNRS, Marseille, France.
Biochemistry. 1988 Apr 5;27(7):2319-23. doi: 10.1021/bi00407a012.
The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.
利用各种磷脂单分子层研究了产气荚膜梭菌纯化的磷脂酶C（α毒素）的酶活性。采用两步反应。磷脂酶C对不溶性卵磷脂膜进行酶促水解，生成1,2 - 二酰基甘油和水溶性磷酸胆碱，并与胰脂肪酶的作用相结合，从而产生脂肪酸和2 - 单酰基甘油，它们会迅速从界面解吸。通过这种新方法，可以以磷脂单分子层为底物，对磷脂酶C催化的反应进行连续且准确的动力学测量。因此，可以避免在先前研究中必要时使用放射性标记底物，并且脂质膜中二酰基甘油积累所导致的困难也降至最低。当使用磷脂酰乙醇胺、磷脂酰丝氨酸或磷脂酰甘油膜作为底物时，未检测到水解现象。通过膜转移技术发现，Ca2+和Zn2+离子发挥着特定且关键的作用。本研究首次明确表明，Ca2+对于酶与脂质膜的结合至关重要，而Zn2+则具体参与底物的催化水解过程。
