Protein synthesis in yeast. Purification of elongation factor 3 from temperature-sensitive mutant 13-06 of the yeast Saccharomyces cerevisiae.
An altered form of the elongation factor 3 (EF-3) has been purified to near homogeneity from a thermolabile yeast mutant ts 13-06. The isolation procedure involved chromatography on DEAE-Sephadex, CM-Sepharose, and hydroxylapatite columns. The final purification of this protein was obtained by affinity chromatography on an ATP-Sepharose column. Because of the extreme lability of the mutant protein, the yield was very poor. Silver stain analysis of the sodium dodecyl sulfate electrophoretograms indicated that the affinity-purified protein was better than 90% pure. From the studies of the physical and biochemical properties, the following characteristics of the purified wild type and the mutant protein have been established. The two proteins were indistinguishable by their molecular weight, amino acid composition, and isoelectric point. Purified mutant EF-3 was rapidly inactivated between 37 and 39 degrees C. Under this condition, wild type EF-3 was completely stable. Ribosome-dependent GTPase and ATPase activities of the mutant EF-3 were heat sensitive; GTPase activity was more labile than the ATPase activity. Mutant EF-3, after exposure to a nonpermissive temperature, failed to stimulate binding of the ternary complex of EF-1 X GTP X aminoacyl-tRNA to ribosome. The wild type protein was fully active under this condition. Other biochemical and physical properties of these two proteins are under current investigation.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D005656:Fungal Proteins / Q000096:biosynthesis; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006358:Hot Temperature; D007526:Isoelectric Point; D008970:Molecular Weight; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000032:analysis* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
D000596:Amino Acids; D005656:Fungal Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; C111584:YEF3 protein, S cerevisiae; D000251:Adenosine Triphosphatases; D020963:GTP Phosphohydrolase-Linked Elongation Factors