Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae.
Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates. The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000. However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed. Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other. In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000. Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates. Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation. When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed. No precursor forms of molecular weight higher than 56,000 were detected in any preparations. These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.
Chakravarti
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Department of Developmental Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461.
Maiti
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Maitra
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D000818:Animals; D000908:Antibodies, Fungal / Q000276:immunology; D015153:Blotting, Western; D002474:Cell-Free System; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D040061:Eukaryotic Initiation Factor-5; D007120:Immunochemistry; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
D000908:Antibodies, Fungal; D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors