Incorporation of amino acids into the protein of isolated mitochondria. A search for optimum conditions and a relationship to oxidative phosphorylation

Abstract

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 ...

Incorporation in vitro of [14C]Leucine into the Mitochondrial Protein of Lucilia cuprina

“…It is tempting to interpret this effect as further support for the antagonistic effect of these ions on adult differentiation in insects. However, a similar effect of varying potassium/sodium ratio has been observed in mitochondrial protein synthesis in rat liver [49] ;…”

Intramitochondrial Fibers With Dna Characteristics

“…Among these we might list (a) whether such fibers are a regular feature of most or all mitochondria, (b) whether there are quantitative variations in the fibers that are related to cell division, differentiation, or neoplastic transformations, I (c) whether there are recognizable differences between cytoplasmic and nuclear DNA's with regard to composition or rate of precursor uptake, (d) what the significance is of the frequently observed nuclearmitochondrial associations (1, 7, 22, 51) which appear to be more frequent during early developmental stages than later (51), (e) what relationship mitochondrial D N A may have to mitochondrial protein synthesis (64,75), and (f) what the relation is between mitochondrial DNA and the reported presence in mitochondria of alkaline DNase (2) as well as some of the enzymes involved in nucleic acid synthesis (34,57).…”

Initial stages in the incorporation of amino acids into protein in rat-liver mitochondria

“…At the end of the incubation period, the reaction was stopped by the addition of 0-5N-HC104, and the protein samples were prepared and their radioactivity was measured as described by Truman & Korner (1962).…”

“…This rules out the possibility that the stimulatory effect of the mitochondrial extract is due to its contamination by cell sap. The contamination of the mitochondria by other cell fractions is very slight when the mitochondria are prepared and washed in the 15-9 manner employed in these experiments (Truman & Korner, 1962). Since cell sap can be replaced by the mitochondrial extract in the ribosome system, it seems possible that the mitochondrial extract is able to activate the amino acids and give rise to a soluble RNA charged with amino acids, which is then utilized by the ribosomes for incorporation into protein, but no direct evidence for the presence of a transfer RNA in mitochondria has been obtained from the work reported above.…”

“…Mitochondria and cell sap were prepared by the method described by Truman & Korner (1962). After isolation the mitochondria were washed by centrifuging through a layer of 0-5M-sucrose as described by Truman & Korner (1962).…”