Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver: Regulation involving inhibition of eukaryotic initiation factor 2α phosphatase activity

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA_i binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNA_i binding, and a 78% reduction in eIF-2B activity. The proportion of the α-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 ± 0.8% in control livers to 52.4 ± 5.5% in response to histidinol. The increase in the amount of eIF-2α in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2α kinase activity in extracts of livers perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2α was associated with an inhibition of eIF-2α phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2α appears to involve an inhibition of eIF-2α phosphatase activity rather than activation of an eIF-2α kinase.