Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors

We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNA_f · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP.     

Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity.

The cleavage of the p220 subunit of eukaryotic initiation factor 4F (eIF-4F) that is induced by the poliovirus protease 2A has been shown previously to require another translation initiation factor, eIF-3. The role of eIF-3 in this cleavage reaction, however, is not known. An antiserum was raised against human eIF-3 and used to analyze the eIF-3 subunit composition in poliovirus-infected and uninfected HeLa cells and after incubation of eIF-3 in vitro with viral 2A protease. No evidence for 2Apro-dependent cleavage of any eIF-3 subunit was detected. Infected cells contain an activity that catalyzes the cleavage of p220 to a specific set of cleavage products. This activity is thought to be an activated form of a latent cellular protease. The p220-specific cleavage activity was partially purified. It was resolved from eIF-3 by both gel filtration and anion-exchange chromatography. Neither intact eIF-3 nor any detectable subunits of eIF-3 were found to copurify with the p220-specific cleavage activity. The latter activity behaves as a protein of 55,000 to 60,000 molecular weight and is inhibited by alkylating agents and metals, which indicates the presence of essential thiol groups. When this activity was incubated with partially purified p220, cleavage occurred only in the presence of eIF-3. Thus, eIF-3 appears to play a role in the p220 cleavage cascade which is subsequent to the 2Apro-induced activation of the p220-specific protease.     

Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25–40% saturation (0°C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation. eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3 sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system. Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.     

Phosphorylation of translational initiation factor 3 (eIF-3) by cyclic AMP-regulated protein kinase.

Translational initiation factor 3 (eIF-3) is phosphorylated by the cyclic AMP-regulated protein kinases from rabbit reticulocytes. eIF-3 is a large molecular weight complex which facilitates binding of the ternary complex containing met tRNA_f, GTP and initiation factor 2 to 40S ribosomal subunits. A single polypeptide with a molecular weight of 130,000 is modified. The phosphorylation is dependent upon the presence of cyclic AMP and is inhibited by the inhibitor protein diagnostic for cyclic AMP-regulated protein kinase. Assuming a molecular weight of 700,000 for eIF-3, one mole of phosphate is incorporated per mole of eIF-3. Thus the phosphorylation of two interacting components of the protein synthesizing system, 40S ribosomal subunits and eIF-3, is controlled by cyclic AMP.     

Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factors 2.

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52 000, 45 000 and 42 000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GPT and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2--3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.     

Identification of a new protein synthesis initiation factor from wheat germ

A previously unidentified factor has been isolated from wheat germ that stimulates globin mRNA-directed polypeptide synthesis in vitro. This factor is separated from eukaryotic initiation factor (eIF)-4B by chromatography on m7GTP-Sepharose. eIF-4B binds to m7GTP-Sepharose, whereas the stimulatory factor does not. Further purification of the factor yields a preparation that contains one major polypeptide with a molecular weight of approximately 59,000, This factor enhances the binding of globin mRNA to 40 S ribosomal subunits in the presence of eIF-2, eIF-3, eIF-4A, and either eIF-4B or eIF-4F and has been designated eIF-4G.     

Factors from wheat germ that enhance the activity of eukaryotic initiation factor eIF-2. Isolation and characterization of Co-eIF-2 beta

A factor has been isolated from wheat germ that enhances the ability of initiation factor 2 (eIF-2) to form a ternary complex with GTP and Met-tRNAf and enhances the binding of Met-tRNAf to 40 s ribosomal subunits. This factor, designated Co-eIF2 beta, is a monomeric protein with a molecular weight of approximately 83,000. Wheat germ eIF-2 forms a stable binary complex with GDP but not with GTP. Co-eIF-2 beta enhances the formation of an eIF-2 . GDP complex, but does not enable eIF-2 to form a stable complex with GTP.     

Characterization of initiation factor 3 from wheat germ. 2. Effects of polyclonal and monoclonal antibodies on activity

Rabbit polyclonal antibodies to wheat germ initiation factor 3 (eIF-3) were obtained and were shown to react strongly with 4 of the 10 subunits of eIF-3 (pp116, pp87, pp56, and pp36). Two mouse monoclonal antibodies were obtained, one of which reacts specifically with pp87 and one of which reacts specifically with pp36. Highly purified anti-pp87 has no effect on the activity of eIF-3. Highly purified polyclonal antibodies and anti-pp36 inhibit the ability of eIF-3 to support polypeptide synthesis in vitro and the ability of eIF-3 to support mRNA binding to 40S ribosomal subunits. These results provide additional evidence that pp116, pp87, and pp36 are in exposed positions in the eIF-3 particle and that pp36 is essential for activity.     

Partial purification and characterization of reticulocyte phosphatase with activity for phosphorylated peptide initiation factor 2

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.     

Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000

Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.     

Purified eukaryotic initiation factor 2 from calf liver consists of two polypeptide chains of 48,000 and 38,000 daltons.

Eukaryotic initiation factor 2 (eIF-2), which specifically binds Met-tRNAMetf and forms stable ternary complexes with GTP, has been purified from ribosomal salt wash proteins from calf liver. The purified factor exhibits only two polypeptide bands of Mr = 48,000 and 38,000 following electrophoresis in 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. Densitometric tracings show the two polypeptides are present in a molar ratio of 1:1. This suggests a Mr = 86,000 for the native enzyme, a value which agrees with the apparent molecular weight determined by physical methods. Less pure preparations of eIF-2 show additional polypeptide bands, including 50,000- and 46,000-dalton bands, all of which can be removed by further purification without affecting the activity of eIF-2.     

The 60 S ribosomal subunit as a carrier of eukaryotic initiation factor 2 and the site of reversing factor activity during protein synthesis

Studies on the recycling of eukaryotic initiation factor 2 (eIF-2) during protein synthesis in normal and heme-deficient reticulocyte lysates indicate that eIF-2 binds physiologically to the 60 S ribosomal subunit. Several findings suggest that the 60 S subunit serves as a carrier for eIF-2 during protein synthesis. The addition of purified eIF-2 (beta-32P) to normal hemin-supplemented lysates results in its binding to polyribosomal 60 S subunits; the binding is temperature-dependent. In lysates inhibited by heme deficiency, phosphorylated eIF-2 alpha can be detected on polyribosomal 60 S subunits early in the initial linear phase of protein synthesis; after polyribosomal disaggregation and shut-off of protein synthesis, phosphorylated eIF-2 alpha accumulates on free 60 S ribosome subunits and on the 60 S subunits of 80 S ribosome couples. The phosphorylated eIF-2 alpha associated with the 60 S subunits in heme-deficient lysates appears to be present as the binary complex [eIF-2 (alpha P) X GDP]; the binding of this complex to the 60 S subunit is tight and is not affected by treatment with 25 mM EDTA or by sedimentation in sucrose gradients. Reversal of the inhibition of protein synthesis in heme-deficient lysates by the addition of reversing factor results in a rapid binding of reversing factor to the 60 S subunits and a concomitant dissociation of [eIF-2(alpha P) X GDP]. These findings suggest that the [eIF-2 X GDP] binary complex formed during the assembly of the 80 S initiation complex binds to the 60 S subunit of polyribosomes and is subsequently released by the action of reversing factor.     

Natural mRNA is required for directing Met-tRNA(f) binding to 40S ribosomal subunits in animal cells: involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation

Two protein factors, eIF-2 as well as a high molecular weight protein complex from reticulocyte ribosomal high-salt wash which we term Co-eIF-2, promote Met-tRNA(f) binding to 40S ribosomes. This binding is dependent on the presence of an AUG codon or natural mRNAs [Roy et al. (1984) Biochem. Biophys. Res. Commun. 122, 1418-1425]. Co-eIF-2 contains two component activities, Co-eIF-2A and Co-eIF-2C. Previously, we have purified an 80-kDa polypeptide containing Co-eIF-2A activity and showed that this polypeptide is a component of Co-eIF-2 and is responsible for Co-eIF-2A activity in Co-eIF-2 [Chakravarty et al. (1985) J. Biol. Chem. 260, 6945-6949]. We now report purification of a protein complex (subunits of Mr 180K, 110K, 65K, 63K, 53K, 50K, 43K, and 40K) containing Co-eIF-2C activity and devoid of Co-eIF-2A activity. In SDS-PAGE, the purified Co-eIF-2C preparation and an eIF-3 preparation (purified in Dr. A. Wahba's laboratory) separated into seven similar major polypeptides (Mr 110K, 65K, 63K, 53K, 50K, 43K, and 40K). The 50-kDa polypeptide in Co-eIF-2C was immunoreactive with a monoclonal antibody against eIF-4A (50 kDa). We have studied the roles of purified Co-eIF-2A and Co-eIF-2C activities in ternary and Met-tRNA(f).40S ribosome complex formation. The results are as follows: (1) At low and presumably physiological factor concentration (30 nM), eIF-2 did not form detectable levels of ternary complex. Moreover, such complex formation was totally dependent on the presence of Co-eIF-2A and/or Co-eIF-2C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

A new purification scheme for elongation factor 1 from rabbit reticulocytes and investigation of the homology of the subunits with those of initiation factor 2.

The aim of this work was to compare the subunits of the elongation factor EF-1 and the initiation factor eIF-2 from rabbit reticulocytes. We devised a simple procedure for the purification of EF-1: stepwise chromatography on heparin-Sepharose, separation of the heavy form by sucrose gradient centrifugation, and a final step of stepwise chromatography on RNA-Sepharose. The heparin-Sepharose column also clearly separated EF-1 and EF-2 within one chromatographic step. The EF-1 was 350-fold puried and the yield was 10%. This preparation showed after electrophoresis on polyacylamide gels in the presence of sodium dodecyl sulfate three bands corresponding to those described by others as the subunits, with Mr of 54000, 49000 and 29200. An additional band of Mr 34000 was present but no others. The 49000-Mr and 34000-Mr bands corresponded exactly in molecular weight to two of three subunits of eIF-2. A more detailed comparison was therefore made of all subunits of EF-1 and eIF-2. This was done by examination of chymotryptic fingerprints on polyacrylamide gel electrophoresis. No evidence for homology between EF-1 and eIF-2 was found. However, the two larger subunits of eIF-2 had a majority of chymotryptic fragments in common, thus indicating some homology between these polypeptides.