Isolation of rabbit reticulocyte initiation factors by means of heparin bound to sepharose.
Passage of cell-free extracts of rabbit reticulocytes through heparin-Sepharose affinity columns results in the loss of the ability of the effluent to initiate protein synthesis. This is shown by the loss of response to added rabbit globin mRNA or to inhibitors of initiation of protein synthesis, such as heparin and aurin tricarboxylic acid, and by recovery of initiation activity by addition of protein retained and subsequently eluted from the columns. The effluent retains, however, the ability to elongate protein chains. Only 0.8% of the applied cell extract protein binds to heparin-Sepharose columns. This bound protein, which can be recovered by increasing the salt concentration of the eluting buffer, has initiation factor activity equal to that of a crude initiation factor preparation obtained from rabbit reticulocyte ribosomes by extraction with 0.5 M KCl. The protein patterns on polyacrylamide gels of the initiation factors prepared by either method are very similar and indicate a protein mixture, which may represent a complex. These data confirm that heparin interacts specifically with initiation factos, and indicate that heparin-Sepharose chromatography will simplify procedures for the preparation of initiation factors.
D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001798:Blood Proteins / Q000096:biosynthesis; D002287:Carcinoma, Krebs 2 / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005914:Globins / Q000096:biosynthesis; D006493:Heparin / Q000494:pharmacology; D007930:Leucine / Q000378:metabolism; D051379:Mice; D010448:Peptide Initiation Factors / Q000097:blood / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate
D001798:Blood Proteins; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012967:Sodium Dodecyl Sulfate; D001312:Aurintricarboxylic Acid; D005914:Globins; D006493:Heparin; D012685:Sepharose; D007930:Leucine