Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-eIF-2C. Also under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes.However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAfr40S-AUG complex to the 60S ribosomal subunit to form Met-tRNAfr80S*AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRIphosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation. During heme deficiency, protein synthesis in reticulocyte lysates is inhibited due to activation of a latent protein synthesis inhibitor, heme-regulated inhibitor (HRI) (7-9). HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of Met-tRNAf binding factor, eIF-2 (10-14). HRI also inhibits the following partial initiation reactions and in each case, the inhibition requires ATP: (i) Co-eIF-2B-promoted dissociation of Met-tRNAf-eIF-2-GTP complex at high Mg2+ and low temperature (0°C) (TDF activity) (3, 15, 16); (fi) CoeIF-2C stimulation of ternary complex formation at 1 mM Mg2+ (5,[16][17][18]; and (iii) Met-tRNAf binding to 40S ribosomes (15,16,19). Based on these results, it has been proposed that HRI phosphorylation causes conformational modification of eIF-2 and that phosphorylated eIF-2 [eIF-2(P)] does not interact with two factors, Co-eIF-2B and Co-eIF-2C, and is inactive in peptide chain intiation. However, these studies do not conclusively establish the precise role of eIF-2(P) in the inhibition process and the possibility that the phosphorylation of other components such as HRI, Co-eIF-2B, and Co-eIF-2C also is involved in this inhibition has not been ruled out. Moreover, Trachsel and Staehelin (20) used prephosphorylated eIF-2 (HRI catalyzed) and observed that both eIF-2(P) and eIF-2 were equally active in all the partial initiation reactions studied including Met-tRNAf binding to 40S ribosomes. Also, the possibility that HRI can inhibit more than one step in peptide chain initiation has been indicated (21).To clearly establish the mechanism of HRI inhibition of peptide chain initiation and the precise role of HRI-phosphorylated eIF-2 in this inhibition process, we prep...

A fluorimetric assay was carried out on the activity of extracellular lipase concentrations obtained from Serratia marcescens and Pseudomonaspuorescens using as substrates fatty acyl esters of 4-methylumbelliferone (4-methylumbelliferone elaidate. 4-methylumbelliferone nonanoate, 4-methylum belliferone butyrate, Cmethylumbelliferone palmitate and Cmethylumbelliferone oleate) at pH 4.0, 6.0, 8.0 and 10.0. The Ser. marcescens and Ps. fluorescens were cultured in Pope and Skerman's basal medium (Skerman 1957) supplemented with 0.5% (w/v) of a commercial medium. The

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Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme (1-3). In heme deficiency, a heme-regulated translational inhibitor (HRI) is activated (4-6), which blocks protein chain initiation (2, 3, 5-10). The inhibition of protein synthesis is associated with the disappearance of the complexes of Met-tRNAfMet (Met-tRNAf) and 40S ribosomal subunits (9). The purified HRI has functional properties that are similar to those of the in situ heme-regulated inhibitor (11,12). The inhibition induced in heme deficiency or by the addition of HRI is overcome or prevented by the Met-tRNAf binding factor (11,(13)(14)(15), which forms a ternary complex with Met-tRNAf and GTP (16)(17)(18)(19)(20)(21)(22). The Met-tRNAf binding factor (IF) is called EIF-1 in the nomenclature of Gupta and coworkers (23) and eIF-2 in the proposed new nomenclature (24); in this paper, it is called IF. The HRI has been shown to be an adenosine 3':5'-cyclic monophosphate (cAMP) independent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37), which specifically phosphorylates the 38,000-dalton subunit and not the 52,000-dalton and 50,000-dalton subunits of IF (12,(25)(26)(27)(28). These findings suggest that the inhibition by HRI involves the phosphorylation of IF. Significant inhibition of the binding of Met-tRNAf to 40S subunits has been shown to occur with high concentrations of HRI (13,29,30), and some inhibition of ternary complex formation has also been reported (31). However, the question of the molecular mechanism by which HRI exerts its inhibitory effect is not fully resolved.In the present studies we have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAf to 40S subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF) of Majumdar et al. (23)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAf to 40S subunits. This inhibitory effect of HRI requires ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of IF.The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also dissociate the ternary complex in the presence of 5 mM Mg2+ at 0°(23). At low concentrations of HRI and in the presence of ATP, this TDF-promoted dissociation of the ternary complex is inhibited. These results are consistent with the view that the phosphorylation of IF by HRI modifies its conformation and thus inhibits the protein-protein interaction of IF with one or more components of TDF in the formation of the Met-tRNAf-40S subunit initiation complex.Recently Das and Gupta (32) reported the isolation of a ternary complex dissociation inhibitor (TDI) from reticulocyte ribosome-free supernate. Like HRI, TDI inhibits ternary complex dissociation and the binding of Met-tRNAf to 40S subunits. In our experiments TDI and purified HRI behave similarly in all the results presented here. The apparent lack of requiremen...

A ribosomal salt (0.5 M KCI) wash factor (RF) that reverses inhibition of protein synthesis in heme-deficient reticulocyte lysates has been resolved from the bulk of Met-

A high molecular weight reticulocyte protein factor, named Co-eIF-2, contains Co-eIF-2A, Co-eIF-2B, and Co-eIF-2C activities and stimulates Met-tRNAf binding to eIF-2 both in the presence and absence of Mg2+. Some characteristics of this stimulation in the absence of Mg2+ are: (1) Stimulation is most pronounced at low eIF-2 levels. (2) Stimulation is partially resistant to heat and NEM treatment, and thus appears to be due to the combined action of both heat and NEM-insensitive Co-eIF-2A, and heat and NEM-sensitive Co-eIF-2C activities. (3) [3H]GDP bound in eIF-2 . [3H]GDP complex is rapidly displaced by unlabelled GTP during ternary complex formation Co-eIF-2 stimulates Met-tRNAf binding to eIF-2 even when added after the [3H]GDP from eIF-2 . [3H]GDP has been completely displaced. This indicates that Co-eIF-2-stimulation is not due to GDP displacement from eIF-2 . GDP. We propose that eIF-2 molecules become inactive in the presence of Mg2+ and at high dilution, and Co-eIF-2 restores the inactive eIF-2 molecules into an active form.

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