Kompartimentierte Verteilung von Enzymen in Rattenlebermitochondrien

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Abstract
Mitochondria with broken outer membrane were prepared from isolated rat liver mitochondria by incubation with digitonin according to the method of Schnaitman et al. [2]. Electron microscopic controls of these preparations revealed results similar to those obtained by a second method which is based on rapid freezing of mitochondria in a slightly hypertonic medium: the external mitochondrial membrane is ruptured or is partially stripped off, whereas the inner membrane remains intact and the mitochondrial matrix shows normal electronmicroscopic density.The activities of the following enzymes were determined in mitochondria which were prepared either by the digitonin or by the freezing method: adenylate‐kinase, monoamine oxidase, malate dehydrogenase, TPN‐specific isocitrate dehydrogenase, condensing enzyme, succinate dehydrogenase, glutamate dehydrogenase, β‐hydroxyacyl‐CoA dehydrogenase.The enzymological data prove that only adenylate‐kinase is eluted from the mitochondria when their outer membrane is opened by one or the other method. The extracted activity of adenylate‐kinase is quantitatively recovered in the supernatant.The soluble activities of citric acid cycle enzymes as well as the activities of β‐hydroxyacyl‐CoA dehydrogenase and glutamate dehydrogenase can only be extracted after mechanical disruption of both mitochondrial membranes.Small amounts of soluble citric acid cycle enzymes, of glutamate dehydrogenase and of β‐hydroxyacyl‐CoA dehydrogenase which are found in the supernatant after digitonin treatment of intact mitochondria result from a small portion of completely destroyed mitochondria as proved electromicroscopically and enzymologically by the detection of the structure bound activity of succinate dehydrogenase in the supernatant of digitonin treated mitochondria. Moreover, the activities of succinate dehydrogenase and soluble citric acid cycle enzymes which are present in the supernatant of digitonin treated mitochondria are found in the same proportions as in homogenates of sedimented intact mitochondria.From the data obtained, it is concluded that citric acid cycle enzymes, glutamate dehydrogenase and β‐hydroxyacyl‐CoA dehydrogenase are located within the mitochondrial matrix and are not present within the outer mitochondrial membrane, that is the intracristal space. On the contrary, the total mitochondrial activity of adenylate kinase is present within the intracristal space.Incubations of mitochondria with phospholipase‐A according to the method of Bachmann et al. [8] reveal no difference between the extractability of adenylate‐kinase and citric acid cycle enzymes. It is therefore suggested that Phospholipase‐A causes an opening of the intracristal space as well as of the mitochondrial matrix.

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