Effects of substrates for oxidative phosphorylation and of respiratory inhibitors on the incorporation of amino acids into protein by isolated beef-heart mitochondria

Abstract

T HE incorporation of amino acids into protein by isolated beef-heart mitochondria has been described by Simpson and his co-workers [5] and evidence has been presented that in this system, and in rat-liver mitochondria, the incorporation utilizes energy produced by oxidative phosphorylation [l, 3, 4, 71. We have studied the effects of various respiratory inhibitors on this system, together with the requirements for the substrates of oxidative phosphorylation. Beef-heart mitochondria were prepared as described by L6w and Vallin [2] and incorporation of amino acids into protein was measured after incubation in tris-salts medium (see Table I) at 35” with vigorous shaking. Radioactive amino acids were present at a concentration of 0.5 &/ml, and although most experiments were with l-W-DL-leucine, specific activity 1.5 mC/mM, similar results were obtained with a mixture of radioactive amino acids, specific activity 0.2 mC/mg. After incubation protein samples were prepared and their radioactivity measured as described earlier [7]. In an attempt to assess the level of free radioactive amino acid in the mitochondria, the radioactivity of an acid-soluble extract was measured after incubation in some experiments. This was done by adding 10 vols of cold 0.25 1M sucrose and centrifuging down the mitochondria which were then treated with 5 per cent trichloroacetic acid. After removing the precipitate of protein by centrifugation, a sample of the supernatant was taken for measurement of radioactivity. This would include activated forms of leucine, such as leucyl-adenylate, but would not include decarboxylated derivatives since only position C-l of the leucine was labelled. When incubated under the conditions described, the mitochondria incorporated amino acids into their protein at a constant rate for about 2 hr. In most experiments incubation was for 30 min by which time the specific activity of the protein in the controls was 54.5 cts/min/mg (136 p&/mg). The results of experiments in which various additions were made to the medium are summarized in Table I. Glutamate and succinate reduced the incorporation of amino acids into protein by about 70 per cent. The addition of AMP or ATP alone had little effect, but AMP reduced the inhibition caused by the addition of succinate or glutamate. Table II compares the effect of succinate on the incorporation and on the content of acid-soluble %-leucine in the mitochondria. It should be noted that the quantity of radioactive amino acid in the pool was only of the same order as that incorporated into protein and that when succinate was added to the medium there was a reduction in the level of acid-soluble radioactivity. It is possible that this reduction of radioactivity of