UNIPROT:Q07644 - FACTA Search

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Query: UNIPROT:Q07644 (polypeptide)
72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A translation inhibitor from wheat germ has been purified more than 400-fold to apparent homogeneity. The inhibitor is a basic protein with a molecular weight of 30 000. This protein effectively blocks protein synthesis in animal cell-free extracts but does not affect protein synthesis in intact cells. Inhibition occurs at a ribosome to inhibitor molar ratio of 100:1, indicating an enzymic mechanism of action. The wheat germ protein inhibits the translation of endogenous mRNA, exogenous mRNA, and poly(uridylic acid) at a step in polypeptide chain elongation and without breakdown of the polysomes. Neither the aminoacylation reaction nor mRNA degradation is affected by the inhibitor. An interesting feature of the inhibition reaction is that it requires, in addition to the wheat germ inhibitor, both ATP and tRNA. The function of these two compounds in the inhibition is presently unknown since neither the hydrolysis of the beta,gamma-pyrophosphate bond of ATP nor a modification of the tRNA can be demonstrated during the reaction.
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PMID:Purification and properties of a translation inhibitor from wheat germ. 25 43

The catalytic subunit of cyclic 3':5'-AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) inhibits translation in Artemia salina and wheat germ extracts. It acts, as in reticulocyte lysates [Datta, A., de Haro, C., Sierra, J. M. & Ochoa, S. (1977) Proc. Natl. Acad. Sci. USA 74, 1463-1467] by catalyzing the conversion of a proinhibitor to an inhibitor of polypeptide chain initiation. Addition of ATP and either cyclic AMP or catalytic subunit promotes the proinhibitor-inhibitor conversion in crude proinhibitor preparations from A. salina embryos. The effect of cyclic AMP is due to stimulation of cyclic AMP-dependent protein kinase, present in such preparations, and is inhibited by hemin. In similar preparations from wheat germ, addition of ATP and catalytic subunit promoted proinhibitor-inhibitor conversion, but addition of ATP and cyclic AMP has little or no effect. As assayed with histone as substrate, wheat germ preparations exhibit a protein kinase activity that is not stimulated by the addition of cyclic AMP or cyclic GMP. Our results suggest that a translational control system, similar to that existing in rabbit reticulocytes and other mammalian cells, is present in organisms evolutionarily far removed from mammals.
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PMID:Translational control by protein kinase in Artemia salina and wheat germ. 27 Jun 77

Phosphorylation of eukaryotic initiation factors was examined both in intact cells and in vitro with purified components. Intact rabbit reticulocytes were incubated in a medium containing[32P]phosphate, and eight initiation factors were isolated and partially purified. The purified factors were analyzed on dodecyl sulfate/polyacrylamide gels and compared with highly purified nonradioactive factors. Significant amounts of radioactivity were found associated with initiation factors eIF-2, polypeptide 2 (molecular weight 53,000); eIF-3, polypeptides 2 and 4 (molecular weights 110,000 and 67,000); and eIF-4B. Purfied initiation factors from rabbit reticulocytes were also treated in vitro with [gamma-32P]ATP and a cyclic AMP-independent protein kinase isolated from rabbit erythrocytes. Only the factor polypeptides phosphorylated intracellularly were phosphorylated in vitro. The results suggest that the cyclic AMP-independent protein kinase is responsible for the phosphorylation of specific initiation factors in cells active in protein synthesis and that it may play a role in regulating translation.
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PMID:Phosphorylation of eukaryotic protein synthesis initiation factors. 27 26

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The 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 promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.
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PMID:Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet.40S ribosomal subunit complexes. 27 38

Incubation of reticulocyte lysates or isolated crude ribosomes with low levels of double-stranded RNA (0.1-10 ng/ml) induces the formation of an inhibitor of protein synthesis initiation similar to that observed in heme deficiency. The inhibitor is associated with a cyclic AMP-independent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylates the small polypeptide (38,000 daltons) of the eukaryotic initiation factor eIF-2. Activation of the inhibitor requires ATP in addition to double-stranded RNA and is accompanied by the phosphorylation of a 67,000-dalton polypeptide of unknown function. The inhibitor remains associated with the ribosomes during high-speed sedimentation. Once formed, the ribosome-associated inhibitor phosphorylates eIF-2 and inhibits protein synthesis in the absence of double-stranded RNA. Inhibition is prevented by exogenous eIF-2. The bound inhibitor can be solubilized by extraction with 0.5 M KCl. The soluble inhibitor preparation retains the ability to phosphorylate the small polypeptide of eIF-2 and to inhibit protein synthesis. Untreated crude ribosomes also contain cyclic AMP-independent protein kinase activities that phosphorylate the middle polypeptide (49,000 daltons) of eIF-2 and several polypeptide subunits of eIF-3 (160,000, 125,000, and 65,000 daltons); these kinase activities are not affected by double-stranded RNA and do not inhibit protein synthesis.
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PMID:Regulation of protein synthesis: activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2. 27 4

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. It migrates 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 20 micron 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 protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.
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PMID:Regulation of protein synthesis in rabbit reticulocyte lysates: purification and characterization of heme-reversible translational inhibitor. 27 81

Oxidized glutathione (GSSG) (0.02-0.5 mM) inhibits reticulocyte lysates by a mechanism similar to that observed in heme deficiency. Incubation of hemin-supplemented postribosomal supernates with GSSG results in the activation of a translational inhibitor [I(GSSG)]. The activation of I(GSSG) is enhanced by the presence of an energy-regenerating system. The simultaneous addition of 1 mM dithiothreitol blocks the activation of the GSSG-induced inhibitor; however, once inhibitor is formed, its activity is not affected by 1 mM dithiothreitol. GSSG-treated postribosomal supernates and partially purified preparations of I(GSSG) inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. Inhibition by I(GSSG) is blocked by cyclic AMP (2-10 mM) and is potentiated by ATP (2 mM). The inhibition is also blocked or reversed by eukaryotic initiation factor eIF-2. The activation of I(GSSG) is accompanied by an increased cyclic AMP-independent protein kinase activity which phosphorylates the 38,000-dalton component (alpha subunit) of eIF-2; however, GSSG treatment of supernates does not alter the activity of the cyclic AMP-independent protein kinase activity that phosphorylates the 49,000-dalton polypeptide component (beta subunit) of eIF-2. These data indicate that GSSG treatment of reticulocyte lysates results in the activation of a protein kinase with inhibitory and phosphorylation properties similar to those of the heme-regulated cyclic AMP-independent protein kinase which is activated in heme deficiency.
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PMID:Inhibition of protein synthesis initiation by oxidized glutathione: activation of a protein kinase that phosphorylates the alpha subunit of eukaryotic initiation factor 2. 27 1

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.
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PMID:Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase. 28 94

Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the alpha polypeptide of eukaryotic initiation factor 2 (eIF-2alpha). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [gamma-(32)P]ATP. The [(32)P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2alpha polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2alpha compared to control lysates, which displayed little or no phosphorylation of eIF-2alpha. In heme-deficient lysates, phosphorylation of eIF-2alpha occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2alpha and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2alpha phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2alpha was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2alpha and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2alpha is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.
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PMID:In situ phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase. 28 50

The ATP-dependent proteolytic cell-free system from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of 125I-labeled bovine serum albumin radioactivity. In addition to the previously reported heat-stable polypeptide [Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105], we now describe a protein of high molecular weight (approximately 450,000) that is labile at 42 degrees C. The extremely heat-labile factors is remarkably stabilized by ATP. GTP and CTP, which do not stimulate protolysis, do not stabilize this factor. Adenylate nucleotides such as ADP or the nonhydrolyzable beta,gamma imido or methylene analogues of ATP cause stabilization although they do not activate proteolysis. A third protein component of the protease system, stable at 42 degrees C, has been separated from the heat-labile species by salt precipitation. All three components are required with ATP for proteolytic activity, but thus far only the heat-labile factor has been shown to interact directly with ATP.
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PMID:Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP. 29 Sep 89

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