THE EFFECTS OF PHENYLALANINE ANALOGUES ON THE METABOLISM OF PHENYLALANINE IN RATS

1967 ◽  
Vol 45 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Claude Godin ◽  
Gail Dolan
Keyword(s):  
Amino Acid ◽  
Radioactive Amino Acid

Rats expired 14CO2 slightly faster when DL-phenylalanine-1-14C rather than DL-phenylalanine-3-14C was injected. An injection of DL-fluorophenylalanine-3-14C produced more radioactive CO2 than did phenylalanine-3-14C; DL-beta-thienylalanine-3-14C produced only trace amounts of 14CO2 but significant levels of radioactivity in the urine. When an excess of one of the nonradioactive phenylalanine analogues was injected at the same time as radioactive phenylalanine, the catabolism of the radioactive amino acid was accelerated. This was shown by an increase in the radioactivity in both the CO2 and urine when beta-thienylalanine or 2-amino-3-phenylbutanoic acid was used, and in the urine alone when fluorophenylalanine was used. The incorporation of radioactive phenylalanine into tissue proteins was decreased when one of the nonradioactive phenylalanine analogues was injected simultaneously. However3 the incorporation into proteins of tyrosine-14C derived from injected phenylalanine-14C was increased under these conditions.

scholarly journals A Label-Free Assay for Aminoacylation of tRNA

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1173
Author(s):  
Howard Gamper ◽  
Ya-Ming Hou
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Cell Growth ◽  
Quantitative Measurement ◽  
Label Free ◽  
Radioactive Amino Acid ◽  
Protein Synthesis Machinery ◽  
Wide Range ◽  
Health And Disease

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3′-32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3′-32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.

scholarly journals Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1971 ◽  
Vol 123 (3) ◽  
pp. 355-365 ◽  
Author(s):  
S. A. M. Khairul Bashar ◽  
J. H. Parish ◽  
Marjorie Brown
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Enzyme Synthesis ◽  
Supernatant Fraction ◽  
Amino Acid Residues ◽  
Radioactive Amino Acid ◽  
Tryptophanase Activity

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).

scholarly journals STUDIES ON PROTEIN SYNTHESIS IN VITRO

1957 ◽  
Vol 41 (1) ◽  
pp. 219-231 ◽  
Author(s):  
A. Korner ◽  
H. Tarver
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Free Amino ◽  
Opposite Effect ◽  
Specific Activity ◽  
Rapid Rate ◽  
Radioactive Amino Acid ◽  
Release Data

The release of radioactive amino acid from the particulate fractions separated from the prelabelled livers of rats by centrifugation has been studied under various conditions. Although pure fractions may not have been obtained, great differences in behavior were observed. In the mitochondria and nuclei dinitrophenol (10–4 M) causes an inhibition of release, but in microsomes the opposite effect is observed. When the incubation medium is fortified with ATP and phosphocreatine, release is inhibited. In microsomes and nuclei the inhibition proceeds to the extent that the incorporation of preformed radioactive amino acid occurs. Protein is synthesized at a rapid rate. In incubations longer than 1 hour there is always a release of radioactive amino acid. It is concluded from these results that the interpretation of release data from slices or systems such as those studied is impossible without further information concerning some of the unknown variables. The most important unknown is the specific activity of the "free" amino acid in the particulates and the effect of carrier amino acid in the medium of this specific activity.

Identification and optimization of the effects of endogenous free amino acids on the translational activity of the wheat germ cell-free system

1983 ◽  
Vol 61 (10) ◽  
pp. 1090-1095 ◽  
Author(s):  
Vasek A. Mezl
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Germ Cell ◽  
Free Amino Acids ◽  
Free Amino ◽  
Wheat Germ ◽  
Free System ◽  
Cell Free System ◽  
Radioactive Amino Acid ◽  
Wheat Germ Extracts

This study identifies factors that are responsible for unpredictable problems associated with the wheat germ cell-free translation system. It is shown that variable amounts of an inhibitor of radioisotope incorporation are routinely included in wheat germ cell-free system preparations, even though this activity can be readily reduced by discarding the appropriate fractions of the wheat germ extracts. This inhibition of the incorporation of radioactive amino acid appears to be due to the production of large amounts of some amino acids by wheat germ extracts. Since the free amino acids that occur at high concentrations are described, the results of this study allow one to predict and to minimize this problem. The results also indicate that the choice of the radioactive amino acid precursor used for a translation should be based not only on the abundance of the amino acid in the protein that is being made, but also on the concentration of this amino acid in the wheat germ preparation.

Radioactive amino acid incorporation into the rat pancreatic juice proteins

1957 ◽  
Vol 13 (4) ◽  
pp. 158-159 ◽  
Author(s):  
Hanna A. Rothschild ◽  
G. C. Hirsch ◽  
L. C. U. Junqueira
Keyword(s):  
Amino Acid ◽  
Pancreatic Juice ◽  
Amino Acid Incorporation ◽  
Radioactive Amino Acid ◽  
Acid Incorporation

The localization of radioactive amino acid taken up into the outer segments of frog (rana pipiens) rods

1969 ◽  
Vol 9 (3) ◽  
pp. 385-IN11 ◽  
Author(s):  
Frederick G. Bargoot ◽  
Theodore P. Williams ◽  
Lloyd M. Beidler
Keyword(s):  
Amino Acid ◽  
Rana Pipiens ◽  
Radioactive Amino Acid ◽  
Outer Segments

scholarly journals Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1971 ◽  
Vol 125 (2) ◽  
pp. 643-653 ◽  
Author(s):  
J. H. Parish ◽  
S. A. M. Khairul Bashar ◽  
N. L. Brown ◽  
Marjorie Brown
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Ribonucleic Acid ◽  
Supernatant Fraction ◽  
23S Rrna ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Radioactive Amino Acid ◽  
Terminal Amino

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.–) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.

scholarly journals Inhibition of Hemoglobin Synthesis by Cyanate In Vitro

Blood ◽  
1974 ◽  
Vol 43 (1) ◽  
pp. 57-68 ◽  
Author(s):  
Blanche P. Alter ◽  
Yuet Wai Kan ◽  
David G. Nathan
Keyword(s):  
Amino Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Hemoglobin Synthesis ◽  
Radioactive Amino Acid ◽  
A Cell ◽  
Red Cell Survival

Abstract Cyanate inhibits sickling and prolongs red cell survival in sickle cell anemia. However, cyanate markedly inhibits hemoglobin synthesis in vitro. Incorporation of radioactive amino acid into hemoglobin by human sickle reticulocytes or bone marrow and by rabbit reticulocytes (whole cell or cell-free lysate) was inhibited by as little as 2 mM cyanate and abolished by 50 mM. Both alpha- and beta-S chains were equally affected. The inhibition was only partially reversible by washing the cells after exposure to cyanate. Transport of radioactive amino acid into the cell was not impaired, and free intracellular amino acid was not carbamylated. Aminoacylation of transfer RNA was not inhibited; the acylated amino acid was not carbamylated. Examination of polysome patterns by sucrose density gradient centrifugation revealed degradation of polysomes to monosomes, suggesting inhibition of initiation of protein synthesis by cyanate. In a cell-free lysate, cyanate prevented hemin stimulation of initiation. We conclude that cyanate profoundly inhibits initiation of hemoglobin synthesis in vitro.

Decreased protein synthesis in rats during a single short-term immobilization

1981 ◽  
Vol 59 (2) ◽  
pp. 145-150 ◽  
Author(s):  
Zsuzsanna Huszti ◽  
Agnes Kenessey
Keyword(s):  
Experimental Data ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Detailed Analysis ◽  
Sprague Dawley ◽  
Short Term ◽  
Radioactive Amino Acid ◽  
Sprague Dawley Rats

The effect of short-term immobilization on protein synthesis was investigated in various tissues of the rat. Male, Sprague-Dawley rats, weighing 140–150 g, were immobilized by wrapping them into plaster bandages for 3 h and treated with [3H]tyrosine or [14C]histidine to measure the rate of incorporation of the amino acid into tissue proteins. The labeling of proteins in various tissues of the immobilized rats, examined after the administration of radioactive amino acid, was markedly decreased. A detailed analysis of the experimental data provided evidence that the depressed incorporation of the radioactivity into proteins reflected a real and generalized decrease in protein synthesis in various tissues.

Rapid purification of mouse L cell interferon labeled with radioactive amino acid by immune precipitation

Virology ◽  
1980 ◽  
Vol 100 (1) ◽  
pp. 125-129 ◽  
Author(s):  
Shin Yonehara ◽  
Yoichiro Iwakura ◽  
Yoshimi Kawade
Keyword(s):  
Amino Acid ◽  
Radioactive Amino Acid ◽  
L Cell ◽  
Immune Precipitation ◽  
Rapid Purification
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