Protein synthesis in mitochondria. Requirements for the incorporation of radioactive amino acids into mitochondrial protein

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Abstract
Washed rat-liver mitochondria incorporated radioactive amino acids into their protein when incubated aerobically in the presence of an oxidizable substrate, inorganic phosphate, AMP, magnesium ions, diphosphopyridine nucleotide and a full complement of amino acids. The system was slightly stimulated by ribonuclease and did not require cell sap or pH 5 enzyme. The incorporation was not due to microsomal contamination, but depended upon the maintenance of oxidative phosphorylation by the mitochondria. The optimum concentrations of AMP, magnesium ions, a-oxoglutarate, succinate and phosphate required for amino acid incorporation were determined. Replenishment of the medium during incubation considerably improved the duration of incorporation. Fractionation of radioactive mitochondria showed the highest rate of labelling to be in the protein of an insoluble fraction rich in ribonucleic acid and phospho-lipid. The soluble proteins were poorly labelled. Significant bacterial contamination was observed in the final mitochondrial fraction and in the livers originally taken. However, this contamination contributed negligible amounts to the incorporation, at least during incubation for 3 hr.