Purification and characterization of eukaryotic initiation factor (eIF)- 2α kinases from Ehrlich ascites tumor cells

E. A. Olmsted, L. O'Brien, E. C. Henshaw, R. Panniers

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

A major mechanism of regulation of mammalian protein synthesis initiation is accomplished by the phosphorylation of the α subunit of eukaryotic initiation factor (eIF) 2. This modification inhibits the activity of another initiation factor, guanine nucleotide exchange factor, preventing conversion of eIF-2 · GDP to eIF-2 · GTP and hence binding of initiator tRNA and formation of ternary complex (eIF-2 · GTP · Met-tRNA(t)). Inhibition of protein synthesis and phosphorylation of eIF-2 occurs in Ehrlich cells when they are amino acid- or serum-deprived or heat-shocked as well as in other nucleated cells under similar conditions. This paper describes the purification of two eIF-2α kinases from Ehrlich cells. Unlike the two well characterized eIF-2α kinases, HRI (heme-regulated inhibitor from reticulocytes) and P68 (double-stranded RNA-dependent kinase found in interferon-treated cells), the Ehrlich cell kinases do not appear to auto- phosphorylate. The two kinases chromatograph differently on DEAE-cellulose and Mono Q. Furthermore, their Michaelis constants (K(m) values) for ATP are different. Both kinases can inhibit purified guanine nucleotide exchange factor (GEF) from stimulating ternary complex formation. However, only one kinase inhibits reticulocyte lysate cell free protein synthesis. The other kinase co-purifies with a factor that suppresses inhibition of protein synthesis in reticulocyte lysates by eIF-2α kinases. This suppressing activity is probably guanine nucleotide exchange factor.

Original languageEnglish (US)
Pages (from-to)12552-12559
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number17
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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