Characterization of mammalian translation initiation factor 5 (eIF-5). Demonstration that eIF-5 is a phosphoprotein and is present in cells as a single molecular form of apparent M(r) 58,000

J. Chevesich, J. Chaudhuri, U. Maitra

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Eukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40 S initiation complex (40 S·mRNA·Met-tRNA(f)·eIF- 2·GTP). Using assays that measure (a) the release of 32P(i) from [γ- 32P]GTP bound to the 40 S initiation complex and (b) the eIF-5-dependent formation of an 80 S initiation complex from a preformed 40 S initiation complex containing bound [35S]Met-tRNA(f), we devised a novel and rapid procedure for purifying eIF-5 from rabbit reticulocyte lysates in high yield. Highly purified eIF-5 is a monomeric protein with a M(r) of about 58,000. However, in partially purified preparations, 58-kDa eIF-5 is associated with other cellular protein(s) and sediments in glycerol gradient centrifugation as a protein of M(r) ≃ 160,000. Chicken antibodies to native eIF-5 were isolated from egg yolks of laying hens immunized with rabbit reticulocyte eIF-5. 35S-Labeled eIF-5, isolated from rat anterior pituitary GH3 cells using affinity-purified anti-eIF-5 antibodies, also has an apparent M(r) of 58,000. eIF-5 immunoprecipitated from extracts of 32P-labeled GH3 cells was phosphorylated on serine residues. Phosphopeptide mapping revealed two major sites of phosphorylation, which are distinct from rabbit reticulocyte eIF-5 sites phosphorylated in vitro by casein kinase II. These results demonstrate that eIF-5 is a phosphoprotein that is present in cells as a single molecular form of apparent molecular weight 58,000.

Original languageEnglish (US)
Pages (from-to)20659-20667
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number27
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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