Ravindranath V, Anandatheerthavarada H K
Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore, India.
Anal Biochem. 1990 Jun;187(2):310-3. doi: 10.1016/0003-2697(90)90461-h.
Microsomes have been conventionally prepared by centrifugation of the postmitochondrial supernatant at 100,000g using an ultracentrifuge. Liver microsomes have been prepared by low speed centrifugation following sedimentation of the microsomal membranes in the presence of calcium ions. However, this method has not been suitable for the preparation of microsomes from extrahepatic tissues as it often results in the loss of cytochrome P450 activity. Brain microsomes prepared by the traditional calcium aggregation method results in the loss of cytochrome P450. We now describe a modification of the calcium aggregation method for the rapid preparation of rat and mouse brain microsomes. This involves the incorporation of glycerol, dithiothreitol, and EDTA in the preparation of microsomes. Such preparations do not differ in their cytochrome P450 content and associated monooxygenase activity from the traditionally prepared microsomes using ultracentrifugation. Electron microscopic analysis also does not reveal any differences between the microsomes prepared by the two methods. As brain microsomes are relatively unstable and are obtained in low yields, rapid isolation of large quantities of microsomes, possible using the present method, should be very useful.
传统上，微粒体是通过使用超速离心机对线粒体后上清液在100,000g下进行离心制备的。肝微粒体是在钙离子存在下，使微粒体膜沉降后通过低速离心制备的。然而，这种方法并不适用于肝外组织微粒体的制备，因为它常常导致细胞色素P450活性的丧失。用传统的钙聚集法制备脑微粒体会导致细胞色素P450的丧失。我们现在描述一种对钙聚集法的改进，用于快速制备大鼠和小鼠脑微粒体。这涉及在微粒体制备过程中加入甘油、二硫苏糖醇和乙二胺四乙酸。这样制备的微粒体在细胞色素P450含量和相关单加氧酶活性方面与使用超速离心法传统制备的微粒体并无差异。电子显微镜分析也未揭示这两种方法制备的微粒体之间存在任何差异。由于脑微粒体相对不稳定且产量较低，使用本方法有可能快速分离大量微粒体，这应该会非常有用。
