Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes.
Rabbit reticulocyte initiation factor M2A has been prepared in homogeneous form.  The final preparation was purified 2,300-fold and ran as a single band on polyacrylamide gel electrophoresis in three different buffer systems: alkaline, sodium dodecyl sulfate, and acidic 6.5 M urea.  IF-M2A also ran as a single band in polyacrylamide gel isoelectric focusing experiments with an apparent pI of 6.45.  The molecular weight of IF-M2A was approximately 125,000 based on determinations by low speed equilibrium centrifugation (118,000), sodium dodecyl sulfate gel electrophoresis (130,000) and s20,w combined with Stokes radius (124,000).  The amino acid composition of IF-M2A revealed three unusual features: a) the basic amino acids represented 19.4 mol %; b) glutamicacid (plus glutamine) constituted 18.8 mol%; c) tryptophan and cysteine residues were only 0.4 and 0.7 mol%, respectively.  Homogeneous IF-M2A was tested in several initiation assays using either natural or artificial mRNAs.  In each assay tested, homogeneous IF-M2A fully substituted for cruder preparations and at concentrations commensurate with its increased purity.  IF-M2A was also examined for ribosome-dependent GTP hydrolysis, an assay requiring ribosomes but no other initiation factors.  Analysis of the data yielded a Km for GTP of 10 muM and a Vmax for hydrolysis of 1.20pmol/mug IF-M2A/min.  In addition, IF-M2A mediated GTP hydrolysis required both 40 S and 60 S subunits for maximal activity.  The possibility that IF-M2A is a factor required for the joining of 40 S and 60 S subunits is discussed.
D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors / Q000302:isolation & purification; D010649:Phenylalanine / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
D000596:Amino Acids; D002250:Carbon Radioisotopes; D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D010761:Phosphorus Radioisotopes; D011072:Poly U; D012967:Sodium Dodecyl Sulfate; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases