The two forms of the β-subunit of initiation factor-2 from reticulocyte lysates arise from proteolytic degradation

Nigel T. Price, Sara F. Nakielny, Stephanie J. Clark, Christopher G. Proud

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32 Citations (Scopus)

Abstract

Dholakia and Wahba (J. Biol. Chem. (1987) 262, 10164-10170) have reported that preparations of purified initiation factor-2 (eIF-2) from rabbit reticulocytes contain two forms of the β-subunit. These forms differ in their apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and are accordingly termed βH (heavy, the slower-migrating species, apparent Mr = 54300) and βL (light, the faster-migrating species, apparent Mr = 53 100). We confirm that two forms of eIF-2β are present in such preparations, but present evidence that the βL is generated from βH during the isolation procedure. Crude reticulocyte lysates contain only the βH species as judged from immunoblotting of reticulocyte proteins resolved by SDS-PAGE using an antiserum against eIF-2β. The βL species appears after the ammonium sulphate fractionation step used early in the purification procedure, but is not apparent if a cocktail of proteinase inhibitors is included in the buffers used during the purification, indicating that it is a proteolytic degradation product generated during the isolation procedure. Cleveland mapping failed to reveal any differences between the two species. Both the βH and the βL forms are phosphorylated by casein kinase-2, and, as judged by one- and two-dimensional peptide mapping, at identical sites in each species. Since casein kinase-2 phosphorylates serine-2 in eIF-2β, the βL form must still contain the N-terminal region and is presumably produced by limited proteolysis at the carboxyl terminus of the β-subunit.

Original languageEnglish
Pages (from-to)177-182
Number of pages6
JournalBBA - Gene Structure and Expression
Volume1008
Issue number2
DOIs
Publication statusPublished or Issued - 7 Jul 1989
Externally publishedYes

Keywords

  • (Rabbit reticulocyte lysate)
  • Initiation factor 2
  • Protein phosphorylation

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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