Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells

Abstract

A method is described for the rapid separation of mitochondria (plus other particulate components) from the soluble cytoplasm of isolated rat-liver cells. The cells were incubated briefly with a low concentration of digitonin. After rapid centrifugation, the pellet contained more than 90% of the total adenylate kinase and glutamate dehydrogenase activities and the supernatant at least 80% of the lactate dehydrogenase activity. About 60% of total adenine nucleotides in hepatocytes were found in the soluble cytoplasm. The ATP/ADP ratio in the particulate fraction 80 s after exposure to digitonin of hepatocytes metabolizing alanine was 2.0-2.4, and that in the soluble cytoplasm 6-19. In the presence of atractyloside, these values were 3.5-4.4 and 1.3-2.2, respectively.