Isolation of a translational inhibitor from wheat germ with protein kinase activity that phosphorylates initiation factor eIF-2

Abstract

A translational inhibitor (WGI) has been partially purified from wheat germ extracts. WGI inhibits protein synthesis in rabbit reticulocyte lysates with inhibition kinetics that are similar to those observed in heme-deficiency or by the addition of purified heme-regulated translational inhibitor (HRI). Initiation factor eIF-2 from rabbit reticulocytes overcomes this inhibition. This finding suggests that WGI inhibits protein chain initiation. WGI induced inhibition is enhanced by ATP (2 mM), and overcome by GTP (2 mM) and cyclic-AMP (10 mM). WGI preparations contain a cyclic-AMP independent protein kinase activity that phosphorylates the 38,000-dalton subunit of rabbit reticulocyte eIF-2. The phosphopeptide analyses of eIF-2 phosphorylated by WGI or HRI show that they phosphorylate the same site(s) of eIF-2. HRI phosphorylates the corresponding 38,000-dalton subunit of wheat germ eIF-2. These results obtained with WGI are similar to that of HRI. HRI has been identified as a cyclic-AMP independent protein kinase that phosphorylates the 38,000-dalton subunit of eIF-2 [for review see Ochoa, S. and de Haro, C. (1979) Ann. Rev. Biochem. 48 , 549]. Hence, these findings with wheat germ-a phylogenetically distant eukaryote, raise further the possibility that phosphorylation-dephosphorylation of eIF-2 may be an important general mechanism in the regulation of eukaryotic protein biosynthesis.