Composition of Mitochondrial Preparations in Relation to Intracellular Localization of Azoprotein in the Mouse. ∗

Abstract

Conclusions and Summary

These studies substantiate previous reports that a relatively large proportion of injected labeled protein is found in the mitochondrial fraction of homogenized liver(8). Failure to demonstrate in this fraction the azoprotein which was added to the whole liver homogenate indicates that an in vivo process is involved and that simple adsorption of the azoprotein by mitochondria does not occur. Results of microscopic study of wafers, made by centrifuging mitochondrial fractions, indicate that the intracytoplasmic granules of azoprotein seen in sections from injected animals(1-3,5) are found predominantly in such mitochondrial fractions. However, the facts that injected India ink particles occur in a similar proportion in such fractions, that the azoprotein granules do not take various mitochondrial stains, and that other impurities can be demonstrated in such preparations, indicate that such mitochondrial fractions do not consist only of mitochondria. This conclusion has also been expressed by previous workers, and such fractions have been subfractionated(16). The differences in re-suits with the various mitochondrial stains not only emphasize the inhomogeneity of these preparations, but indicate that such stains do not provide adequate characterization of mitochondria. It is apparent that much more work must be done on the composition of mitochondrial fractions prepared in this way, and on the significance of the results of mitochondrial stains, before cytological and biochemical data on such preparations can be equated with assurance. Since intraeytoplasmic azo-protein granules apparently differ in some respects, both physically and histochemically, from other granules in these preparations which fulfill more of the criteria for mitochondria, the occurrence of labeled proteins in mitochondrial fractions does not as yet seem to justify the conclusion that these proteins are attached to mitochondria, or that mitochondria are the site of antibody formation.

The results of colorimetric and microscopic studies on cell fractions prepared after intravenous administration of variousapoproteins to mice are presented. The azoprotein granules seen intracellularly in the tissues of such animals are present predominantly in the mitochondrial fraction. Evidence that such granules are not labeled mitochondria is presented, and the significance of the variation an the results of different staining technics on mitochondrial fractions is discussed with regard to the homogeneity of these preparations.