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Previous studies have shown that eIF-2B purified from rabbit reticulocytes binds ATP and that the binding is prevented by NADP+. Because NADP+inhibits the activity of eIF-2B in in vitroreactions we have examined whether or not the activity of eIF-2B is modulated by ATP. In these studies, eIF-2B, purified from rat liver, was incubated with ATP prior to assay. We found that the activity of eIF-2B was inhibited with an IC50of approximately 0.8 mM. The inhibition was not due to phosphorylation of the factor. However, the inhibition of eIF-2B activity caused by ATP could be prevented by coincubation with either NADPH or fructose-1,6-bisphosphate. The activity of eIF-2B was also inhibited following addition of either ATP or AMPPNP to a postmitochondrial supernatant prepared from rat liver. Therefore, it is possible that the activity of eIF-2B might be allosterically regulated in vivonot only by changes in the redox state of pyridine dinucleotides but also by changes in the relative amounts of NADPH and ATP.

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.