The Isolation and Analysis of a Mitochondrial Membrane Fraction

There exists in the literature indication that some mitochondrial enzymes, particularly succinoxidase and cytochrome oxidase, are part of the mitochondrial structure and thus are insoluble when the mitochondria are disrupted. I t has further been supposed in particular by Cleland and Slater (1) that this insoluble fraction consists of mitochondrial membranes. However, no direct test of the membrane hypothesis has been reported. The present note describes briefly findings which strongly support the idea that certain insoluble enzymes are structurally linked to membranes of mitochondria. Rat liver mitochondria were isolated in 0.44 • sucrose and treated in suspension with 0.3 per cent deoxycholate. This treatment resulted in the solubilization of 80 to 90 per cent of the protein N. Of various fractions obtained in the centrifuge and examined in the electron microscope by sectioning techniques, the final pellet obtained at 105,000 g is pertinent here. Fig. 1 shows this fraction to consist mostly of systems of vesicles bounded by single or multiple layers of membranes. The dimensions of these systems are in the order of mitochondrial dimensions. We were not clear as to the source of these elements since they may have arisen not only from mitochondrial membranes, but possibly also from interactions between deoxycholate, the various non-membranous components present, and osmium tetroxide. They might also represent microsomal contamination. In order to follow the interaction of mitochondria with deoxycholate, a pellet of isolated mitochondria was layered in the centrifuge tube with 0.3 per cent deoxycholate in sucrose, allowed to stand for a while, and then fixed and embedded. The pellet was then sectioned transversely through the depth of the pellet, and mitochondria in various stages of dissolution from the bottom to the top of the pellet could be followed. Fig. 2 shows, near the bottom of the pellet, mostly intact mitochondria plus a number of swollen ones. Microsomal contamination present in this particular preparation can be identiffed by the granules adhering to the membranes. In the middle of the pellet layered with deoxycholate are found numerous much swollen mitochondria (Fig. 3). These still contain most of their matrix. In Fig. 4 is shown the appearance of mitochondria at the top of the pellet. These elements have lost most of their matrix, and are somewhat smaller than those encountered in 379