Preparation of brain microsomes with cytochrome P450 activity using calcium aggregation method

Abstract

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.