We have reported that treatment of mouse L cells with homologous interferon results in a reduction in the ability of initiation factor (eIF-2) to bind with initiator tRNA (Met-tRNAf) and GTP (1). In the rabbit reticulocyte lysate-protein synthesizing systems, polypeptide chain initiation is inhibited by a heme-regulated translation inhibitor (HRI) in the presence of ATP. Inhibition is induced also by a low concentration of doublestranded RNA (dsRNA), but prevented by the addition of either excess eIF or cAMP and GTP (2,3). Recent evidence showed that the inhibition of polypeptide chain initiation induced by HRI involves the phosphorylation of eIF-2 by HRI associated protein kinase (4-9). In the interferon system it has also been reported that protein synthesis in cellextracts from interferon-treated cells is decreased by low concentrations of dsRNA (10, 11) which activates certain protein kinases, a nuclease and production of a low molecular inhibitor of protein synthesis (1 1-17).The present study was undertaken to study the effect of these nucleotides, high and low concentrations of dsRNA, and other control substances on the eIF-2 inhibitory mechanism induced by interferon in mouse L cells. The results provide evidence that substances which affect the activity of different protein kinases strongly influence the level of eIF-2 activity as shown in the rabbit reticulocyte lysate system (4-9).Materials and Methods. Interferon treatment. Exponentially growing mouse L cells (monolayer strain L929) were treated with 300 reference units/ml of mouse interferon (specific activity: lo7 units/mg) for 24 hr at 37" in the presence of 2% fetal calf serum. After treatment, the cells were harvested and washed with 10 mM Tris-HC1 buffer (pH 7.5) containing 10 mM KC1, 5 mM MgC12 and 2 mM dithiothreitol.35S-Met-tRNAfpreparation. Initiator tRNA (tRNAf-Met) was purified from rat liver us-ing DEAE-cellulose and BD-cellulose, successively, and then charged with 35S-methionine (22.3 Ci/mmol) using Met-tRNA synthetase purified from Escherichia coli as previously reported (1).Preparation of eIF-2. The cells treated or untreated with interferon (about 4 X 10' cells) were homogenized in 20 mM Tris-HC1 buffer (pH 7.5) containing 10 mM KCl, 5 mM MgClz and 2 mM dithiothreitol (DTT), then centrifuged for 20 min at 15,000 rpm. Ribosomes in this supernatant were further purified by 60% sucrose cushion gradient centrifugation as previously reported (1). The purified ribosomes were suspended in 10 mM Tris-HC1 (pH 7.5) containing 0.25 M sucrose, 1 mM EDTA, 2 mM DTT and 0.5 M KC1, and then gently stirred for 60 min at 4". After centrifugation (45,000 rpm for 3 hr), solid ammonium sulfate (0.361 g/ml) was added to the supernatant. The precipitate was redissolved in 1.0 ml of 20 mM Tris-HC1 (pH 7.5) containing 0.1 M KC1, 2 mM DTT and 5% glycerol. After dialysis against the same buffer, the crude eIF-2 preparation was stored at -20" and used within 1 month.Assay of eIF-2 activity. Each 0.1 ml of the reaction mixture contained 50 mM Tris-HC1 (pH 7.5), ...