Most of the protein biosynthesis occurring in ratliver cells takes place in the microsomes (Keller, Zamecnik & Loftfield, 1954), but some amino acid incorporation into protein also occurs in nuclei (Rees & Rowland, 1961) and in mitochondria (McLean, Cohn, Brandt & Simpson, 1958; Roodyn, Reis & Work, 1961). The amount of amino acid incorporation into protein which occurs in isolated mitochondria is very small compared with that found with microsomes (McLean et al. 1958), but nevertheless the mitochondrial system is of special interest because of the importance of the mitochondria in the general metabolism of the cell. Since only a slight amount ofincorporation occurs in mitochondria it seemed desirable to confirm the earlier reports on mitochondrial amino acid incorporation (McLean et al. 1958) and to establish that the mitochondria themselves are responsible for the incorporation of amino acids into protein. The optimum conditions for the incorporation of amino acids into the proteins of isolated rat-liver mitochondria and the effect of variation of the conditions on the rate of incorporation were also investigated. METHODS Animal&. Female albino rats, weighing about 200 g., were used in the experiments. Radioactive comqounds. DL-[1-14C]Leucine and generally (G) labelled L-[G-14C]leucine were used, and were obtained from The Radiochemical Centre, Amersham, Bucks. Material&. Free acid AMP, the sodium salts of ATP, ADP, and AMP, tris and NAD were obtained from the Sigma Chemical Co. Ribonuclease (recrystallized five times) was obtained from both the Sigma Chemical Co. and Nutritional Biochemicals Corp. Phosphocreatine was prepared by the method of Ennor & Stocken (1948), and phosphocreatine kinase by the method of Kuby, Noda & Lardy (1954). Bovine serum albumin prepared by Armour Pharmaceuticals was used and rat serum albumin was prepared by the method of Korner & Debro (1956). All other reagents were of AnalaR grade, except amino acids, which were obtained from Roche Products Ltd. Sucrose was further purified by passing solutions through a column of Amberlite MB-1 mixed-bed ion-exchange resin, and the solutions were then boiled to remove dissolved carbon dioxide and to reduce bacterial contamination. All solutions were made in glass-distilled water. Preparation of mitochomdria. The rats were killed by decapitation and were bled. The liver was rapidly removed and transferred to ice-cold 0 25M-sucrose, in which it was cut into small pieces with scissors. All subsequent operations in the preparation of the mitochondria were carried out between O0 and 20. The pieces of liver were blotted, the volumes measured by displacement in fresh 0 25M-sucrose and the liver was homogenized in 0 25M-sucrose, a handoperated homogenizer of the type described by Dounce, Witter, Monty, Pate & Cottone (1955), which was kept in an ice bath, being used. Homogenization was carried out in three stages as described by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955). The liver was first homogenized in 2-5 vol. of sucrose, with three ...
