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Biomedical subjects

R Benne

Publications and source records attributed to R Benne.

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

Purification and characterization of protein synthesis initiation factor eIF-3 from wheat germ.

eIF-3 from wheat germ is a large multicomponent factor. It sediments at 15 S and is comprised of ten different polypeptides with an Mr value ranging from 26 000 to 135 000; five out of the ten seem to be present in a 1 : 1 stoichiometric ratio, whereas the others appear to occur approximately in a 0.5 to 1 ratio. The factor is active in a partially purified cell-free system derived from wheat germ and in a mammalian model assay system for the synthesis of methionyl puromycin.

Cell-Free System↗
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

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

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

Phosphorylation of eukaryotic protein synthesis initiation factors.

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.

Animals↗