A previous study demonstrated that the translational inhibitor isolated from lysates of heme-deficient rabbit reticulocytes is associated with a protein kinase activity. Chromatography of this inhibitor preparation on phosphocellulose yields two distinct protein kinase activities, PCI and PC2. PCI, which .In lysates of heme-deficient rabbit reticulocytes, the rate of protein synthesis proceeds linearly for several minutes and then declines abruptly (1-3). The shut-off of protein synthesis results from an inhibition that occurs at the level of protein chain initiation (4-6); inhibition is due to the action of a translational inhibitor that is activated in the absence of heme (2, 3, 7-9); addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition similar to that observed in heme deficiency (8, 10, 11). We reported that a partially purified inhibitor preparation contained a protein kinase activity (12). This inhibitor preparation (designated DS2 in this report) has been resolved by chromatography on phosphocellulose into two distinct protein kinase activities, designated PC1 and PC2. We examined these activities utilizing as putative substrates 40S ribosomal subunits and the initiation factor (IF) that binds initiator Met-tRNAf in the ternary complex [IF, Met-tRNAf, GTP] (13).We present the following evidence here: (i) PC1 is a 3':5'-cyclic AMP(cAMP)-dependent protein kinase that does not inhibit protein synthesis and does not phosphorylate IF; however, PC1 does phosphorylate 40S subunits at one major site.(ii) PC2 inhibits protein synthesis and phosphorylates the 38,000-dalton subunit (14) of IF in a reaction that does not require cAMP; PC2 does not act on 40S subunits. MATERIALS AND METHODSRabbit reticulocyte lysates were prepared as described (8).Salt-washed (0.5 M KCl) 40S ribosomal subunits and the initiation factor that binds Met-tRNAf in the ternary complex [IF, Met-tRNAf, GTP] were prepared from reticulocyte polyribosomes as described for the corresponding components from mouse fibroblasts (13, 15); IF was further purified on phosphocellulose to approximately 50% purity. Reticulocyte protein kinases DS1 and DS2 are DEAE-Sephadex fractions that correspond to PK1 and PK2 previously described (12,16,17). Beef heart muscle protein kinase (18) was a gift of Dr.

To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under, nondenaturing conditions.Itmigrates as~a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20o M hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of prY.in synthesis in the lysate due to phosphorylation of eIF-2 ?pear to be related. These findings suggest that hemin acts on the heme-reversible inhibitor.Protein synthesis in rabbit reticulocytes and reticulocyte lysates is dependent on the presence of hemin (1-5 (12,16,27). However, the heme-reversible form of the inhibitory kinase has not been purified to a high degree of purity. We describe now the isolation and purification of the the heme-reversible form of the inhibitory kinase from rabbit reticulocyte lysates. We call this inhibitory kinase reversible HRI to distinguish it from the stable form (irreversible HRI), which was earlier called HRI (16).Further, we present evidence that self-phosphorylation of the purified reversible HRI accompanies its activation in the absence of hemin.

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37 degrees C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60 degrees C. With Na-caseinate, the K(m) of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue.

In the absence of added hemin, protein synthesis in rabbit reticulocyte lysates proceeds at maximal linear rates for several minutes and then ceases abruptly. Inhibition involves the action of a translational inhibitor whose formation is regulated by hemin. Addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition of protein chain initiation similar to that observed in heme-deficiency. The inhibitor has been purified over 300-fold and contains a protein kinase activity that copurifies with the inhibitory function. With calf thymus histone II as the phosphate receptor, the inhibitor-associated protein kinase requires ATP as the phosphorylating agent. Cyclic AMP stimulates kinase activity 5-to 8-fold; the concentration of cyclic AMP required for half-maximal activity is 4 'X 10-8 M. Preincubation of the inhibitor in the presence of cyclic AMP significantly reduces cyclic AMP-dependent phosphorylation and inhibitory activity. The corresponding protein' kinase activity from hemin-supplemented lysates displays reduced cyclic AMP-dependency and little or no inhibitory'activity.These findings suggest that the protein kinase activity associated with the purified translational inhibitor is involved in

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme. In heme-deficient reticulocyte lysates, protein synthesis proceeds at the initial rate for several minutes and then declines abruptly. Inhibition of protein synthesis is due to the activation of a heme-regulated translational inhibitor (HRI) which blocks the initiation of protein synthesis. Addition of the isolated HRI to hemin-supplemented lysates causes inhibition of initiation similar to that observed in heme-deficiency. HRI has been shown to be a protein kinase that specifically phosphorylates the Met-tRNAf binding factor (eIF-2). We have isolated an inhibitor (LI) of protein chain initiation from rat liver which displays properties similar to those of HRI: (i) the chromatographic behavior of LI on DEAE-Sephadex, DEAE-cellulose, and phosphocellulose is similar to that of HRI; (ii) both LI and HRI inhibit protein chain initiation in rabbit reticulocyte lysates with the same kinetics of inhibition-i.e., an initial period of synthesis for several minutes at the control rate followed by an abrupt decline in the rate of initiation; (iii) both inhibitions are prevented or reversed by eIF-2; (iv) GTP (2 mM) prevents, and ATP (2 mM) potentiates, the inhibition of protein synthesis induced by either inhibitor; (v) LI is associated with a protein kinase that also phosphorylates the 38,000-dalton subunit of eIF-2. These findings indicate that a mechanism for the regulation of protein synthesis similar to that found in rabbit reticulocytes may be present in rat liver.