72b] Isolation and purification of the outer membrane and inner membrane of liver mitochondria

Abstract

This chapter describes the isolation and purification of the outer membrane and inner membrane of liver mitochondria. The preparations can be satisfactorily checked for cross contamination by two methods. First, the color of the outer membrane pellet is examined by transmitted light. If free of inner membrane, they are clear and light brown in color. In contrast, contaminated outer membrane preparations resemble the inner membrane in being yellow and opalescent. Secondly, the outer membrane fraction can be negatively stained by surface spreading in 3% potassium phosphotungstate (pH 6.8). The inner membrane contains all the oxidative phosphorylation linked cytochromes. Hence, outer membrane preparations can be checked for inner membrane contamination by spectrophotometric or enzymatic assay of cytochrome oxidase. The outer membrane is the site of a NADH2 cytochrome b, reductase. Hence, endoplasmic reticulum contamination is conveniently estimated by assay of NADPH2-cytochrome c reductase.