Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000.
Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.
Raychaudhuri
P
P
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Chevesich
J
J
Ghosh
S
S
Maitra
U
U
D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D040061:Eukaryotic Initiation Factor-5; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D040061:Eukaryotic Initiation Factor-5; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D017886:Durapatite