Control of eIF-2 phosphatase activity in rabbit reticulocyte lysate.

A sensitive assay requiring picomole amounts of [32P]eIF-2 to measure eIF-2 phosphatase activity has been developed. Dephosphorylation of [32P]eIF-2 alpha (38,000-dalton subunit) is extremely rapid (t 1/2 = 20 sec) and occurs at the same rate in both hemin-supplemented and hemin-depleted lysates. In contrast, [32P]eIF-2 beta phosphate is stable under all conditions studied. At concentrations required to produce a transient inhibition of protein synthesis, GDP prevents dephosphorylation of half of the phosphate introduced on eIF-2 alpha by the hemin-controlled repressor. Equimolar GTP is without effect. The concept that the energy charge of the guanylate pool may regulate accessibility of a phosphorylated site on eIF-2 alpha to its phosphatase and the implication of this to the mechanism of hemin-regulated translational control are discussed.