Association of initiation factor eIF-2 with a rapidly sedimenting fraction from Ehrlich ascites-tumour cells

Clemens, M J; Pain, Virginia M.

doi:10.1042/bj1940357

Cited by 7 publications

(3 citation statements)

References 26 publications

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“…All cells were routinely cultured either at 34'C or 37°C as appropriate, in 75-cm2 tissue-culture flasks or in stirred spinner culture vessels in a-minimal essential medium plus 8 % (by vol.) foetal calf serum [33].…”

Section: Cell Culturementioning

confidence: 99%

A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation

Pollard

Galpine

Clemens

1989

European Journal of Biochemistry

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Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH to the non-permissive temperature of 39.5"C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2 . GTP . Met-tRNAf ternary complexes and thus in the formation of 43s ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2a subunit. It has previously been shown in other systems that phosphorylation of eIF-2a slows the rate of recycling of eIF-2 . GDP to eIF-2. GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2a by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsHl cells. Thus the observed increased phosphorylation of eIF-2a at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo.Using a single-step temperature revertant of fsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5"C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALe" in vivo in tsHl cells at 39.5"C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging p e r se is not involved in the regulatory mechanism.Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the a subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous elF-2a, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5"C.Exposure of Chinese hamster ovary (CHO) cells containing temperature-sensitive aminoacyl-tRNA synthetase mutations to non-permissive conditions produces a rapid inhibition of the initiation of protein synthesis [l]. Using a temperature-sensitive leucyl-tRNA synthetase mutant, t s H l , we have shown that this is due to inhibition of the ability of initiation factor eIF-2 to form eIF-2 . GTP . Met-tRNA, ternary complexes and 43s pre-initiation complexes on native small ribosomal subunits [2]. Such impairment of eIF-2 activity is a consequence of a reduction in the rate of regeneration of the binary complex, eIF-2 . GTP, from eIF-2 . GDP by the guanine nucleotide exchange factor, eIF-2B (sometimes known as GEF). This reduces the availability of eIF-2 . GTP to participate in ternary complex formation with the initiator Met-tRNAY" and consequently in the formation of the 43s ribosomal complexes necessary to initiate mRNA binding [3]. The inh...

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Section: Cell Culturementioning

confidence: 99%

A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation

Pollard

Galpine

Clemens

1989

European Journal of Biochemistry

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“…The activity of the eIF-2 factor was determined by the formation of a ternary complex between eIF-2, GTP and [35S]methionyl-tRNA. The procedure was that described by Gupta et al [42], as modified by Clemens & Pain [43]. Extracts were prepared from livers of rats weighing 120 g. Either 0.2 ml of 0.9 % NaCl or 10 nM-vasopressin dissolved in the same solution was slowly injected into the portal vein; 5 min thereafter, two liver biopsies (approx.…”

Section: Assay Of Eif-2 Activitymentioning

confidence: 99%

“…The composition of the incubation mixture for the assay of eIF-2 activity was as described in ref. [43], and contained 5 % of postribosomal supernatant. This concentration was found to give a linear response in the formation of ternary complexes.…”

Section: Assay Of Eif-2 Activitymentioning

confidence: 99%

Effect of vasopressin on the regulation of protein synthesis initiation in liver cells

Menaya

Parrilla

Ayuso

1988

Biochemical Journal

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Vasopressin was found to be an effective inhibitor of protein labelling in isolated liver cells. Its effect shows the following distinct characteristics: (1) in contrast with alpha-adrenergic agonists, its effect is observable under a wide range of cellular Ca2+-loading conditions; (2) it is not influenced by the nutritional state of the animal. The lack of vasopressin effect on valine production, and its ability to decrease protein labelling from near-saturation concentrations of [3H]valine, indicate that the observed variations in protein labelling reflect actual changes in the rate of protein synthesis. The action of vasopressin is primarily exerted on the initiation step of protein synthesis and this effect is accompanied by a decreased activity of eukaryotic initiation factor 2. Activators of protein kinase C showed similar but not additive effects on protein synthesis, as did vasopressin. It seems plausible to conclude that protein kinase C activation may play an important regulatory role in hepatic protein synthesis as a transducer of hormonal and perhaps other type of signals.

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Function of eukaryotic ribosomes

Bielka¹

1982

The Eukaryotic Ribosome

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Association of initiation factor eIF-2 with a rapidly sedimenting fraction from Ehrlich ascites-tumour cells

Cited by 7 publications

References 26 publications

A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation

A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation

Effect of vasopressin on the regulation of protein synthesis initiation in liver cells

Function of eukaryotic ribosomes

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