TY - JOUR
T1 - Characterization of mammalian translation initiation factor 5 (eIF-5)
T2 - Demonstration that eIF-5 is a phosphoprotein and is present in cells as a single molecular form of apparent Mr 58,000
AU - Chevesich, Jorge
AU - Chaudhuri, Jayanta
AU - Maitra, Umadas
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1993/9/25
Y1 - 1993/9/25
N2 - 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.
AB - 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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M3 - Article
C2 - 8376415
AN - SCOPUS:0027198021
SN - 0021-9258
VL - 268
SP - 20659
EP - 20667
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -