Malmqvist T, Malmqvist M, Möllby R
Acta Pathol Microbiol Scand B. 1981 Oct;89(5):357-61. doi: 10.1111/j.1699-0463.1981.tb00200_89b.x.
Through hydrophobic interaction, sphingomyelin was adsorbed to agarose beads containing octyl groups by a stepwise dilution procedure. This immobilized lipid was used as a substrate for three bacterial phospholipases C (E.C. 3.1.4.3.). The degradation with time of this substrate showed two different fractions of the substrate according to hydrolysing velocity in the early part of the time-curve when phospholipases C from Bacillus cereus and Clostridium perfringens were used. The early fractions could be predigested by the enzymes, a procedure which resulted in linear time-curves. The corresponding early part of the time-curve for phospholipase C from Staphylococcus aureus was linear, indicating a comparatively large early fraction of the substrate for this enzyme. The stock gel of the immobilized lipid substrate could be stored for months. It was easily and reproducibly handled as a water suspension. After enzymatic hydrolysis the substrate was rapidly separated from enzyme and product by filtration. The enzyme assay presented thus represents a convenient way to avoid the difficulties connected with the use of temporary sonicated suspensions as substrate for bacterial phospholipases C.
通过疏水相互作用，鞘磷脂通过逐步稀释程序吸附到含有辛基的琼脂糖珠上。这种固定化脂质被用作三种细菌磷脂酶C（E.C. 3.1.4.3.）的底物。当使用蜡状芽孢杆菌和产气荚膜梭菌的磷脂酶C时，根据时间曲线早期的水解速度，该底物随时间的降解显示出两种不同的底物部分。早期部分可以被酶预先消化，这一过程导致时间曲线呈线性。金黄色葡萄球菌磷脂酶C时间曲线的相应早期部分是线性的，表明该酶的底物早期部分相对较大。固定化脂质底物的储备凝胶可以储存数月。它作为水悬浮液易于操作且可重复。酶促水解后，通过过滤可迅速将底物与酶和产物分离。因此，所呈现的酶分析代表了一种方便的方法，可以避免使用临时超声处理的悬浮液作为细菌磷脂酶C底物所带来的困难。
