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1.
Eukaryotic initiation factors are found in the postribosomal supernatant as well as bound to the 40S ribosomal subunits. We have analyzed the factor activities from the supernatant by means of zonal centrifugation followed by Sepharose-heparin affinity chromatography. They exist both as free factors, sedimenting in a broad range from 4 to 7S, and complexed with other protein(s) with a sedimentation value of 16–20S. This complexed fraction contains besides eIF-2 another activity which exhibits a profound stimulation on amino acid incorporation in crude lysates and appears to counteract the heme-regulated inhibitor.Abbreviations eIF-2, eIF-3, eIF-4A and eIF-4B are eukaryotic initiation factors, see FEBS Letters 76, 1-10 (1977).  相似文献   

2.
From a rabbit reticulocyte postpolysomal supernatant a fraction has been isolated which is enriched in ribosomal particles sedimenting at 50S. This fraction is efficiently in vitro translated predominantly into α-globin. Besides the RNAs and proteins of the small ribosomal subunit the 50S particle contains α-globin mRNA and additional high molecular weight proteins, most of which correspond to polypeptides of the initiation factors eIF-2 and eIF-3. The 50S particle may represent a native [mRNA·40S·eIF′s·Met-tRNAf·GTP] complex which may occur in vivo as a translatable intermediate in the initiation sequence.  相似文献   

3.
We have examined the association of an F' plasmid and an R plasmid in Proteus mirabilis with a rapidly sedimenting material that is generated by sodium dodecyl sulfate lysis and low speed centrifugation. Virtually all of the chromosomal deoxyribonucleic acid (DNA) and the F' plasmid DNA are associated with the rapidly sedimenting material after gentle lysis and centrifugation. A portion of R plasmid NR1 DNA (usually 5 to 25%) is not bound to the rapidly sedimenting material and is recovered in the supernatant fraction. This difference in binding is not related to the size of the plasmid DNA, since F' plasmids and R plasmids of different molecular weights showed the same behavior. R plasmid DNA labeled by a brief pulse of [(3)H]thymine is recovered in the supernatant fraction to a lower extent than the total R plasmid DNA. It would appear that R plasmid replication takes place in association with the rapidly sedimenting material. With prolongation of the [(3)H]thymine pulse, the [(3)H]thymine-labeled R plasmid DNA is recovered in the supernatant fraction with the same probability as the total R plasmid DNA. This finding indicates that a change in R plasmid attachment to the rapidly sedimenting material occurs some time after its replication. The differences observed in the replication of F' plasmids and R plasmids in P. mirabilis may be related to their different modes of association with the rapidly sedimenting material.  相似文献   

4.
(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.  相似文献   

5.
Eukaryotic protein synthesis initiation factor 2 (eIF-2) from rat liver has been resolved into two subfractions by anion-exchange chromatography on DEAE-cellulose. One of these contained all three components (eIF-2 alpha, eIF-2 beta, eIF-2 gamma) characteristic of mammalian eIF-2, whilst the other fraction contained only two. By a number of criteria these were shown to be eIF-2 alpha and eIF-2 gamma. The absence of eIF-2 beta from this fraction was not due to its proteolytic degradation during purification since it was unaffected by the inclusion of a range of proteinase inhibitors in the isolation media. The properties of eIF-2 containing or lacking eIF-2 beta have been directly compared. It was found that eIF-2 beta was not required for the binding of guanine nucleotides to eIF-2 or for formation of ternary initiation complexes with GTP and the initiator tRNA. eIF-2 lacking eIF-2 beta was able to form 40 S initiation complexes and the presence of eIF-2 beta was also unnecessary for the stimulation of eIF-2 activity by the recycling factor, eIF-2B. Some of these findings are at variance with previous reports in which eIF-2 beta was removed proteolytically. The role of eIF-2 beta in the overall physiological function of eIF-2 remains to be elucidated.  相似文献   

6.
Eukaryotic protein synthesis initiation factor 2 (eIF-2) from rat liver has been resolved into two subfractions by anion-exchange chromatography on DEAE-cellulose. One of these contained all three components (eIF-2α, eIF-2β, eIF-2γ) characteristic of mammalian eIF-2, whilst the other fraction contained only two. By a number of criteria these were shown to be eIF-2α and eIF-2γ. The absence of eIF-2β from this fraction was not due to its proteolytic degradation during purification since it was unaffected by the inclusion of a range of proteinase inhibitors in the isolation media. The properties of eIF-2 containing or lacking eIF-2β have been directly compared. It was found that eIF-2β was not required for the binding of guanine nucleotides to eIF-2 or for formation of ternary initiation complexes with GTP and the initiator tRNA. eIF-2 lacking eIF-2β was able to form 40 S initiation complexes and the presence of eIF-2β was also unnecessary for the stimulation of eIF-2 activity by the recycling factor, eIF-2B. Some of these findings are at variance with previous reports in which eIF-2β was removed proteolytically. The role of eIF-2β in the overall physiological function of eIF-2 remains to be elucidated.  相似文献   

7.
Our experiments were designed to identify the source of the increased number of cells forming colonies in culture (CFU-C) detected after short-term culture of mouse marrow cells over 'feeders' of renal tubules. Accordingly, marrow cell suspensions were fractionated by velocity sedimentation and aliquots of each fraction cultured for 2 days over tubule 'feeders'. We found a greater increase of CFU-C in suspensions of slowly sedimenting cells as compared to rapidly sedimenting cells. Colcemid and vinblastine were used to alter the peak sedimentation velocity of marrow suspensions by changing the distribution of cells in the cell cycle. After such manipulations, the peak increase in CFU-C continued to be associated with fractions containing slowly sedimenting cells. Such fractions also contained most of the pluripotent stem cells identified by the spleen colony technique.  相似文献   

8.
Recent observations have indicated that eukaryotic initiation factor (eIF)-2 and GTP or GDP normally bind to 60 S ribosomal subunits in rabbit reticulocyte lysate and that when eIF-2 alpha is phosphorylated and polypeptide chain initiation is inhibited, eIF-2 X GDP accumulates on 60 S subunits due to impaired dissociation that is normally mediated by the reversing factor (eIF-2B). Current findings now indicate that inhibition due to phosphorylation of eIF-2 alpha is mediated, at least in part, by the inability to dissociate eIF-2 X GDP from the 60 S subunit of complete initiation complexes. At the onset of inhibition, there is an accumulation of Met-tRNA(f) and eIF-2 on the polysomes, despite a marked reduction in Met-tRNA(f) bound to 40 S subunits and Met-peptidyl-tRNA bound to the polysomes. This initial effect is not associated with the formation of "half-mers" (polysomes containing an extra unpaired 40 S subunit), and the 40 S X Met-tRNA(f) complexes, though reduced, still sediment at 43 S. When inhibition is maximal and the polysomes are largely disaggregated, there is an accumulation of 48 S complexes consisting of a 40 S subunit and Met-tRNA(f) bound to globin mRNA as well as small polysomal half-mers, such that residual protein synthesis occurs to about the same degree on "1 1/2"s and "2 1/2"s as on mono-, di-, and triribosomes. Exogenous eIF-2B increases protein synthesis on mono-, di-, and triribosomes and decreases that on half-mers. This is associated with reduced binding of Met-tRNA(f) and eIF-2 to ribosomal particles sedimenting at 80 S and greater and a shift from 48 S to 43 S complexes. These results suggest that eIF-2B must normally promote dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes before they can elongate but cannot when eIF-2 alpha is phosphorylated, resulting in the accumulation of these complexes, some of which dissociate into Met-tRNA(f) X 40 S X mRNA and 60 S X eIF-2 X GDP.  相似文献   

9.
Envelope fraction I prepared from a ?X174 sensitive host, KD4301, showed a strong eclipsing activity, while the lipopolysaccharide (LPS) fraction showed a weak activity. The eclipsing activity in envelope fraction I was sensitive to heat treatment, while that in the LPS fraction was insensitive. When the complete phage particles (114S) were treated with envelope fraction I, the eclipsed particles (70S) and a rapidly sedimenting component were obtained, but when they were treated with LPS, only 70S eclipsed particles were obtained. Electron microscopic observation showed that there were two types of eclipsed particles formed on treatment with fraction I; in one of them phage DNA was extruded from the phage particles as a thick bundle, and in the other more than 95% of the phage DNA was extruded from the phage particles. The rapidly sedimenting component was the membrane-eclipsed particle complex. LPS gave only one type of eclipsed particles in which DNA was extruded as a thick bundle. These results indicate that a heat labile component in the cell envelopes other than LPS is involved in the extrusion of ?X174 DNA.  相似文献   

10.
In unfractioned reticulocyte lysate, interaction of eukaryotic initiation factor 2 (eIF-2) with other components regulates the accessibility of phosphatases and kinases to phosphorylation sites on its alpha and beta subunits. Upon addition of eIF-2 phosphorylated on both alpha and beta subunits (eIF-2(alpha 32P, beta 32P) to lysate, the alpha subunit is rapidly dephosphorylated, but the beta subunit is not. In contrast, both sites are rapidly dephosphorylated by the purified phosphatase. The basis of this altered specificity appears to be the association of eIF-2 with other translational components rather than an alteration of the phosphatase. Formation of an eIF-2(alpha 32P,beta 32P) Met-tRNAi X GTP ternary complex prevents dephosphorylation of the beta subunit, but has no effect on the rate of alpha dephosphorylation. eIF-2B, a 280,000-dalton polypeptide complex required for GTP:GDP exchange, also protects the beta subunit phosphorylation site from the purified phosphatase. However, the dephosphorylation of eIF-2(alpha 32P) is inhibited by 75% while complexed with eIF-2B. The altered phosphatase specificity upon association of eIF-2 with eIF-2B also affects the access of protein kinases to these phosphorylation sites. In the eIF-2B X eIF-2 complex, the alpha subunit is phosphorylated at 30% the rate of free eIF-2. Under identical conditions, phosphorylation of eIF-2 beta can not be detected. These results illustrate the importance of substrate conformation and/or functional association with other components in determining the overall phosphorylation state allowed by alterations of kinase and phosphatase activities.  相似文献   

11.
The suggestion that a rapidly sedimenting rough endoplasmic reticulum fraction in close association with mitochondria, is the preferred site of cytochrome P-450 synthesis has been examined. The rate of cytochrome P-450 synthesis in the different subcellular fractions has been evaluated invivoandinvitro, using the immunoprecipitation technique. The results indicate that the conventional microsomal fraction (100,000 X g sediment) is the major site of cytochrome P-450 synthesis and that the rapidly sedimenting rough endoplasmic reticulum fraction associated with mitochondria is not a preferred site for the hemoprotein synthesis.  相似文献   

12.
An enzyme fraction containing phosphatase activity for phosphorylated eukaryotic peptide initiation factor 2 (eIF-2) has been isolated from rabbit reticulocytes and partially characterized. The enzyme efficiently catalyzes release of phosphate from the small subunit of eIF-2 (eIF-2 alpha) that has been phosphorylated by the hemin-controlled repressor. It is shown to restore activity of this phosphorylated eIF-2 for binding of methionyl-tRNAf to 40 S ribosomal subunits in a partial reaction of peptide initiation. The enzyme fraction also has phosphatase activity for eIF-2 phosphorylated in its largest subunit and for the 100,000-dalton peptide associated with the eIF-2 alpha kinase activity of the hemin-controlled repressor. The phosphoprotein phosphatase has been isolated by a procedure involving precipitation with ethanol at room temperature and has an apparent molecular weight in the order of 76,000. Its phosphatase activity for eIF-2 alpha is stimulated about 3-fold by optimal concentrations of Mn2+, but is not stimulated by Ca2+ or Mg2+. The enzyme is strongly inhibited by Fe2+ and by purine nucleoside diphosphates.  相似文献   

13.
Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) is a major mechanism regulating protein synthesis in rabbit reticulocytes. To determine whether phosphorylation of eIF-2 alpha is a likely regulatory mechanism in the Ehrlich cell, we have measured the percent of cellular eIF-2 alpha which is phosphorylated in cells exposed to heat shock, 2-deoxyglucose, or amino acid deprivation, conditions which rapidly decrease the concentration of 40 S initiation complexes and inhibit protein synthesis. eIF-2 alpha and eIf-2 alpha (P) were separated by isoelectric focusing and were detected by immunoblotting with a monoclonal antibody we developed for this purpose. Under the above three inhibitory conditions, phosphorylation of eIF-2 alpha increased rapidly, and this increase correlated in time with the rapid inhibition of protein synthesis. In heat-shocked cells which were returned to 37 degrees C, both phosphorylation and protein synthesis remained unchanged for 10 min and then returned toward control values slowly and in parallel. The close temporal correspondence between changes in protein synthesis and phosphorylation supports an important regulatory role for phosphorylation in protein synthesis. An increase of 25-35 percentage points, to 50-60% phosphorylation from control levels of 20-30% phosphorylation, correlated with an 80-100% inhibition of protein synthesis. This steep curve of inhibition is consistent with a mechanism in which eIF-2 alpha (P) saturates and inhibits the guanine-nucleotide exchange factor.  相似文献   

14.
A 10-50-fold, biphasic increase in the rate of 32Pi labeling of eIF-4E was closely correlated with the induction of protein and glycoprotein biosynthesis when resting murine splenic B lymphocytes (B cells) were activated by bacterial lipopolysaccharide or the combination of phorbol 12-myristate 13-acetate and ionomycin. The fraction of eIF-4E which was phosphorylated only increased from 46% in resting cells to 83% in lipopolysaccharide-activated cells. This discrepancy between the increase in the fraction of phosphorylated eIF-4E and the increase in 32Pi labeling suggested that the phosphoryl group of eIF-4E turns over slowly in resting B cells compared with activated cells. The turnover rate for the eIF-4E phosphate moiety in lipopolysaccharide-activated cells was rapid (t1/2 = 2 h) in comparison to the eIF-4E polypeptide chain, which did not turn over detectably in 6 h. Neither protein kinase C nor a cyclic nucleotide-dependent protein kinase appeared to be involved in eIF-4E phosphorylation in B cells, based on the observations that the metabolic labeling of eIF-4E by 32Pi was insensitive to the protein kinase inhibitors H-7 and HA1004, and that maximal labeling occurred after protein kinase C activity was "down-regulated" to very low levels in phorbol 12-myristate 13-acetate/ionomycin-activated cells. Dephosphorylation in vivo was blocked by okadaic acid (IC50 = 200 nM). These results indicate that a rapid phosphorylation-dephosphorylation of eIF-4E is associated with high translation rates during the activation of B cells, and implicate protein phosphatase-1 (or possibly-2A) in the dephosphorylation of the initiation factor.  相似文献   

15.
Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.  相似文献   

16.
Nuclear ribonucleoprotein particles were isolated from chick erythroblast nuclei. The particles were found to sediment as heterogeneous material. The major fraction of the rapidly synthesized RNP sedimented at 30 S, whereas the nuclei were found to contain a major, apparently more stable, RNP component sedimenting at about 40 S. The RNA isolated from the RNP particles was assayed for globin messenger activity in a wheat germ cell-free system. RNP sedimenting at relatively low S values (approx. 15 S) as well as RNP-particles of larger size code for globin. In addition to globin, the RNA of the particles codes also for other, not yet identified, proteins.  相似文献   

17.
Kinetics of dephosphorylation of eIF-2(alpha P) and reutilization of mRNA   总被引:4,自引:0,他引:4  
Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) causes mRNA to accumulate in 48 S complexes containing Met-tRNAf and eIF-2(alpha P). When the eIF-2 alpha kinase is inhibited by 2-aminopurine, the mRNA is slowly transferred from 48 to 80 S initiation complexes after an initial lag. The cause of this lag was examined by investigating whether mRNA and Met-tRNAf dissociated from 48 S complexes before binding to 80 S. Both compounds were quantitatively transferred from 48 to 80 S complexes after addition of 2-aminopurine and the eIF-2(alpha P) bound to 48 S complexes was dephosphorylated after an initial lag more slowly than unbound eIF-2(alpha P), which was rapidly dephosphorylated. the eIF-2(alpha P) in isolated 48 S complexes was slowly dephosphorylated by partially purified lysate phosphatases, whereas free eIF-2(alpha P) was readily dephosphorylated. These results indicated that 48 S complexes could directly join to a 60 S ribosomal subunit after eIF-2(alpha P) dephosphorylation. The lag and slow kinetics of dephosphorylation of eIF-2(alpha P) bound to 48 S complexes accounted for the slow transfer of mRNA from 48 to 80 S complexes. Moreover, the mRNA bound to 48 S complexes was more susceptible to cleavage by an endonuclease than mRNA in polyribosomes, as shown by activating the (2'-5')oligo(A)-dependent endonuclease. This finding is discussed in view of the possible role of eIF-2 alpha kinase and endonuclease in the inhibition of viral mRNA translation in interferon-treated cells.  相似文献   

18.
The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes. 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP. 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP. 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y. 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+. 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y. 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+. However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP. 7) eIF-2y bound [3H]GDP and this binding was significantly enhanced in the presence of Mg2+. Also, [3H]GDP in the preformed eIF-2y X [3H]GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+. Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions. 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y. However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.  相似文献   

19.
Mutants in genes 46 and 47 of bacteriophage T4 exhibit early cessation of DNA synthesis, inability to form a normal rapidly sedimenting DNA intermediate (200S), reduced genetic recombination, and reduced viable phage production. A gene-specific suppressor mutation called das partially restores many of the pleiotropic effects of gene 46-47 mutants (13). Our results indicate that this partial suppression by das is associated with (i) the synthesis of a small fraction of DNA containing long single chains not detectable in 46-47 infection and (ii) a decrease in an "early" function which participates in the degradation of DNA synthesized in the absence of 46-47 functions. However, das does not restore the formation of a normal rapidly sedimenting (200S) DNA intermediate.  相似文献   

20.
Phosphocellulose chromatography of initiation factor eIF-2 from rat liver separates it from a protein fraction which is highly stimulatory for [eIF-2.GTP.Met-tRNAf] ternary complex formation. Evidence is presented which indicates that this stimulatory fraction contains a specific GDPase activity. eIF-2 dependent formation of 40S ribosomal initiation complexes is also enhanced by the GDPase preparation. The enzyme may play a role in the recycling of eIF-2 by removing inhibitory GDP which is generated during 80S initiation complex formation.  相似文献   

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