NITROGEN METABOLISM IN P. GRISEOFULVUM 289 this fraction. Incorporation into glutamic acid was sufficient to account for the synthesis of at least 85% of the total mycelial ao-amino nitrogen content. The redistribution of label observed after the exhaustion of exogenous nitrogen confirmed previous non-isotopic evidence that a rapid turnover of insoluble nitrogen, involving breakdown to the amino acid level, occurred during nitrogen starvation. Extracellular organic nitrogen released after exhaustion of ammonia arose from the breakdown of insoluble mycelial material. Thanks are due to Mr ID. H. W. Scott for skilled technical assistance in the work describecL in this and the two preceding papers. I am also grateful to Mr R. G. Harrison for carrying out determinations with the mass spectrometer, and to Mr A. F. Henson for help in interpreting the isotopic data.

1.A cell-free system from mouse Krebs I1 ascites cells is described which responds by increased amino acid incorporation into protein on addition of encephalomyocarditis virus (EMC-RNA). RNA from other sources does not produce this response.2 . The stimulation by EMC-RNA occurs over a narrow range of Mg2f concentrations and is maximal a t 5mM, which is optimal for the endogenous incorporation and lower than that required in the presence of poly(U).3. The EMC-RNA-directed product is distinguished from the endogenous products by its size and composition.4. After an initial lag of 4 min, during which the nascent chains are too short to be acidinsoluble, EMC-RNA-directed protein synthesis is active for more than 1 h.5. EMC-RNA-directed synthesis is particularly sensitive to the inhibitors cycloheximide and dextran sulphate. Using the latter, it has been shown that the initiation of protein synthesis is completed during the first 15 min of incubation.6. Only 20-3001, of the product of the cell-free system, whether endogenous or EMC-RNAdirected, is released from the ribosomes. Despite the size of the viral RNA, it did not appear to promote the formation of exceptionally large polysomes.7. It is concluded that the EMC-RNA is acting as a message for viral proteins in the ascites cell-free system. The demonstration that bacteriophage RNA can act as a messenger RNA in cell-free preparations from Escherichia coli [l] has greatly facilitated the study of several aspects of protein biospthesis in bacteria. We wished to extend this approach to mammalian systems using a homologous viral RNA. It is known that the RNA extracted from encephalomyocarditis virus (EMC) [2,3] and from several other mammalian viruses [4-61 is capable of stimulating amino acid incorporation when added to extracts of certain mammalian or avian cells and we have used the EMClKrebs I1 ascites cell system in this work. We report here the development of a highly sensitive cell-free preparation from mouse Krebs I1 ascites cells and describe some of the characteristics of the stimulation of protein synthesis elicited by encephalomyocarditis virus RNA (EMC-RNA). The fidelity of this system has recently been established by the demonstration that the product formed Unusual Abbreviations. EMC, encephalomyocarditis; Guo-5'-P2-CH,-P, 5'-guanylyl-methylenediphosphate; poly-(U), polyuridylic acid; S-30, the 30000 x g supernatant.Enzymes. Creatine phosphokinase (EC 2.7.3.2); pancreatic ribonuclease (EC 2.7.7.16).under the direction of EMC-RNA corresponds to material present in EMC-infected cells [7].

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.

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1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg(2+) ions. The degradation in the absence of Mg(2+) ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2.4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na(+) and K(+) ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.

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 ...