scholarly journals Seed extracts inhibiting protein synthesis in vitro

1980 ◽  
Vol 186 (2) ◽  
pp. 439-441 ◽  
Author(s):  
A Gasperi-Campani ◽  
L Barbieri ◽  
P Morelli ◽  
F Stirpe
Keyword(s):  
Protein Synthesis ◽  
Rabbit Reticulocyte Lysate ◽  
Intact Cells ◽  
Euonymus Europaeus ◽  
Lower Extent ◽  
Reticulocyte Lysate ◽  
Dialysis Membranes ◽  
Inhibiting Protein ◽  
Seed Extracts

Of 33 seed extracts examined, 12 inhibited protein synthesis in a rabbit reticulocyte lysate. This activity seems to be due to a protein, since (i) it was recovered with the (NH4)2SO4 precipitate, (ii) it was retained by dialysis membranes, and (iii) in all cases but one was destroyed by boiling. Only the extracts from the seeds of Adenia digitata and, to a lower extent, of Euonymus europaeus inhibited protein synthesis in intact cells.

scholarly journals Ribosome-inactivating proteins from plant cells in culture

1989 ◽  
Vol 257 (3) ◽  
pp. 801-807 ◽  
Author(s):  
L Barbieri ◽  
A Bolognesi ◽  
P Cenini ◽  
A I Falasca ◽  
A Minghetti ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Phytolacca Americana ◽  
Pokeweed Antiviral Protein ◽  
Antiviral Protein ◽  
Ribosome Inactivating Protein ◽  
Intact Cells ◽  
Ribosome Inactivating Proteins ◽  
Reticulocyte Lysate ◽  
Ic50 Concentration

1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the ‘pokeweed antiviral protein’ (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg.

scholarly journals Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases.

1988 ◽  
Vol 8 (2) ◽  
pp. 993-995 ◽  
Author(s):  
V K Pathak ◽  
D Schindler ◽  
J W Hershey
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Mutant Form ◽  
Two Dimensional Gel Electrophoresis ◽  
Reticulocyte Lysate

The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.

scholarly journals Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells

1980 ◽  
Vol 188 (3) ◽  
pp. 941-944 ◽  
Author(s):  
P N Dalrymple ◽  
L L Houston
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
Indirect Immunofluorescence ◽  
Cultured Cells ◽  
Sodium Dithionite ◽  
Eukaryotic Cells ◽  
Protein Synthesis Inhibition ◽  
Inhibiting Protein ◽  
A Chain

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases

1988 ◽  
Vol 8 (2) ◽  
pp. 993-995
Author(s):  
V K Pathak ◽  
D Schindler ◽  
J W Hershey
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Mutant Form ◽  
Two Dimensional Gel Electrophoresis ◽  
Reticulocyte Lysate

The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.

scholarly journals Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena

1987 ◽  
Vol 244 (2) ◽  
pp. 331-335 ◽  
Author(s):  
P H Andreasen ◽  
H Dreisig ◽  
K Kristiansen
Keyword(s):  
Codon Usage ◽  
Genetic Code ◽  
Subcellular Fraction ◽  
Tetrahymena Thermophila ◽  
Rabbit Reticulocyte Lysate ◽  
Reading Frame ◽  
Universal Genetic Code ◽  
Reticulocyte Lysate ◽  
Translational Termination

The codon usage of Tetrahymena thermophila and other ciliates deviates from the ‘universal genetic code’ in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.

scholarly journals cAMP induces co-translational modification of proteins in IPC-81 cells

1999 ◽  
Vol 342 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Randi HOVLAND ◽  
Anne P. DØSKELAND ◽  
Thor S. EIKHOM ◽  
Bernard ROBAYE ◽  
Stein O. DØSKELAND
Keyword(s):  
Protein Synthesis ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Leukaemia Cell Line ◽  
Camp Analogue ◽  
Dependent Protein ◽  
Induced Apoptosis

An elevated cAMP concentration results in growth arrest and protein synthesis-dependent apoptosis in the promyelocytic leukaemia cell line IPC-81. A comparison of two-dimensional gels of extracts from these cells labelled with [35S]methionine revealed that five distinct protein spots were induced by cAMP in a protein-synthesis-dependent manner. The spots seemed to result from the acidic shift of a precursor protein. The most abundant spot was phospho-actin. The spots induced by cAMP in intact cells were induced by cAMP-dependent protein kinase (cAPK) during the translation in vitro of mRNA from the leukaemia cells. The effect of cAPK was strictly co-translational, none of the spots being induced when cAPK was added after translation. This suggested that the protein spots arose by co-translational phosphorylation catalysed by cAPK. Two of the protein spots, phospho-actin and a protein with a molecular mass of 30 kDa and an isoelectric point of 4.5, were studied further with respect to expression. They were produced during the whole pre-apoptotic period, had cellular half-lives of several hours and were induced by the same concentrations of cAMP analogue that induced apoptosis. It is suggested that the accumulation of co-translationally modified proteins could be important for long-term cAMP signalling.

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