Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II.
Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.
Ghosh
S
S
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Chevesich
J
J
Maitra
U
U
D000818:Animals; D000918:Antibody Specificity; D047388:Casein Kinases; D002458:Cell Fractionation; D040061:Eukaryotic Initiation Factor-5; D007106:Immune Sera; D015151:Immunoblotting / Q000379:methods; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis*
D040061:Eukaryotic Initiation Factor-5; D007106:Immune Sera; D010448:Peptide Initiation Factors; D011494:Protein Kinases; D047388:Casein Kinases