guanosine-triphosphate and Carcinoma--Krebs-2

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.

Topics: Adenosine Triphosphate; Animals; Ascitic Fluid; Carcinoma, Krebs 2; Casein Kinases; Cytoplasm; Guanosine Triphosphate; Mice; Phosphoproteins; Phosphorylation; Protein Kinases; Ribosomal Proteins

Topics: Animals; Carcinoma, Krebs 2; Cell Line; Encephalomyocarditis virus; Eukaryotic Initiation Factor-2; Guanosine Triphosphate; Magnesium; Mice; Peptide Initiation Factors; Proteins; Ribosomes; RNA, Transfer, Amino Acyl

A factor having activity similar to that described in other systems for the eukaryotic elongation factor eEF-Ts was isolated from the heavy, aggregate form of eEF-TH (formally named EF-1H). This protein has a molecular weight of 52000 under native conditions and of 25500 under denaturing conditions. It has been shown to stimulate eEF-Tu-dependent aminoacyl-tRNA binding to ribosomes and therefore eEF-Tu/eEF-G-dependent polyphenylalanine synthesis by ribosomes and was found to stimulate GDP-GTP exchange in eEF-Tu . GDP complexes. In the course of this work, it was also demonstrated that the removal of deacylated tRNA from the ribosome is a GTP-dependent process. This report, therefore, adds further support to the concept that a third elongation factor, eEF-Ts, may be common to all systems in the eukaryotic domain.

Topics: Animals; Carcinoma, Krebs 2; Cells, Cultured; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Mice; Molecular Weight; Peptide Biosynthesis; Peptide Elongation Factors; Phenylalanine; Poly U; Ribosomes; RNA, Transfer

Topics: Animals; Carcinoma, Krebs 2; Guanosine Triphosphate; Kinetics; Magnesium; Methionine; Mice; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Rabbits; Reticulocytes; Ribonucleotides; Ribosomes; RNA, Transfer; Saccharomyces cerevisiae

By means of affinity chromatography of lysates from Krebs II ascites cells and rabbit reticulocytes on Sepharose-heparin an active fraction of initiation factors has been obtained. The fraction is eluted from the column at 350 mM KCl using a linear gradient and displays a number of activities, i.e. binding of Met-tRNAfMet to form a ternary complex with GTP; transferring this complex to 40-S subunits in an A-U-G-independent step and finally coupling of the 40-S initiation complex to the 60-S subunit, a reaction which is completely A-U-G-dependent. Moreover, MettRNA is bound into the P-site as is indicated by its puromycin sensitivity. The method is very suitable for large-scale preparation. Further purification and characterization of the factors have been carried out on DEAE-cellulose and phosphocellulose columns. Evidence is presented that the polysomes present in a lysate that has been passed through the Sepharose-heparin column can only complete their nascent chains, initiation of new polypeptides is completely dependent on addition of initiation factors.

Topics: Animals; Binding Sites; Carcinoma, Krebs 2; Chromatography, Affinity; Guanosine Triphosphate; Heparin; Kinetics; Methionine; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Potassium Chloride; Puromycin; Rabbits; Reticulocytes; Ribosomes; RNA, Transfer

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.

Topics: Animals; Binding Sites; Carcinoma, Krebs 2; Encephalomyocarditis virus; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Magnesium; Methionine; Mice; Osmolar Concentration; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Potassium Chloride; Protein Binding; Protein Biosynthesis; Ribosomes; RNA, Transfer; RNA, Viral

An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments in which mixtures of [3H]EF-1, ribosomes from Drebs II ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a tRNA in their P-site. On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound.

Topics: Animals; Carcinoma, Krebs 2; Guanosine Triphosphate; Mice; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Phenylalanine; Ribosomes; RNA, Transfer

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.

Topics: Abrin; Adenosine Triphosphatases; Animals; Carcinoma, Krebs 2; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Kinetics; Mice; Mice, Inbred BALB C; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Plant Proteins; Protein Biosynthesis; Puromycin; Rabbits; Reticulocytes; Ribosomes; Ricin

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Carbon Radioisotopes; Carcinoma, Krebs 2; Cell Fractionation; Cell-Free System; Centrifugation, Density Gradient; Culture Techniques; Depression, Chemical; DNA-Directed RNA Polymerases; Encephalomyocarditis virus; Guanosine Triphosphate; RNA, Viral

Topics: Animals; Binding Sites; Carbon Radioisotopes; Carcinoma, Krebs 2; Chromatography, Gel; Dialysis; Escherichia coli; Guanine Nucleotides; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Macromolecular Substances; Mice; Peptide Elongation Factors; RNA, Bacterial; RNA, Neoplasm; RNA, Transfer; Species Specificity; Tritium

Topics: Animals; Binding Sites; Carbon Radioisotopes; Carcinoma, Krebs 2; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Hydroxyapatites; Kinetics; Mice; Molecular Weight; Organ Specificity; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Phenylalanine; Phosphorus Radioisotopes; Protein Binding; Ribosomes; Sodium Dodecyl Sulfate; Species Specificity; Spectrophotometry, Ultraviolet; Sucrose; Tritium

Topics: Animals; Carbon Isotopes; Carcinoma, Krebs 2; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Globins; Guanosine Triphosphate; Guinea Pigs; Leucine; Liver; Methionine; Mice; Models, Biological; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Protein Binding; Rabbits; Reticulocytes; Ribosomes; RNA, Messenger; RNA, Transfer; Templates, Genetic; Tritium