PURIFICATION AND CHARACTERIZATION OF EUKARYOTIC TRANSLATIONAL INITIATION-FACTOR EIF-2B FROM LIVER

Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein cons isted of five nonidentical subunits with apparent molecular weights ra nging from 30.9 to 89.1 kDa. The holoprotein was characterized in term s of its Stokes radius and frictional coefficient. The isoelectric poi nts for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximate to 6.0, respectively; the alpha- and delta-subuni ts did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater th an 8.5. The purified protein was used as antigen to generate monoclona l antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal ant ibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were t hen used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found t o be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, sup porting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2 eIF-2B complex. The purified protein was also used as s ubstrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here ar e the first to show a direct quantitation of eIF-2 and eIF-2B in diffe rent tissues. They also provide evidence that the epsilon-subunit of e IF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.