AN IMPROVED PROCEDURE FOR PREPARATION OF INNER MEMBRANE VESICLES FROM RAT LIVER MITOCHONDRIA BY TREATMENT WITH DIGITONIN

A method for the preparation of inner membrane vesicles from rat liver mitochondria that represents a substantial improvement over one previously described (Hoppel, C. and Cooper, C., Biochem. J. 107, 367 (1968)) is presented. The inner membrane preparation is further fractionated by differential centrifugation and although all of the fractions are qualitatively similar with regard to enzyme content only the fraction sedimenting between 50-100,000g shows considerable structural uniformity. Electron micrographs obtained by both sectioning and negative staining are presented and the former show that the bounding membrane is trilaminar and that material appears to be present within the vesicles. Certain inner membrane enzymes are recovered in high yields from mitochondria with a large increase in specific activity. The specific activity of succinic dehydrogenase is increased 7.4-fold and the recovery from the mitochondria is 61% while rotenone-sensitive NADH-cytochrome c reductase showed a 12.2-fold increase in specific activity and the recovery was 98%. The specific activity for phosphate uptake with 3-hydroxybutyrate as substrate is approximately 80% of that of the intact mitochondria. The preparation is virtually free of adenylate kinase and nucleoside diphosphokinase and the Pi-ATP and ADP-ATP exchange reactions have the same rates. © 1969.