Restore Protein Synthesis Research Articles

Stress-induced gene expression requires programmed recovery from translational repression.

Active repression of protein synthesis protects cells against protein malfolding during endoplasmic reticulum stress, nutrient deprivation and oxidative stress. However, long-term adaptation to these conditions requires synthesis of new stress-induced proteins. Phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation in diverse stressful conditions. GADD34 is a stress-inducible regulatory subunit of a holophosphatase complex that dephosphorylates eIF2alpha, and has been hypothesized to play a role in translational recovery. Here, we report that GADD34 expression correlated temporally with eIF2alpha dephosphorylation late in the stress response. Inactivation of both alleles of GADD34 prevented eIF2alpha dephosphorylation and blocked the recovery of protein synthesis, normally observed late in the stress response. Furthermore, defective recovery of protein synthesis markedly impaired translation of stress-induced proteins and interfered with programmed activation of stress-induced genes in the GADD34 mutant cells. These observations indicate that GADD34 controls a programmed shift from translational repression to stress-induced gene expression, and reconciles the apparent contradiction between the translational and transcriptional arms of cellular stress responses.

Baculovirus infection blocks the progression of fat body degradation during metamorphosis in Bombyx mori.

The effect of baculovirus infection into silkworm pupa particularly on programmed fat body degradation during metamorphosis was investigated. Pupal fat body degradation did not occur following infection with Bombyx mori nucleopolyhedrovirus (BmNPV). There were no histolytic differences between the fat body tissues of mock and BmNPV infected papae until 48 h postinfection (p.i.). Between 48 and 72 h p.i., significant differences were observed. In order to determine whether the histolysis of fat body was due to apoptosis, genomic DNAs were purified at various p.i. times and analyzed. Rapid genomic DNA fragmentation was observed at 24 and 48 h after pupation in both mock and BmNPV infected pupae. However, BmNPV infection clearly inhibited DNA fragmentation and ladder formation at 72 h and later times p.i. Furthermore, pulse-labeling analysis showed that BmNPV infection restored protein synthesis in fat body cells. These results suggested that fat body degradation during pupal-adult development was due to apoptosis, and that BmNPV was able to evoke a cellular response in these cells.

Translational Defects of Escherichia coli Mutants Deficient in the Um2552 23S Ribosomal RNA Methyltransferase RrmJ/FTSJ

We recently identified RrmJ (alias FtsJ), the first encoded protein of the rrmJ-hflB heat shock operon, as an Um2552 methyltransferase of the 23S rRNA. We now report that the rrmJ-deficient strain exhibits growth and translational defects compared to the wild-type strain. Growth rates of the rrmJ mutant are decreased at both low and high temperatures. Protein synthesis activity is reduced up to 65% when S30rrmJ mutant extracts are tested in a coupled in vitro transcription/translation assay. In vitro methylation of these extracts by RrmJ partially restores protein synthesis activity. Polysome profile analysis of the rrmJ strain reveals an increase in the proportion of free 30S and 50S subunits at both 30 and 42°C. These results suggest that the RrmJ-catalyzed methylation of Um2552 in 23S RNA strengthens ribosomal subunit interactions, increases protein synthesis activity, and improves cell growth rates even at non-heat shock temperatures.

Orally Administered Leucine Stimulates Protein Synthesis in Skeletal Muscle of Postabsorptive Rats in Association with Increased eIF4F Formation

We investigated the protein synthetic response of skeletal muscle to an orally administered dose of leucine given alone or in combination with carbohydrate. Male rats were freely fed (F) or food deprived for 18 h; food-deprived rats were then administered saline (S), carbohydrate (CHO), leucine (L) or a combination of carbohydrate plus leucine (CL). CHO and CL meals were isocaloric and provided 15% of daily energy requirements. L and CL meals each delivered 270 mg leucine. Muscle protein synthesis in S was 65% of F (P<0.01) 1 h after meal administration. Concomitant with lower rates of protein synthesis, phosphorylation of the translational repressor, eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), was less in S, leading to greater association of 4E-BP1.eIF4E, and reduced formation of the active eIF4G.eIF4E complex compared with F (P<0.01). Oral administration of leucine (L or CL), but not CHO, restored protein synthesis equal to that in F and resulted in 4E-BP1 phosphorylation that was threefold greater than that of S (P<0.01). Consequently, formation of 4E-BP1.eIF4E was inhibited and eIF4G.eIF4E was not different from F. The amount of eIF4E in the phosphorylated form was greater in S and CHO (P<0.01) than in all other groups. In contrast, no differences in the phosphorylation state of eIF2alpha or the activity of eIF2B were noted among treatment groups. Serum insulin was elevated 2.6- and 3.7-fold in CHO and CL, respectively, but was not different in L, compared with S (P<0.05). These results suggest that leucine stimulates protein synthesis in skeletal muscle by enhancing eIF4F formation independently of increases in serum insulin.

Protein synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock.

Oxygen-limiting conditions are critical to the survival of the bacteria in tuberculosis. Mycobacterium tuberculosis can survive anaerobiosis in vitro for long periods of time only after a gradual transition to a microaerophilic stationary phase. The underlying mechanism behind stationary phase adaption needs to be elucidated. The protein profiles of Mycobacterium tuberculosis during long-term stationary phase growth and under strict anaerobic incubation were monitored by [35S]methionine labelling, SDS-PAGE and fluorography. These experiments have established that protein synthesis gradually decreased over 50 days in the long-term stationary phase cultures which were considered to be microaerophilic. There was an 80% linear decrease in the level of total protein synthesis during the first 40 days of microaerophilic growth and then the rate of protein synthesis faded quickly. For the first time we have shown that total protein synthesis shutdown occurred when bacilli were incubated under further anaerobic conditions. Viability, estimated by cfu counts, remained constant during stationary phase growth and under anaerobic incubation. Furthermore, when oxygen was introduced into the anaerobic culture, protein synthesis restarted. Also heat shock at 45 degrees C, 48 degrees C and 50 degrees C rapidly induced protein synthesis in stationary and anaerobic cultures. These data indicate that dormant bacteria shut down protein synthesis but remain responsive to specific stimuli which restore protein synthesis. In addition the dormant bacilli induced by anaerobiosis developed more heat resistance than nondormant organisms.

Protein synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock

Oxygen-limiting conditions are critical to the survival of the bacteria in tuberculosis. Mycobacterium tuberculosis can survive anaerobiosis in vitro for long periods of time only after a gradual transition to a microaerophilic stationary phase. The underlying mechanism behind stationary phase adaption needs to be elucidated. The protein profiles of Mycobacterium tuberculosis during long-term stationary phase growth and under strict anaerobic incubation were monitored by [ 35S]methionine labelling, SDS-PAGE and fluorography. These experiments have established that protein synthesis gradually decreased over 50 days in the long-term stationary phase cultures which were considered to be microaerophilic. There was an 80% linear decrease in the level of total protein synthesis during the first 40 days of microaerophilic growth and then the rate of protein synthesis faded quickly. For the first time we have shown that total protein synthesis shutdown occurred when bacilli were incubated under further anaerobic conditions. Viability, estimated by cfu counts, remained constant during stationary phase growth and under anaerobic incubation. Furthermore, when oxygen was introduced into the anaerobic culture, protein synthesis restarted. Also heat shock at 45°C, 48°C and 50°C rapidly induced protein synthesis in stationary and anaerobic cultures. These data indicate that dormant bacteria shut down protein synthesis but remain responsive to specific stimuli which restore protein synthesis. In addition the dormant bacilli induced by anaerobiosis developed more heat resistance than non-dormant organisms.

Protein synthesis under conditions of anoxia and changing workload in ventricle strips from turtle heart.

An earlier study determined that protein synthesis in isolated perfused turtle (Trachemys [= Pseudemys] scripta elegans) hearts was three-fold lower under conditions of anoxia than under conditions of normoxia. However, the earlier study did not attempt to define the role of work in the isolated perfused preparation. In this study, the effects of varying workload, as defined by changing frequency of contraction, and anoxia on protein synthesis were examined. The ventricle strip preparation allows for comparison of multiple strips from a single heart, which aids in eliminating the variability found between individuals chosen from wild populations. Ventricle strips forced to contract at 24 contractions.min-1 under anoxic conditions failed more rapidly than strips forced to contract at 24 contractions.min-1 under normoxic conditions. Protein synthesis decreased by 32% when compared to normoxic controls. When stimulation was terminated after 2 hr of contraction, the rate of protein synthesis in strips under anoxic conditions was similar to that in strips under normoxic conditions. Also, returning strips to normoxic conditions after 2 hr of anoxia restored protein synthesis to the level of the normoxic controls. A significant correlation between pacing rate and protein synthesis was found under normoxic conditions but not under anoxic conditions when strips were paced at 12, 18 and 24 contractions.min-1. Protein synthesis increased by 30% at the 18 contractions.min-1 frequency and 45% at the 24 contractions.min-1 frequency over the rate at 12 contractions.min-1 frequency. Force-frequency studies revealed that under normoxic conditions force generation did not change until above 24 contractions.min-1, but under anoxic conditions there was a significant negative inotropic effect (20% decrease in force) at 24 contractions.min-1 and fell to 50% of initial at 36 contractions.min-1. These studies indicate that, in the turtle heart, anoxia per se is not the only determinant of protein synthesis but rather that work plays an important role in protein synthesis, as in the mammalian heart.

Type 1 Phosphatase Inhibitors Reduce the Restoration of Guanine Nucleotide Exchange Activity of Eukaryotic Initiation Factor 2B in Inhibited Reticulocyte Lysates Rescued by Hemin

In heme-deficient reticulocyte lysates, the α-subunit of eukaryotic initiation factor-2 (eIF-2α) is phosphorylated due to the activation of the heme-regulated eIF-2α kinase (HRI). Phosphorylation of eIF-2α impairs the guanine nucleotide exchange activity of eIF-2B and thereby inhibits or shuts off protein synthesis. Delayed addition of hemin to shut-off lysates inhibits the eIF-2α kinase activity of HRI and restores protein synthesis; under those conditions, the endogenous phosphatase of the lysate dephosphorylates phosphorylated eIF-2α and restores eIF-2B activity. In this report we present evidence that the restoration of eIF-2B activity is dependent on the concentration of added hemin and is related to HRI activity in lysates. The recovery of eIF-2B activity is not affected by protein synthesis inhibitors such as cycloheximide, pactamycin, and puromycin, which do not affect the eIF-2α phosphorylation. Also, the functional eIF-2B activity that is available in hemin-supplemented lysates is not affected by phosphatase inhibitors such as okadaic acid and heat-stable inhibitor-2. However, the recovery of eIF-2B activity that is observed by the delayed addition of hemin to inhibited heme-deficient lysates is reduced by inhibitor-2 and high concentrations of okadaic acid. These findings suggest that a type 1 phosphatase is involved in the recovery of eIF-2B activity and protein synthesis upon delayed addition of hemin to heme-deficient lysates.

Protein synthesis inhibition induces cytoresistance in cultured human proximal tubular (HK-2) cells.

After sublethal injury, proximal tubular cells acquire resistance to further attack. This study evaluated whether this could be a possible consequence of decreased protein synthesis, a potential correlate of cell damage. To this end, cultured human proximal tubular cells (HK-2) were subjected to 0-24 h of protein synthesis inhibition (> 98%), either by adding protein synthesis inhibitors [cycloheximide (CH) or verrucarin A] or by inducing sublethal ATP depletion (antimycin A + 2-deoxyglucose). After 24 h of these treatments, significant resistance to Ca2+ ionophore/ATP depletion-induced attack was noted (assessed by vital dye exclusion, compared with normal cells). That < or = 6 h of protein synthesis inhibition caused no cytoresistance implied the importance of evolving protein depletion rather than nonspecific drug effects or protein synthesis inhibition per se. CH plus ATP depletion did not induce additive benefits, suggesting a common mechanism. Cytoresistance was dissociated from the extent of free Ca2+ loading and ATP depletion but was associated with a decrease in membrane deacylation. CH removal promptly restored protein synthesis and cytoresistance was lost; conversely, ATP recovery did not restore protein synthesis and cytoresistance persisted. The emergence of cytoresistance correlated with the disappearance/dephosphorylation of an unidentified 130-kDa tyrosine-phosphorylated protein/protein complex (denoted pp-130). The functional significance of this change was suggested by the fact that tyrosine phosphatase inhibition with orthovanadate maintained pp-130 expression and prevented the cytoresistant state. We conclude that protein synthesis inhibition in HK-2 cells can induce a cytoresistant state. Suppression in phospholipase activity and altered tyrosine phosphorylation events may have functional significance in this regard.

Cytosolic Nucleoside Diphosphate Kinase Associated with the Translation Apparatus May Provide GTP for Protein Synthesis

Elongation of nascent polypeptides in a Dictyostelium discoideum in vitro translation system did not require the addition of ATP and GTP when creatine phosphate and creatine phosphokinase were present. However, depletion of the exogenous energy supply completely abolished incorporation of amino acids. Addition of dTTP, a nucleoside triphosphate that can be utilized by nucleoside diphosphate kinase (NDP kinase) to phosphorylate endogenous ADP and GDP, partially restored protein synthesis. Dictyostelium ribosomes were found to contain NDP kinase activity that could not be released by 1 M KCl. Thermal denaturation studies, specific inhibition with antibodies, and Western blotting identify the activity as cytosolic NDP kinase. These data support the idea that GTP can be fed into the translation machinery efficiently by NDP kinase associated with active ribosomes.

Control of protein synthesis by hemin. Purification of a rabbit reticulocyte hsp 70 and characterization of its regulation of the activation of the hemin-controlled eIF-2(alpha) kinase.

We have purified a soluble rabbit reticulocyte protein, previously termed the supernatant factor, that reverses the inhibition of protein synthesis in hemin-deficient lysate by promoting the inactivation of the hemin-controlled eIF-2 alpha kinase (HCR) mediating the effect of hemin deficiency. We have identified the supernatant factor as a member of the heat shock protein 70 family, denoted hsp 70(R), based upon its size (72 kDa), specific reaction to a monoclonal antibody against eukaryotic hsp 70, strong binding affinity for ATP, and endogenous ATPase activity. We have investigated the role of hsp 70(R) and hemin in the regulation of the activation of HCR from its latent precursor (ProHCR) and the translational control of protein synthesis in rabbit reticulocyte lysate. We find that autophosphorylation of Pro-HCR is reduced by about 75% by adding saturating hsp 70(R) and almost completely reduced by adding either saturating hemin or limiting hemin plus limiting hsp 70(R). In contrast, autophosphorylation of HCR, which is similar in magnitude to that of ProHCR, is unaffected by adding either saturating hsp 70(R), saturating hemin, or limiting amounts of both. The activation of HCR (measured by inhibition of protein synthesis) from isolated ProHCR is completely prevented by hsp 70(R) in the presence, but not absence, of dithiothreitol. This suppression appears to be due to the association of hsp 70(R) with ProHCR, since hsp 70(R) action is prevented by ATP/Mg2+ and because activation of HCR from less purified ProHCR, that has associated hsp 70(R), is suppressed by dithiothreitol alone. This association is confirmed by sucrose gradient centrifugation, which shows co-sedimentation of some hsp 70(R) with ProHCR following preincubation together that is prevented by ATP/Mg2+ and does not occur after conversion of ProHCR to HCR. Limiting hsp 70(R) reduces the concentration of hemin required to prevent activation of HCR from isolated ProHCR from 0.75 to 0.15 microM and the optimal hemin concentration needed to maintain protein synthesis in reticulocyte lysate from 25 to 10 microM. Limiting hsp 70(R) also allows the delayed addition of hemin to suppress activation of HCR from ProHCR and to reverse inhibition of protein synthesis in hemin deficient lysate. The association of hsp 70(R) with ProHCR also underlies the observation that much more protein is synthesized in reticulocyte lysate in the absence of hemin at 25 degrees C than at temperatures of 30 degrees C or greater. These observed effects may be specific to hsp 70(R), since they are not observed with rabbit reticulocyte eIF-2 or eIF-2B, and since the comparable hsp 70 from bovine brain is incapable of maintaining or restoring protein synthesis in hemin-deficient lysate.

Overexpression of the Short Form of the Growth Hormone Receptor in 3T3-L1 Mouse Preadipocytes

In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHRL) and a smaller isoform, GHRs that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHRs may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHRs was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHRs in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHRL in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHRL. The transfected cDNA for GHRs directs the synthesis of a 50 kDa protein. Cross-linking of [125I]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHRs[125I]hGH complex. In the presence of 10% newborn calf serum, incorporation of [35S]methionine into cellular proteins was similar in transfected clones and control cells. Deprivation of serum for 2 hr decreased protein synthesis by approximately 70% in control cells, but in the transfected cells, protein synthesis was reduced only by approximately 50% or 30% in cells exhibiting 2x or 3x increases in GH binding. In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level. Similarly, although 3H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by approximately 70% in control cells, but only by approximately 30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr or serum deprivation. IGF-1 had no effect after only 2 hr of serum deprivation. Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHRs produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHRs. We conclude that GHRs contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHRs in 3T3-L1 cells altered cell function in the absence of GH.

The effect of γ-Aminobutyric acid on hepatic regenerative activity following partial hepatectomy in rats

Background: γ-Aminobutyric acid (GABA) is a potent inhibitory neurotransmitter with growth-regulatory properties. In fulminant hepatic failure, a condition in which hepatic regeneration may be impaired, systemic serum GABA concentrations are markedly elevated. The present study was designed to determine whether increased amounts of circulating GABA interfere with hepatic regenerative activity. Methods: Exogenous GABA or isotonic saline was administered to adult male rats (n = 6 − 12/group) 16 hours before partial hepatectomy and twice daily for 1–3 days thereafter. The hypertrophic and hyperplastic components of hepatic regeneration were determined by calculation of the restitution of liver mass, [14C]leucine incorporation into protein (protein synthesis), and [3H]thymidine incorporation into hepatic DNA (DNA synthesis). Results: Exogenous GABA impaired restitution of liver mass (GABA vs. controls, day 3: 76% ± 7% vs. 90% ± 9%, mean ± SD) (P < 0.005) and the rate of protein synthesis (GABA vs. controls, day 1: 379 ± 39 dpm/mg protein vs. 564 ± 67 dpm/mg protein) (P < 0.01) without interfering with DNA synthesis. Supplemental administration of corticosterone and putrescine restored protein synthesis rates to normal in GABA-treated rats. Conclusions: These results indicate that elevated serum GABA concentrations interfere with the hypertrophic component of hepatic regeneration following partial hepatectomy in rats.

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.

Cellular mono(ADP-ribosyl) transferase inhibits protein synthesis

A reticulocyte translation system was depleted of functional EF-2 by treatment with diphtheria toxin (DT) fragment A and NAD. After dialysis to remove NAD, the system was reconstituted using preparation of EF-2 derived from pyBHK cells. Untreated and reconstituted lysates permitted similar rates of translation. As expected, when DT-treated EF-2 was used to reconstitute the system, no translation occurred. Furthermore EF-2, reacting with the endogenous ADP-ribosyl transferase from pyBHK cells, was also unable to restore protein synthesis in the reconstituted system. These studies suggest that eukaryotic cellular ADP-ribosyl transferases may play a role in regulating protein synthesis.

Chloroquine inhibits heme-dependent protein synthesis in Plasmodium falciparum.

A cell-free protein-synthesizing system has been reconstituted using the S-30 fraction or ribosomes and the S-100 fraction from Plasmodium falciparum. Addition of heme in vitro stimulates cell-free protein synthesis strikingly. Chloroquine inhibits the heme-dependent protein synthesis in the parasite lysate. The drug has also been found to inhibit parasite protein synthesis in situ at therapeutic concentrations soon after addition to parasite cultures. Ribosomes as well as the S-100 fraction isolated from such chloroquine-treated cultures are defective in protein synthesis. Addition of hemin plus glucose 6-phosphate or high concentrations of GTP, cAMP, and an active preparation of eIF-2 to the parasite cell-free system restores protein synthesis to a significant extent in chloroquine-treated cultures. Under conditions of inhibition of protein synthesis in situ by chloroquine in the culture, the parasite eukaryotic initiation factor 2 alpha- (eIF-2 alpha) is phosphorylated in the parasite lysate to a greater extent than that observed in the control culture. Addition of hemin in vitro suppresses this phosphorylation. eIF-2 alpha kinase activity is present in the parasite lysate and is not a contaminant derived from the human erythrocytes used to culture the parasite. The heme-chloroquine interactive effects can also be demonstrated with purified eIF-2 alpha kinase from rabbit reticulocyte lysate. It is proposed that chloroquine inhibits heme-dependent protein synthesis in the parasite and this is an early event mediating the growth-inhibitory effects of the drug.

Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap.

Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Recently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:1 molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat-shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine triphosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5' mRNA cap. We have ruled out proteolysis and competitive inhibitors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non-competitive inhibitor blocks complex binding to cap. We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, p220.p28 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.

Peptides containing 2‐aminopimelic acid

Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2-aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L-Lys-ambo-Apm, ambo-Apm-L-Lys, L-Lys-L-Ala-ambo-Apm, ambo-Apm-L-Ala-L-Lys, L-Ala(Cl)-ambo-Apm and ambo-Apm-L-Ala(Cl). In the two latter cases, Apm was associated with antibacterial amino acid beta-chloro-L-alanine [L-Ala(Cl)], an inhibitor of alanine racemase and transaminase B. The peptides displayed weak or no antibacterial activities; nevertheless, those containing L-Ala(Cl) had low MIC values in the presence of amino acids restoring protein synthesis. When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L-Ala(Cl)-containing peptides, when the growth medium was supplemented with specific amino acids. It was demonstrated that the weak or nil effect of the L-lysine-containing peptides was due to a poor uptake.

Cap binding protein complex that restores protein synthesis in heat-shocked Ehrlich cell lysates contains highly phosphorylated eIF-4E.

Cell-free protein-synthesizing systems derived from Ehrlich ascites tumor cells that have been exposed to elevated temperatures retain the inhibition of translation that is seen at the cellular level. A multisubunit cap binding protein complex able to restore protein synthesis in these cell free systems was purified from Ehrlich ascites tumor cells via affinity chromatography using m7GTP-Sepharose and fast protein liquid chromatography on Mono Q. The purified complex contains an Mr 220,000 polypeptide (p220) and an Mr 28,000 polypeptide (p28), both of which are components of eukaryotic initiation factor 4F (eIF-4F). p28 is identical to eIF-4E. Restoring activity was relatively free of the Mr 46,000 polypeptide (p46) that is the third component of eIF-4F and does not appear to be dependent on its presence. p28 associated in a complex with p220 is 85% phosphorylated; however, the majority of p28 is not associated with p220, and this free form is only about 50% phosphorylated. The correlation between association of p28 with p220 and high levels of p28 phosphorylation suggests a possible role for phosphorylation in association of p220 with p28.

Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha.

Previous studies from other laboratories, using rabbit reticulocyte lysate filtered through Sephadex G-25 or G-50, have demonstrated that glucose 6-phosphate is required to maintain active rates of translation, but its mechanism of action is currently unsettled. We have tested whether glucose 6-phosphate is required to prevent activation of the hemin-controlled translational repressor and the phosphorylation of the smallest or alpha subunit of eukaryotic initiation factor 2 (eIF-2). We have found that antibody to the hemin-controlled translational repressor can completely restore protein synthesis in reticulocyte lysate, filtered through Sephadex G-25, that is incubated in the absence of hemin and presence of glucose 6-phosphate, but cannot restore protein synthesis in such lysate incubated in the presence of hemin and absence of glucose 6-phosphate. We have also found, using a modification of the method of Matts and London [1984) J. Biol. Chem. 259, 6708-6711) to measure the ability of gel-filtered lysate to dissociate and exchange GDP from eIF-2.GDP, that this endogenous eIF-2B activity is reduced to the same low level in the presence of hemin and absence of glucose 6-phosphate as it is in the absence of hemin and presence of glucose 6-phosphate. Although there is a low level of phosphorylation of eIF-2 alpha in gel-filtered lysate given hemin but no glucose 6-phosphate, it cannot account for the loss of eIF-2B activity, since this phosphorylation is removed by antibody to the hemin-controlled translational repressor or isocitrate, which do not restore protein synthesis or eIF-2B activity, and not by fructose 1,6-diphosphate, which does partially restore protein synthesis and eIF-2B activity. These findings suggest that sugar phosphates may exert a direct effect on eIF-2B and may be required for its proper function. Additional support for this conclusion is our finding that protein synthesis and eIF-2B activity in partially hemin-deficient lysate can be restored by high levels of glucose 6-phosphate or fructose 1,6-diphosphate without a reduction in the level of phosphorylated eIF-2 alpha, suggesting that such levels of sugar phosphate may permit restoration of normal function with a limiting amount of eIF-2B.