Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae

Debabrata Chakravarti, Tapan Maiti, Umadas Maitra

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Abstract

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 Mr = 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 Mr = 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 Mr > 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 Mr = 54,000) and a minor band (apparent Mr = 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 Mr = 50,000-56,000.

Original languageEnglish (US)
Pages (from-to)5754-5762
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number8
StatePublished - Mar 15 1993

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Eukaryotic Initiation Factor-5
Eukaryotic Initiation Factors
Yeast
Saccharomyces cerevisiae
Yeasts
Cells
Sodium Dodecyl Sulfate
Peptides
Gel Chromatography
Gels
Proteins
Guanosine Triphosphate
Molecular Structure
Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae. / Chakravarti, Debabrata; Maiti, Tapan; Maitra, Umadas.

In: Journal of Biological Chemistry, Vol. 268, No. 8, 15.03.1993, p. 5754-5762.

Research output: Contribution to journalArticle

Chakravarti, Debabrata ; Maiti, Tapan ; Maitra, Umadas. / Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 8. pp. 5754-5762.
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abstract = "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 Mr = 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 Mr = 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 Mr > 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 Mr = 54,000) and a minor band (apparent Mr = 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 Mr = 50,000-56,000.",
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