THE IN SITU PHOSPHORYLATION OF THE α-SUBUNIT OF eIF-2 IN RETICULOCYTE LYSATES INHIBITED BY HEME DEFICIENCY, dsRNA, GSSG, OR THE HEME-REGULATED INHIBITOR

Abstract

Publisher Summary This chapter analyzes in situ phosphorylation of the α-subunit of eIF-2 in reticulocyte lysates inhibited by heme deficiency, dsRNA, GSSG, or the heme-regulated inhibitor. The initiation of protein synthesis in reticulocyte lysates is inhibited in the absence of hemin, or in the presence of GSSG (50–500 μM) or dsRNA (1–50 ng/ml). All three conditions activate cAMP-independent protein kinases that phosphorylate the α-subunit (38,000 daltons) of eIF-2 (elF-2α). In this study, this phosphorylation is examined directly in lysates by labeling for brief periods with a pulse of high specific activity [γ-32P]ATP. The [32P]phosphoprotein profiles were analyzed by one-dimensional SDS-PAGE and autoradiography under conditions in which the eIF-2α polypeptide could be clearly distinguished. All modes of inhibition produced a rapid and significant increase in the phosphorylation of eIF-2α, compared to uninhibited control incubations which displayed little or no phosphorylation of eIF-2α. In heme-deficient lysates, phosphorylation of eIF-2α was detectable within 30 seconds after the start of incubation, and at all times subsequent both before and after the shut-off of protein synthesis. The restoration of synthesis by the delayed addition of hemin is accompanied by a decrease in the phosphorylation of eIF-2α.