The Biosynthesis of Rat-Liver Cytochrome c. 1. Subcellular Distribution of Cytochrome c

A radioimmunoassay for cytochrome c is described. It has been applied successfully to crude subcellular preparations with a very low content in cytochrome c. Quantitative fractionation of rat liver homogenates by differential centrifugation has shown that, on the basis of the distribution patterns of cytochrome oxidase and cytochrome c, the overwhelming bulk of the cytochrome c recovered in the nuclear and large granule fractions may be assigned to mitochondria. In contrast, the cytochrome c of the microsomal and the supernatant fractions, which amounts to about 6% of the total cytochrome c content of liver, cannot be accounted for only by mitochondrial contamination. Analysis of microsomes by isopycnic centrifugation in sucrose gradient indicates that this subcellular fraction contains three pools of cytochrome c. Part of the microsomal cytochrome c belongs to contaminating mitochondria; another part is redistributed cytochrome c adsorbed to the ribosomes; a third part is associated with small vesicles of low density, enzymically characterized by monoamine oxidase activity, and presumably derived from, or related to, the outer mitochondrial membranes.