Two elongation factors from the extremely halophilic archaebacterium Halobacterium cutirubrum. Assay systems and purification at high salt concentrations.
Two peptide chain elongation factors from Halobacterium cutirubrum were purified nearly to homogeneity. They were identified and characterized by three assay systems working at very high salt concentrations: poly(Phe) synthesis, GDP binding and ADP-ribosylation. The purification procedure consisted of Sepharose 4B chromatography with a decreasing ammonium sulfate gradient, gel filtration on Sephadex G-100 in the presence of (NH4)2SO4 and, for each of the two separated factors, an independent adsorption chromatography on hydroxyapatite. The factors were absolutely dependent on high salt concentrations for stability and activity. Both factors (I and II) complement each other to give fully active poly(Phe) synthesis, which is totally inhibited by puromycin and anisomycin. Factor I (Mr 51 000) is a major protein of the cell-free extract. It binds GDP, which can be displaced by GTP only to a small extent. The function of factor I in poly(Phe) synthesis is not impaired by high concentrations of kirromycin. The observed characteristics resemble partly those of prokaryotic EF-Tu and partly those of eukaryotic EF-1. Factor II (Mr 111 000) can be ADP-ribosylated by diphtheria toxin, and thus was identified as an EF-2-type elongation factor.
D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006217:Halobacterium / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D010445:Peptide Elongation Factors / Q000302:isolation & purification / Q000378:metabolism*; D012492:Salts
D010445:Peptide Elongation Factors; D012492:Salts; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate