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H Mellor

Publications and source records attributed to H Mellor.

31 records · Page 2Linked to original sources
PubMed1995

Structure and sequence of the gene encoding the alpha-subunit of rat translation initiation factor-2B.

Eukaryotic initiation factor-2B (eIF-2B) is a heteropentameric guanine nucleotide exchange factor involved in the recycling of eIF-2 during the translation initiation phase of protein synthesis. The activity of eIF-2B is controlled by diverse mechanisms under a wide range of conditions. As part of our studies into the control of the expression of eIF-2B subunit mRNAs, we have isolated and characterized the rat gene encoding the alpha-subunit of eIF-2B. The gene is comprised of 9 exons which are contained within 8.5 kb of genomic DNA. A comparison of the 5'-flanking region of the gene with that of the previously isolated eIF-2B epsilon gene reveals that both possess a potential binding site for the alpha-Pal transcription factor as well as several other regions of homology.

Animals↗
PubMed1994

Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver.

Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein consisted of five nonidentical subunits with apparent molecular weights ranging from 30.9 to 89.1 kDa. The holoprotein was characterized in terms of its Stokes radius and frictional coefficient. The isoelectric points for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximately 6.0, respectively; the alpha- and delta-subunits did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater than 8.5. The purified protein was used as antigen to generate monoclonal antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal antibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were then used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found to be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, supporting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2.eIF-2B complex. The purified protein was also used as substrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here are the first to show a direct quantitation of eIF-2 and eIF-2B in different tissues. They also provide evidence that the epsilon-subunit of eIF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.

Animals↗
PubMed1994

Cloning and characterization of a cDNA encoding rat PKR, the double-stranded RNA-dependent eukaryotic initiation factor-2 kinase.

We have isolated and sequenced a full-length cDNA encoding the double-stranded RNA-dependent protein kinase PKR from rat. The derived amino acid sequences of the protein kinase and RNA-binding domains are highly conserved between the rat, human and mouse enzymes. In addition, sequence elements in the 5'- and 3'-untranslated regions are also conserved between species.

Animals↗
PubMed1994

Brefeldin A inhibits protein synthesis through the phosphorylation of the alpha-subunit of eukaryotic initiation factor-2.

Brefeldin A is a fungal metabolite which disrupts protein traffic through the Golgi apparatus and thereby inhibits protein secretion. Recently, it has been shown that Brefeldin A also causes a marked decrease in the rate of protein synthesis in cells in culture [1992, FEBS Lett. 314, 371-374]. We show here that treatment of rat GH3 pituitary cells with Brefeldin A leads to an inhibition of protein synthesis at the level of peptide-chain initiation through a mechanism involving the phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha).

Animals↗
PubMed1994

Cloning and characterization of cDNA encoding rat hemin-sensitive initiation factor-2 alpha (eIF-2 alpha) kinase. Evidence for multitissue expression.

Reticulocytes contain an eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase that is negatively regulated by hemin. This protein kinase, which has been termed heme-regulated inhibitor and heme-controlled repressor (HCR), has a strong inhibitory effect on the initiation of protein synthesis and plays an important role in translational control in these cells. Previous evidence has suggested that HCR may be an erythroid-specific enzyme. As reported herein, we have cloned and characterized the cDNA encoding an eIF-2 alpha kinase from rat brain. The predicted amino acid sequence of the kinase from rat brain shows 82% homology to rabbit reticulocyte HCR with the greatest variation concentrated in a central region of approximately 135 amino acids located between protein kinase subdomains IV and VI. We have expressed the rat brain cDNA in Escherichia coli and have demonstrated that the recombinant enzyme is a hemin-sensitive eIF-2 alpha kinase. We have also shown that mRNA recognized by the rat brain cDNA is expressed in a wide range of non-erythroid rat tissues at a lower but significant level compared with reticulocytes. These findings raise the possibility of additional, uncharacterized roles for HCR in non-erythroid cells.

Amino Acid Sequence↗
PubMed1993

Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.

Adenoviridae↗
PubMed1993

Purification and characterisation of an initiation-factor-2 kinase from uninduced mouse erythroleukaemia cells.

Mouse erythroleukaemia (MEL) cells, which have not been induced into erythroid development, contain a protein kinase (MKu) which phosphorylates the alpha subunit of protein-synthesis-initiation factor 2 (eIF-2 alpha). In this paper, we show that this kinase phosphorylates both eIF-2 alpha and a synthetic peptide based on the phosphorylation site in eIF-2 alpha at Ser51, the target residue for other eIF-2 alpha kinases. Consistent with this, prior treatment of eIF-2 with MKu impaired the exchange of bound GDP for GTP which is catalysed by the exchange factor eIF-2B. Using a modified cell-free translation system, we have shown that MKu inhibits translation, consistent with the above observations concerning the site of phosphorylation and the effect of phosphorylation on eIF-2B-mediated guanine-nucleotide exchange. MKu has been purified and its properties have been compared with those of the haem-controlled repressor eIF-2 alpha kinase (HCR) from rabbit reticulocytes. Its behaviour on gel filtration is similar to that of HCR, while its behaviour on anion exchange resembles that of certain phosphorylated species of HCR. Highly purified preparations of MKu contain a protein with an apparent molecular mass of 98 kDa which comigrates with HCR on SDS/PAGE. This protein undergoes phosphorylation when incubated in the presence of Mg(2+)-ATP, and both this apparent autophosphorylation and the activity of the kinase against eIF-2 alpha are inhibited by the same, low, (10 microM) concentrations of haemin. Phosphorylation of the 98-kDa components present in the MEL-cell kinase preparation and in purified rabbit reticulocyte HCR occurs on serine and threonine residues. Analysis of these phosphoproteins by peptide mapping reveals significant differences in their structures, indicating that they may be closely related, but are certainly not identical.

Amino Acid Sequence↗
PubMed1991

A synthetic peptide substrate for initiation factor-2 kinases.

A peptide P(45-56) corresponding to residues 45-56 (sequence: ILLSELSRRRIR) of eIF-2 alpha was synthesised. It was phosphorylated by both of the well characterised eIF-2 alpha kinases viz.; the heme-controlled repressor (HCR) and the double stranded RNA-dependent inhibitor (dsI). Of four other protein kinases tested only protein kinase C (PKC) phosphorylated P(45-56), with complete dependence on phosphatidylserine. Only the residue corresponding to serine-51 in eIF-2 alpha was phosphorylated by HCR, dsI or PKC. The phosphorylation of the peptide by dsI and the phosphorylation of eIF-2 alpha by dsI or HCR showed sigmoidal kinetics with respect to substrate concentration.

Amino Acid Sequence↗
PubMed1989

Whole blood procoagulant activity in breast and colorectal cancer.

Whole blood procoagulant activity was determined by measuring the recalcification time of citrated blood, with and without the addition of bacterial endotoxin, in patients with breast cancer (n = 39), colorectal cancer (n = 20), benign breast disease (n = 15), benign colorectal disease (n = 11), normal volunteers (n = 15) and inpatients with non-malignant disease (n = 22). The median clotting times of those samples incubated with endotoxin were significantly shorter in the patients with breast and colorectal cancer compared with normal controls. Furthermore, significant differences between the median clotting times of stimulated and unstimulated samples within each subject group were observed only in the two cancer groups. There was no correlation between whole blood procoagulant activity and absolute monocyte counts, with histological staging or with plasma concentrations of plasma fibrinopeptide A. The results suggest that blood from patients with cancer is more sensitive to endotoxin stimulation than that from normal or benign controls, but that in its present form the technique cannot be used to distinguish between malignant and non-malignant disease.

Adult↗
PubMed1983

Oxygen affinity of the respiratory chain of Acanthamoeba castellanii.

Apparent Km values for O2 for the soil amoeba Acanthamoeba castellanii determined polarographically and by bioluminescence gave similar values (0.37 and 0.41 microM respectively). Mitochondria oxidizing succinate or NADH in the presence or absence of ADP gave values in the range 0.21-0.36 microM-O2. Oxidation of respiratory-chain components to 50% of the aerobic steady states in intact cells was observed at the following O2 concentrations: cytochrome aa3, 0.1-0.25 microM; cytochrome c, 0.3-0.6 microM; cytochrome b, 0.35-0.45 microM; flavoprotein, 2 microM. In isolated mitochondria corresponding values for a-, c- and b-type cytochromes were 0.007, 0.035-0.05 and 0.06-0.09 microM-O2. It is concluded that an O2 gradient exists between plasma membrane and mitochondria in A. castellanii.

Amoeba↗