Kinetics of Phosphorylation of eIF-2 by the hemin-controlled repressor and casein kinase II. Inhibition by hemin and 2,3-diphosphoglyceric acid.

The kinetics of phosphorylation of eukaryotic initiation factor 2 (eIF-2) by two cyclic nucleotide-independent protein kinases from rabbit reticulocytes have been studied. The hemin-controlled repressor (HCR) and casein kinase II phosphorylate the alpha and beta subunits of eIF-2, respectively. The apparent Km for eIF-2 with HCR and casein kinase II was determined to be in the range of 0.5-1.0 microM. Using fully dephosphorylated eIF-2, it was possible to show that the phosphorylation state of the beta subunit did not affect the apparent Km of either HCR or casein kinase II for eIF-2. The effects of two inhibitory compounds, hemin and 2,3-diphosphoglyceric acid (2,3-DPG), were studied also. Hemin acted as a noncompetitive inhibitor of HCR with respect to eIF-2 with an apparent Ki of 3 microM, while inhibition of casein kinase II by 2,3-DPG was competitive with respect to eIF-2. The apparent inhibitory constant was determined to be 1 mM, a concentration within the physiological range of 2,3-DPG in red blood cells. From the kinetic values obtained with hemin and 2,3-DPG, it appears that these compounds could directly regulate the respective protein kinases in vivo.