ABSTRACT
Ribosomal subunits were prepared from rat liver and skeletal muscle after incubation with puromycin and treatment at low concentrations of Mg(2+). After isolation the resulting subunits could be recombined and the particles effected the synthesis of polyphenylalanine in the presence of polyuridylic acid. Hybrid particles formed from subunits of liver and muscle respectively were also active. The homogeneity of the isolated subunits was checked by polyacrylamide-gel-agarose electrophoresis. The method is shown to be a reliable and comparatively simple way of preparing active ribosomal subunits from skeletal muscle.
Subject(s)
Liver/cytology , Muscles/cytology , Ribosomes/metabolism , Acrylates , Animals , Carbon Isotopes , Female , Gels , Magnesium , Male , Peptide Biosynthesis , Polymers , Polysaccharides , Puromycin , RNA/analysis , Rats , Ribosomes/analysis , Uracil NucleotidesABSTRACT
The increase in the incorporation of amino acids into protein in vitro by preparations obtained from protein-fed rats as compared with preparations obtained from carbohydrate-fed rats has been described previously. After molecular sieving through Sephadex G-25 of cell-free preparations, the difference in incorporating activity between the two types of rats was diminished in systems containing ATP, phosphoenolpyruvate, pyruvate kinase, GTP, and a mixture of amino acids. When, after molecular sieving, a mitochondrial (15,000 g) supernatant was incubated for 4 min at 35 degrees C the polysomal pattern of the preparations was unchanged. In the presence of ATP, phosphoenolpyruvate, and pyruvate kinase the polysomal incorporating activity was low and the polysomal pattern was only slightly changed. Addition of GTP increased the activity markedly, and a more pronounced activity was observed when a mixture of amino acids was added as well. As the amino acid incorporation ability increased, monosomes were formed from the polyribosomes. The activity of the polyribosomes was severalfold higher than that of non-Sephadex-treated preparations, indicating an activation of polysomal aggregates which under the usually applied conditions of incubation and prior to molecular sieving show little or insignificant activity. It was possible to activate polyribosomes from carbohydrate-fed and protein-fed rats to almost the same extent.