The Isolation of Morphologically Intact Mitochondria from Rat Liver.

In previous communications it was reported that the respiratory enzyme systems, cytochrome oxidase and succinoxidase, were associated, probably exclusively, with the "large granule" fraction isolated by differential centrifugation either from water homogenates or from saline extracts of rat liver. The identification of the components making up the large granule fraction presented an important and difficult problem. Microscopical examination of the fraction prepared in isotonic saline revealed the presence of spherical bodies ranging between 0.5 to 2 μ in diameter. The corresponding preparation obtained from a water homogenate of liver contained much larger and paler spheres. It was suggested, mainly on the basis of a comparison of the size of the isolated granules with that of known cellular inclusions, that the large granule fraction consisted mostly of mitochondria., It was realized, however, that this evidence was not conclusive proof for the hypothesis that large granules were mitochondria, since the isolated large granules did not possess the generally accepted properties of mitochondria, namely, a rod-like shape and the ability to stain vitally with Janus green.Subsequent investigations showed that isotonic saline solutions were undesirable media for reasons other than the above cytological considerations, in that sodium chloride, as well as certain other electrolytes, caused agglutination of large granules. This agglutination was reflected in the finding that irregular and usually great amounts of succinoxidase activity were sedimented during the removal, by low-speed centrifugation, of nuclei from saline homogenates. When water was used as the medium, no agglutination was visible, and the loss of succinoxidase in the nuclear fraction was much less.