Isolation of eukaryotic initiation factor 2 from yeast Saccharomyces cerevisiae.
Protein synthesis initiation factor eIF-2 has been isolated from the high speed supernatant fraction of the yeast Saccharomyces cerevisiae. This purification steps include ammonium sulfate fractionation, column chromatography on Sephacryl 300 and hydroxyapatite, and glycerol gradient sedimentation. Electrophoresis of the purified factor on sodium dodecyl sulfate polyacrylamide gels reveals three polypeptides with a total Mr of 127,000. The molecular weights of the subunits are 31,000, 46,500, and 49,600. The pI of each of these subunits is 4.5, 7.3, and 8.6, respectively. The stoichiometry of the subunits varies from 1:1:1 to 1:0.25:1 suggesting that the active factor may be composed of only two subunits with total Mr of 80,000. The factor is part of a high molecular weight complex during the first steps of the purification. Prior to chromatography on Sephacryl, this complex is broken up in high salt. The activity of the factor is stabilized by inclusion of GDP in all buffers during the preparation and is stimulated by magnesium ion.
D004355:Drug Stability; D015852:Eukaryotic Initiation Factor-2; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008274:Magnesium