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H O Voorma

Publications and source records attributed to H O Voorma.

At least 73 records · Page 4Linked to original sources
PubMed1981

Shutoff of neuroblastoma cell protein synthesis by Semliki Forest virus: loss of ability of crude initiation factors to recognize early Semliki Forest virus and host mRNA's.

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing "pH5" system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (+/-80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5' terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M(r), 80,000) and cap-binding protein (M(r), 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.

Animals↗
PubMed1980

The role of eIF-4C in protein synthesis initiation complex formation.

eIF-4C has a pronounced stimulatory effect on initiation complex formation with native 80-S ribosomes (80-Sn) as the only source of ribosomal subunits, but only a small effect when washed 40-S subunits are used. eIF-4C is accessary to eIF-3 in dissociating 80-Sn ribosomes. eIF-4C is present on 40-Sn but absent on 40-Sn dimers, which occur in preparations of native ribosomes and are as such inactive in protein synthesis. eIF-4C dissociates 40-Sn dimers into active monomers. These results can be explained by assuming that the presence of eIF-4C on 40-Sn prevents: (a) premature association with 60-S ribosomal subunits and (b) dimerisation, thus increasing the rate and extent of initiation complex formation.

Animals↗
PubMed1980

Purification of free eukaryotic initiation factors eIF-4A and eIF-4D on Cibacron Blue F3G-A.

The eukaryotic initiation factors eIF-4A and eIF-4D are almost exclusively found in the supernatant of a reticulocyte lysate. The basic steps of general purification schemes failed because these factors do not bind to Sepharoseheparin or, in the of eIF-4A, to phosphocellulose and new procedure had to be devised. Cibacron Blue F3G-A proved to be a successful alternative in the purification of these factors from the excessive load of proteins found in the supernatant.

Animals↗
PubMed1980

The mechanism of action of eukaryotic initiation factor 4C in protein synthesis.

Binding of eukaryotic initiation factor 4C (eIF-4C) to a 40-S initiation complex could be detected by analysis of assay mixtures for 40-S initiation complex formation on columns of Sepharose 6B and on sucrose gradients. On Sepharose 6B columns an almost stoichiometric binding of [14C]eIF-4C was observed, provided that eIF-1, eIF-2, eIF-3, Met-tRNAf and mRNA were included in the assay mixtures. On sucrose gradients the presence of 0.5 microM edeine was an additional prerequisite for stable binding of eIF-4C. The presence of eIF-4C on the 40-S initiation complex resulted in a pronounced increase of its stability, which might explain the observed stimulatory effect of the factor on the binding of Met-tRNAf into initiation complexes. The interaction between eIF-4C and the small ribosomal subunit presumably occurs at an early stage of the initiation process, since eIF-4C was capable of dissociating 80-S ribosomes into subunits. Protein synthesis in a 'pH-5' assay was almost totally dependent on the addition of eIF-4C, when the assay was performed with purified, nearly homogeneous, initiation factors.

Animals↗
PubMed1979

A comparison of the initiation factors of eukaryotic protein synthesis from ribosomes and from the postribosomal supernatant.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernantant. A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.

Animals↗
PubMed1979

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.

Animals↗
PubMed1979

Postribosomal complexes containing eukaryotic initiation factor eIF-2.

Eukaryotic initiation factors are found in the post-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.

Animals↗
PubMed1979

The activity of eukaryotic initiation factor eIF-2 in ternary complex formation with GTP and Met-tRNA.

Ternary complex formation between [3H]Met-tRNAf, [14C]H3-eIF-2, and GTP was measured on nitrocellulose filters. It is shown that [3H]Met-tRNAf and [14C]H3-eIF-2 are present on the filter in equimolar amounts when ATP, creatine phosphate, and creatine kinase are included in the reaction mixture. Under these conditions the factor is 100% active. With small amounts of factor significant losses occur due to adsorption to the wall of the reaction vessels, resulting in seemingly low activities of eIF-2. These losses can be prevented by the presence of "stimulatory" proteins, which enhance the recovery of both [3H]Met-tRNAf and [14C]H3-eIF-2 on the filter but do not alter their ratio.

Animals↗
PubMed1978

Initiation factor IF-3 and the binary complex between initiation factor IF-2 and formylmethionyl-tRNA are mutually exclusive on the 30-S ribosomal subunit.

The formation of 30-S initiation complexes depends strongly on initiation factor IF-3; at molar ratios of IF-3 to 30-S ribosomes up to one a stimulation is observed, whereas at ratios higher than one, initiation complex formation declines strongly. The target of the observed inhibition of fMet-tRNA binding at high concentrations of IF-3 is the 30-S initiation complex itself. On the one hand addition of IF-3 to preformed 30-S initiation complexes leads to a release of bound fMet-tRNA which is linear with the amount of factor added, whereas no effect on isolated 70-S initiation complexes is seen. The release of fMet-tRNA from preformed 30-S initiation complexes is accompanied by a release of IF-2 in a one-to-one molar ratio which is in agreement with our previous findings showing that binding of fMet-tRNA takes place via a binary complex: IF-2 . fMet-tRNA (Eur. J. Biochem. 66, 181--192 and 77, 69--75). On the other hand increasing amounts of both IF-2 and fMet-tRNA relieve the IF-3-induced inhibition of 30-S initiation complex formation. From these findings it is concluded that IF-3 and the IF-2 . fMet-tRNA complex are mutually exclusive on the 30-S ribosome. This implies that under our experimental conditions MS2 RNA binding precedes fMet-tRNA binding if one accepts that the presence of IF-3 on the 30-S subunit is obligatory for messenger binding.

Escherichia coli↗
PubMed1978

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.

Escherichia coli↗
PubMed1977

The involvement of a complex between formylmethionyl-tRNA and initiation factor IF-2 in prokaryotic initiation.

A complex between initiation factor IF-2 and fMet-tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde. The IF-2 - FMet-tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di of triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF-2 - fMet-tRNA complex formation is not influenced by initiation factors IF-1 and IF-3. The binary complex can be bound to the 30-S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF-2 - fMet-tRNA complex and the nucleotide moiety to the 30-S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF-2 - fMet-tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate had no effect.

Aurintricarboxylic Acid↗