CHARACTERIZATION OF GTP-DEPENDENT MET-TRANSFER-RNAF BINDING-PROTEIN

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 252  (11) , 3843-3847
Abstract
Purified GTP-dependent Met-tRNAf binding protein (EIF2) prepared from the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes was successfully resolved into its 3-component subunits by isoelectric focusing in the presence of urea. The 37,000-dalton subunit focused at a pH of 5.8 and was resolved into 2 spots; the 48,000-dalton subunit focused at a pH of 6.6 and was resolved into 3 spots; the 52,000-dalton subunit exhibited an isoelectric point of 8.9 and migrated as a single spot. When isolated 37,000- and 48,000-dalton subunits were assayed for functional activity, the 48,000-dalton subunit possessed Met-tRNAf and mRNA binding activities while the 37,000-dalton subunit possessed GDP binding activity. Phosphorylation of EIF2 by protein kinases present in reticulocyte lysates was demonstrated using [.gamma.-32P]GTP or [.gamma.-32P]ATP as the phosphate donor. The 37,000-dalton subunit was preferentially phosphorylated when [.gamma.-32P]ATP was used as substrate; the 48,000-dalton subunit was preferentially phosphorylated when the [.gamma.-32P]GTP was used as phosphate donor, although some phosphorylation of the 37,000-dalton subunit was also observed. The 37,000-dalton subunit of ribosome-associated EIF2 was present predominantly in a dephosphorylated form following purification.

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