Effect of hemin on site-specific phosphorylation of eukaryotic initiation factor 2.

Initiation factor 2 (eIF-2) is phosphorylated in vitro by two different cyclic nucleotide-independent protein kinases. As previously shown, a protein kinase activity that comigrates with the major casein kinase activity from rabbit reticulocytes phosphorylates eIF-2beta. In addition, a second protein kinase that specifically phosphorylates eIF-2alpha has been identified. Both protein kinase activities demonstrate cyclic nucleotide-independent activity and are not inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase activities. Phosphorylation of eIF-2alpha is almost completely inhibited by 20--35 muM hemin, whereas phosphorylation of eIF-2beta is only partially inhibited. Hemin acts by decreasing the rate of incorporation of phosphate into eIF-2alpha. The protein kinase activity that modifies eIF-2alpha has been shown to have inhibitory activity in the cell-free protein-synthesizing system, whereas the protein kinase for eIF-2beta has no effect. The identity of the former enzyme with the hemin-controlled repressor and role of hemin in the control of initiation are discussed.

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.