FIG. 5.

Autoradiographic images of electrophoretically separated proteins from reaction mixtures containing activated or nonactivated purified PKR, eIF-2, and either GST or GST-U_S11. PKR, partially purified from rabbit reticulocyte ribosomes as indicated in Material and Methods, was preincubated with 0.10 mM ATP and 2.5 mM MgCl₂, either without (nonactivated PKR) or with poly(I-C) (0.1 μg/ml), for 20 min at 34°C. The contents of the reaction mixtures are given above the autoradiogram. The additions were 5 pmol of eIF-2, 22 pmol of GST-U_S11 or GST, and a final concentration of 0.09 mM [γ-³²P]ATP in a final volume of 10.5 μl of TKM buffer. The reaction was begun by addition of activated (lanes 1 to 8) or nonactivated (lanes 9 to 14) PKR and incubation at 34°C. Aliquots (5.0 μl) were removed from each reaction mixture and placed into disruption buffer after 6 and 15 min, boiled, and subjected to electrophoresis in denaturing gels and autoradiography. Positions of the U_S11 protein, M_r-39,000 phosphoprotein, and eIF-2α bands are shown. The position of GST is indicated, although it was not labeled.