scholarly journals Synthesis of phosphoenolpyruvate carboxykinase (guanosine triphosphate) by isolated liver polyribosomes

1974 ◽  
Vol 144 (2) ◽  
pp. 199-207 ◽  
Author(s):  
F J Ballard ◽  
M F Hopgood ◽  
L Reshef ◽  
S Tilghman ◽  
Richard W. Hanson
Keyword(s):  
Protein Synthesis ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Sodium Dodecyl ◽  
Enzyme Synthesis ◽  
Mrna Synthesis ◽  
Synthesis System ◽  
Isolated Liver ◽  
Protein Synthesis System ◽  
Specific Mrna

1. Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was synthesized by postmitochondrial supernatants of rat liver in the presence of appropriate salts, an energy supply and [3H]leucine. Synthesis of enzyme released from polyribosomes was detected by immunoprecipitation with specific antibody followed by electrophoresis of the dissolved antibody–antigen precipitates on sodium dodecyl sulphate–polyacrylamide gels in the presence of a14C-labelled enzyme marker. 2. Enzyme synthesis in vitro occurs predominantly on free rather than bound polyribosomes. 3. Starved animals in which de-induction of phosphoenolpyruvate carboxykinase (GTP) had been initiated by re-feeding for 2h had a markedly decreased rate of enzyme synthesis, whether the measurements were made after injection of radioactive leucine into the intact animal or if synthesis was determined in vitro. 4. The low rate of enzyme synthesis by liver polyribosomes from re-fed animals was not due to the absence of soluble factors, nor could it be increased by the addition of cyclic AMP to the protein synthesis system. 5. Phosphoenolpyruvate carboxykinase (GTP) synthesis in vitro is diminished relative to total protein synthesis when the postmitochondrial supernatant is kept at 0°C for several hours before measurement of protein synthesis. Since this effect is blocked by heparin, it is probably caused by selective ribonuclease attack on enzyme mRNA. 6. De-induction of phosphoenolpyruvate carboxykinase (GTP) is tentatively explained as being due to a transcriptional block in specific mRNA synthesis, followed by rapid degradation of existing message.

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system

2010 ◽  
Vol 32 (7) ◽  
pp. 897-902 ◽  
Author(s):  
Satoshi Mikami ◽  
Tominari Kobayashi ◽  
Kodai Machida ◽  
Mamiko Masutani ◽  
Shigeyuki Yokoyama ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Human Cell ◽  
Synthesis System ◽  
In Vitro Protein Synthesis ◽  
Protein Synthesis System ◽  
Vitro Protein

scholarly journals Identification of a gene that makes a protein by using a cell free protein synthesis system

2021 ◽  
Vol 8 (4) ◽  
pp. 124-125
Author(s):  
Umair Masood
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Living Cell ◽  
Molecular Size ◽  
Synthesis System ◽  
Free Protein ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

A living cell could be genetically modified to perform a function such as the production of a protein. However, these genetic modifications often conflict with normal cellular function and result in a mutation. Defects can be overcome through removing the bacterial membrane which leaves the lysate that is performing both transcription and translation. The cell free-protein synthesis is also known as in vitro protein synthesis and is the production of a protein without using a living cell. The gene is acting as instructions to make the protein. If we can isolate a gene and then apply a cell free protein synthesis system after synthesis the protein and run on gel-electrophoresis we can identify a gene on the basis of the protein. Gel electrophoresis is a laboratory technique used to ______ contrasting proteins according to molecular size and charge.

scholarly journals Synthesis of δ-aminolaevulinate synthase by isolated liver polyribosomes

1976 ◽  
Vol 158 (2) ◽  
pp. 391-400 ◽  
Author(s):  
M J Whiting
Keyword(s):  
Protein Synthesis ◽  
Chick Embryo ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Synthesis ◽  
Pulse Labelling ◽  
General Protein Synthesis

1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta-aminolaevulinate synthase in vitro. These results suggest that haemin represses the synthesis of delta-aminolaevulinate synthase by decreasing the amount of mRNA for the enzyme available for translation.

scholarly journals A factor converting monoribosomes into polyribosomes during protein synthesis in vitro

1968 ◽  
Vol 110 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Brian B. Cohen
Keyword(s):  
Protein Synthesis ◽  
Potassium Chloride ◽  
Active Component ◽  
Synthesis System ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described previously (Miller, Hamada, Yang, Cohen & Schweet, 1967). The participation of the extract in cell-free protein synthesis was studied. Purified polyribosomes were isolated and converted into monoribosomes by incubation in the cell-free protein-synthesis system. The monoribosomes were isolated and found to be unable to synthesize protein in the cell-free system. The addition of the ribosomal extract to the system stimulated protein synthesis. This was accompanied by the conversion of some of the monoribosomes into polyribosomes. The active component or components of the extract were shown to be protein.

scholarly journals De novo expression and antibacterial potential of four lactoferricin peptides in cell-free protein synthesis system

2021 ◽  
Vol 29 ◽  
pp. e00583
Author(s):  
Nawal Abd El-Baky ◽  
Maie Ahmed Elkhawaga ◽  
Eman Shawky Abdelkhalek ◽  
Mona Mohammed Sharaf ◽  
Elrashdy Mustafa Redwan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
De Novo ◽  
Synthesis System ◽  
Free Protein ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis ◽  
Antibacterial Potential

scholarly journals Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves

1975 ◽  
Vol 146 (3) ◽  
pp. 675-685 ◽  
Author(s):  
S G Siddell ◽  
R J Ellis
Keyword(s):  
Energy Source ◽  
Protein Synthesis ◽  
Large Subunit ◽  
Sodium Dodecyl ◽  
Supernatant Fraction ◽  
Pisum Sativum L ◽  
Fraction I ◽  
Plastid Ribosomes

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic ‘map’ of its L-(35S)methionine-labelled peptides with the tryptic ‘map’ of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.

Effects of prolactin on testosterone-induced growth and protein synthesis in rat accessory sex glands

1983 ◽  
Vol 96 (3) ◽  
pp. 407-416 ◽  
Author(s):  
R. Jones ◽  
P. R. Riding ◽  
M. G. Parker
Keyword(s):  
Protein Synthesis ◽  
Total Protein ◽  
Sodium Dodecyl ◽  
Chronic Administration ◽  
Specific Protein ◽  
Ventral Prostate ◽  
Plasma Prolactin ◽  
Accessory Sex Glands ◽  
Caput Epididymidis

The relative importance of testosterone and prolactin in regulating growth and protein synthesis in rat accessory sex glands has been investigated. Protein synthesis was measured by incubating tissue minces in vitro with [35S]methionine and analysing labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Plasma prolactin was assayed by radioimmunoassay. Results showed that castration for 8 days significantly reduced wet weights and total protein synthesis in the ventral prostate, dorsolateral prostate and caput epididymidis, but that these effects could be reversed by exogenous testosterone. Similarly, the specific incorporation of [35S]methionine into four polypeptides in the ventral prostate, two polypeptides in the dorsolateral prostate and two polypeptides in the caput epididymidis was lowered by castration but markedly stimulated by testosterone. Acute or chronic administration of 2-bromo-α-ergocryptine to animals in combination with testosterone had no significant effect on any of the parameters measured, although the drug reduced circulating prolactin to undetectable levels. In addition, exogenous prolactin given alone, or in combination with testosterone, to hypophysectomized rats had no effect on general or specific protein synthesis. The induction of hyperprolactinaemia in immature or mature rats with pituitary homographs had no effect on testosterone-stimulated growth of any accessory gland, although it caused a significant stimulation of total protein synthesis in the dorsolateral prostate and coagulating glands. However, this was a generalized effect as it did not increase the specific incorporation of [35S]methionine into androgen-dependent proteins. The results do not indicate a major role for prolactin in regulating androgen responsiveness of male accessory sex glands in the rat.

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