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 Mr 58,000

Jorge Chevesich, Jayanta Chaudhuri, Umadas Maitra

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Abstract

Eukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40 S initiation complex (40 S·mRNA·Met-tRNAf·eIF-2· GTP). Using assays that measure (a) the release of 32Pi from [γ-32P]GTP bound to the 40 S initiation complex and (6) the eIF-5-dependent formation of an 80 S initiation complex from a preformed 40 S initiation complex containing bound [35S]Met-tRNAf, 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 Mr 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 Mr ≈ 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 GHs cells using affinity-purified anti-eIF-5 antibodies, also has an apparent Mr 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 - Sep 25 1993

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Eukaryotic Initiation Factor-5
Eukaryotic Initiation Factors
Peptide Initiation Factors
Phosphoproteins
Demonstrations
Reticulocytes
Guanosine Triphosphate
Rabbits
Somatotrophs
Casein Kinase II
Phosphopeptides
Phosphorylation
Proteins
Egg Yolk
Centrifugation
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

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 Mr 58,000. / Chevesich, Jorge; Chaudhuri, Jayanta; Maitra, Umadas.

In: Journal of Biological Chemistry, Vol. 268, No. 27, 25.09.1993, p. 20659-20667.

Research output: Contribution to journalArticle

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abstract = "Eukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40 S initiation complex (40 S·mRNA·Met-tRNAf·eIF-2· GTP). Using assays that measure (a) the release of 32Pi from [γ-32P]GTP bound to the 40 S initiation complex and (6) the eIF-5-dependent formation of an 80 S initiation complex from a preformed 40 S initiation complex containing bound [35S]Met-tRNAf, 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 Mr 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 Mr ≈ 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 GHs cells using affinity-purified anti-eIF-5 antibodies, also has an apparent Mr 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.",
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