Isolation of mitochondria, lysosomes, and microsomes from the rat ventral prostate with a note on inverted microsomal vesicles

Abstract

A procedure is presented for the isolation of lysosomes, mitochondria, and microsomes from the rat ventral prostate with relatively good yield. Homogenization was performed with a Polytron homogenizer or in combination with the Potter-Elvehjem device. Reasonably pure mitochondria and lysosomes could only be obtained using a Metrizamide gradient, whereas it was possible to prepare pure microsomal fractions by differential centrifugation in sucrose. The purity of the lysosomes and mitochondria was 90 and 85%, respectively, as judged by the presence of different marker enzymes. These findings were confirmed by ultrastructural analyses. Electron micrographs of the isolated lysosomes showed intact lysosomes surrounded by a single membrane. The lysosomes contained intramatrical vesicles with lipid-like material. Vesicles derived from the endoplasmic reticulum in the microsomal fraction ranged from 70 to 90% depending on the centrifugal force used to sediment the mitochondrial fraction. Electron micrographs of the microsomal fraction showed that about 40% of the vesicles were inverted and turned “inside-out”, i.e., having their ribosomes attached to the inside of the vesicles. By fractionation of ethylenediaminetetraacetate treated microsomes on a sucrose gradient a partially purified fraction was isolated which consisted of 65% of inverted microsomes.