BRAIN HEXOKINASE - PREPARATION OF INNER AND OUTER MITOCHONDRIAL MEMBRANES

Three different methods were used to prepare inner and outer membranes of brain mitochondria. The digitonin and phospholipase A methods were relatively unsuccessful. In the former case, the outer membrane marker enzyme, monoamine oxidase, was not released into the outer membrane fraction over a range of concentrations from 0.11 to 0.66 mg of digitonin per mg of mitochondrial protein. In the latter case, 60% of the outer membrane marker rotenone-insensitive reduced diphosphopyridine nucleotide-cytochrome c reductase was lost during the incubation with phospholipase A and the portion of the enzyme which was recovered remained in the inner membrane fraction even after prolonged digestion. On the other hand, the combined swelling, shrinking, and sonication method gave more satisfactory results. The sonication procedure resulted in a 1.5-2-fold activation of hexokinase activity which could be duplicated by diluting the mitochondrial suspension in 1 % bovine serum albumin. Biochemical assay revealed that between 70 and 75 % of the outer membranes were removed. Approximately 63 % of the total hexokinase activity was associated with the outer membrane fraction while 12% of the activity was solubilized during the process. The increase in the specific activity of hexokinase was the same as that for the outer membrane marker, rotenone insensitive reduced diphosphopyridine nucleotide-cytochrome c reductase. Biochemical and electron microscopic data are presented which indicate that rotenone insensitive reduced diphosphopyridine nucleotide-cytochrome c reductase is not a microsomal contaminant. The results are discussed with reference to the problems and limitations of enzyme localization. © 1969, American Chemical Society. All rights reserved.