Jergil B, Sommarin M
Biochim Biophys Acta. 1983 Jul 5;758(1):10-6. doi: 10.1016/0304-4165(83)90003-x.
A method has been developed to measure the Ca2+- and phospholipid-dependent protein kinase in membrane fractions. The method is based on the fact that this enzyme is resistant to comparatively high concentrations of octylglycoside. Rat liver membranes were treated with octylglycoside and the phosphate incorporation from [gamma-32P]ATP was measured in the presence of histone H1. The enzyme activity was determined as the difference between the incorporation obtained after addition of Ca2+ and phosphatidylserine and the incorporation obtained without these additions but with EGTA. The endogenous incorporation of phosphate to membrane components was constant under these incubation conditions. The conditions for determination of the membrane-bound enzyme were optimized. Two thirds of the total enzymic activity was attached to membranes in rat liver cells. A highly purified plasma membrane preparation had the highest specific activity, while most of the bound enzyme was found in microsomes, and only traces were found in mitochondria.
已开发出一种测量膜组分中钙和磷脂依赖性蛋白激酶的方法。该方法基于这样一个事实，即这种酶对相对高浓度的辛基糖苷具有抗性。用辛基糖苷处理大鼠肝膜，并在组蛋白H1存在的情况下测量来自[γ-32P]ATP的磷酸掺入。酶活性通过添加Ca2+和磷脂酰丝氨酸后获得的掺入量与未添加这些物质但添加EGTA时获得的掺入量之间的差异来确定。在这些孵育条件下，膜组分中磷酸盐的内源性掺入是恒定的。优化了膜结合酶的测定条件。大鼠肝细胞中总酶活性的三分之二附着于膜上。高度纯化的质膜制剂具有最高的比活性，而大部分结合酶存在于微粒体中，线粒体中仅发现微量。
