scholarly journals Misincorporation of free m-tyrosine into cellular proteins: a potential cytotoxic mechanism for oxidized amino acids

2006 ◽  
Vol 395 (2) ◽  
pp. 277-284 ◽  
Author(s):  
Hande Gurer-Orhan ◽  
Nuran Ercal ◽  
Suneetha Mare ◽  
Subramaniam Pennathur ◽  
Hilmi Orhan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Trna Synthetase ◽  
Cellular Proteins ◽  
Alternative Mechanism ◽  
Tandem Mass Spectrometric

In vitro studies demonstrate that the hydroxyl radical converts L-phenylalanine into m-tyrosine, an unnatural isomer of L-tyrosine. Quantification of m-tyrosine has been widely used as an index of oxidative damage in tissue proteins. However, the possibility that m-tyrosine might be generated oxidatively from free L-phenylalanine that could subsequently be incorporated into proteins as an L-tyrosine analogue has received little attention. In the present study, we demonstrate that free m-tyrosine is toxic to cultured CHO (Chinese-hamster ovary) cells. We readily detected radiolabelled material in proteins isolated from CHO cells that had been incubated with m-[14C]tyrosine, suggesting that the oxygenated amino acid was taken up and incorporated into cellular proteins. m-Tyrosine was detected by co-elution with authentic material on HPLC and by tandem mass spectrometric analysis in acid hydrolysates of proteins isolated from CHO cells exposed to m-tyrosine, indicating that free m-tyrosine was incorporated intact rather than being metabolized to other products that were subsequently incorporated into proteins. Incorporation of m-tyrosine into cellular proteins was sensitive to inhibition by cycloheximide, suggesting that protein synthesis was involved. Protein synthesis using a cell-free transcription/translation system showed that m-tyrosine was incorporated into proteins in vitro by a mechanism that may involve L-phenylalanine-tRNA synthetase. Collectively, these observations indicate that m-tyrosine is toxic to cells by a pathway that may involve incorporation of the oxidized amino acid into proteins. Thus misincorporation of free oxidized amino acids during protein synthesis may represent an alternative mechanism for oxidative stress and tissue injury during aging and disease.

scholarly journals Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1971 ◽  
Vol 121 (5) ◽  
pp. 817-827 ◽  
Author(s):  
R. C. Hider ◽  
E. B. Fern ◽  
D. R. London
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Extensor Digitorum Longus ◽  
Extensor Digitorum ◽  
Longus Muscle ◽  
Kinetics Of

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

scholarly journals Action of growth hormone in vitro on the net uptake and incorporation into protein of amino acids in muscle from intact rabbits given protein-deficient diets

1976 ◽  
Vol 35 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. R. Turner ◽  
P. J. Reeds ◽  
K. A. Munday
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

1. Net amino acid uptake, and incorporation into protein have been measured in vitro in the presence and absence of porcine growth hormone (GH) in muscle from intact rabbits fed for 5 d on low-protein (LP), protein-free (PF) or control diets.2. In muscle from control and LP animals GH had no effect on the net amino acid uptake but stimulated amino acid incorporation into protein, although this response was less in LP animals than in control animals.3. In muscle from PF animals, GH stimulated both amino acid incorporation into protein and the net amino acid uptake, a type of response which also occurs in hypophysectomized animals. The magnitude of the effect of GH on the incorporation of amino acids into protein was reduced in muscle from PF animals.4. The effect of GH on the net amino acid uptake in PF animals was completely blocked by cycloheximide; the uptake effect of GH in these animals was dependent therefore on de novo protein synthesis.5. It is proposed that in the adult the role of growth hormone in protein metabolism is to sustain cellular protein synthesis when there is a decrease in the level of substrate amino acids, similar to that which occurs during a short-term fast or when the dietary protein intake is inadequate.

scholarly journals Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1970 ◽  
Vol 119 (4) ◽  
pp. 629-634 ◽  
Author(s):  
M. J. Clemens ◽  
A. Korner
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Mechanism Of Action ◽  
Plasma Concentrations ◽  
Liver Slices ◽  
Hypophysectomized Rats ◽  
Stimulation Of

1. Incorporation of [14C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [3H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.

Amino Acid and Protein Metabolism During Differentiation (Sclerotization) of the Myxomycete Physarum flavicomum

1975 ◽  
Vol 53 (8) ◽  
pp. 834-843 ◽  
Author(s):  
Henry R. Henney Jr. ◽  
Glenna Maxey
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Metabolism ◽  
Cellular Proteins ◽  
Transport Systems ◽  
Differentiation Process ◽  
Cell Pool ◽  
Intracellular Amino Acids ◽  
Differentiation Period

Protein synthesized by growing plasmodia of Physarum flavicomum was steadily degraded when the plasmodia were induced to differentiate (form sclerotia). Protein synthesis occurred during the initial one-fifth (9 h) of the 48 h differentiation period, but most of this protein was also degraded shortly after its synthesis. Amino acids were primary catabolites during the differentiation process, and catabolism was extensive, even in the presence of dextrose. Glutamic acid was catabolized at a rate about two and a half or three times greater, respectively, than that observed for valine and arginine. Active transport systems for amino acids appeared to be present and to remain functional in P. flavicomum during differentiation. Amino acids included in the sclerotization media were rapidly accumulated into the cell pool and protein fractions. Intracellular amino acids were actively retained and were not released into the medium during differentiation.Differentiation of this Myxomycete, therefore, is characterized by a change in the metabolism of the sclerotizing plasmodium to an autolytic type, as cellular proteins and amino acids are actively catabolized during the formation of the dormant sclerotia.

scholarly journals Oxidation of cellular amino acid pools leads to cytotoxic mistranslation of the genetic code

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Tammy J Bullwinkle ◽  
Noah M Reynolds ◽  
Medha Raina ◽  
Adil Moghal ◽  
Eleftheria Matsa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Genetic Code ◽  
Trna Synthetase ◽  
Control Mechanisms ◽  
Cellular Toxicity ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
The Cost

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.

scholarly journals Effects of p-chlorophenylalanine and α-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells

1978 ◽  
Vol 174 (3) ◽  
pp. 931-938 ◽  
Author(s):  
C J Kelly ◽  
T C Johnson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Animal Models ◽  
Phenylalanine Hydroxylase ◽  
Present Report ◽  
Neuroblastoma Cells ◽  
Acid Uptake

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.

EFFECT OF IODIDE AND THYROTROPHIN ON IN VITRO 14C-AMINO ACID INCORPORATION INTO RAT THYROID PROTEINS

1974 ◽  
Vol 76 (2) ◽  
pp. 273-285 ◽  
Author(s):  
Lubomir J. Valenta
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Relative Concentration ◽  
Total Radioactivity ◽  
Amino Acid Incorporation ◽  
Control Value ◽  
Acid Incorporation ◽  
Rat Thyroid

ABSTRACT Thyroid lobes from rats on normal (NID) or low iodine (LID) intake were incubated for 4 hours in vitro in the presence of 14C-amino acids. The 14C-amino acid incorporation into thyroid protein was significantly higher in thyroids from LID than from NID fed rats, 7.82 ± 1.01 % (mean ± sd) of total radioactivity of the incubation mixture per 100 mg tissue compared to 3.74 ± 0.60 % respectively. Thyrotrophin (TSH) in vitro did not influence the 14C-amino acid incorporation. Iodide in concentration 10−7 m and higher decreased 14C-radioactivity incorporation into protein by 19.40 ± 3.06 and 26.59 ± 4.06 % of the control value for NID and LID rats respectively. This effect of iodide did not depend on iodine organification and was not influenced by the changes of free amino acids pool. There were no significant differences in the relative concentration of 14C-labelled thyroglobulin and total 14C-thyroid protein. Differential fragility demonstrable by unfolding or dissociation was observed between different classes of thyroglobulin. The fragility was increasing from the old non-labelled molecules to newly iodinated and newly synthesized ones. It is concluded that iodide has a direct intrathyroidal blocking effect on thyroid protein synthesis which may contribute to its antigoitrogenic action. The lack of in vitro stimulation of protein synthesis by TSH remains unexplained.

scholarly journals The Origin of Prebiotic Information System in the Peptide/RNA World: A Simulation Model of the Evolution of Translation and the Genetic Code

Life ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 25 ◽  
Author(s):  
Sankar Chatterjee ◽  
Surya Yadav
Keyword(s):  
Amino Acids ◽  
Information System ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Genetic Code ◽  
Rna World ◽  
Trna Synthetase ◽  
Darwinian Evolution ◽  
Universal Genetic Code ◽  
Translation Machine

Information is the currency of life, but the origin of prebiotic information remains a mystery. We propose transitional pathways from the cosmic building blocks of life to the complex prebiotic organic chemistry that led to the origin of information systems. The prebiotic information system, specifically the genetic code, is segregated, linear, and digital, and it appeared before the emergence of DNA. In the peptide/RNA world, lipid membranes randomly encapsulated amino acids, RNA, and peptide molecules, which are drawn from the prebiotic soup, to initiate a molecular symbiosis inside the protocells. This endosymbiosis led to the hierarchical emergence of several requisite components of the translation machine: transfer RNAs (tRNAs), aminoacyl-tRNA synthetase (aaRS), messenger RNAs (mRNAs), ribosomes, and various enzymes. When assembled in the right order, the translation machine created proteins, a process that transferred information from mRNAs to assemble amino acids into polypeptide chains. This was the beginning of the prebiotic <i>information</i> age. The origin of the genetic code is enigmatic; herein, we propose an evolutionary explanation: the demand for a wide range of protein enzymes over peptides in the prebiotic reactions was the main selective pressure for the origin of information-directed protein synthesis. The molecular basis of the genetic code manifests itself in the interaction of aaRS and their cognate tRNAs. In the beginning, aminoacylated ribozymes used amino acids as a cofactor with the help of bridge peptides as a process for selection between amino acids and their cognate codons/anticodons. This process selects amino acids and RNA species for the next steps. The ribozymes would give rise to pre-tRNA and the bridge peptides to pre-aaRS. Later, variants would appear and evolution would produce different but specific aaRS-tRNA-amino acid combinations. Pre-tRNA designed and built pre-mRNA for the storage of information regarding its cognate amino acid. Each pre-mRNA strand became the storage device for the genetic information that encoded the amino acid sequences in triplet nucleotides. As information appeared in the digital languages of the codon within pre-mRNA and mRNA, and the genetic code for protein synthesis evolved, the prebiotic chemistry then became more organized and directional with the emergence of the translation and genetic code. The genetic code developed in three stages that are coincident with the refinement of the translation machines: the GNC code that was developed by the pre-tRNA/pre-aaRS /pre-mRNA machine, SNS code by the tRNA/aaRS/mRNA machine, and finally the universal genetic code by the tRNA/aaRS/mRNA/ribosome machine. We suggest the coevolution of translation machines and the genetic code. The emergence of the translation machines was the beginning of the Darwinian evolution, an interplay between information and its supporting structure. Our hypothesis provides the logical and incremental steps for the origin of the programmed protein synthesis. In order to better understand the prebiotic information system, we converted letter codons into numerical codons in the Universal Genetic Code Table. We have developed a software, called CATI (Codon-Amino Acid-Translator-Imitator), to translate randomly chosen numerical codons into corresponding amino acids and vice versa. This conversion has granted us insight into how the genetic code might have evolved in the peptide/RNA world. There is great potential in the application of numerical codons to bioinformatics, such as barcoding, DNA mining, or DNA fingerprinting. We constructed the likely biochemical pathways for the origin of translation and the genetic code using the Model-View-Controller (MVC) software framework, and the translation machinery step-by-step. While using AnyLogic software, we were able to simulate and visualize the entire evolution of the translation machines, amino acids, and the genetic code.

scholarly journals Influence of amino acid supply on ribosomes and protein synthesis of perfused rat liver

1969 ◽  
Vol 111 (5) ◽  
pp. 703-712 ◽  
Author(s):  
L. S. Jefferson ◽  
A. Korner
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Plasma Concentration ◽  
Liver Protein ◽  
Perfusion Medium ◽  
Initial Concentration ◽  
Normal Plasma ◽  
Polysome Profile

1. The livers of rats were perfused in situ with medium containing mixtures of amino acids in multiples of their concentration in normal rat plasma. The incorporation of labelled amino acid into protein of the liver and of the perfusing medium increased with increasing amino acid concentration. During 60min. perfusions, labelling of liver protein reached a plateau, and labelling of medium protein was inhibited when the initial concentration of the amino acid mixture was more than ten times the normal plasma value. 2. Examination of polysome profiles derived from livers perfused without amino acids in the medium showed that the number of large aggregates was decreased and the number of small aggregates, particularly monomers and dimers, was increased with time of perfusion. The addition of amino acids to the perfusion medium reversed this polysome shift to an extent that was dependent on the initial concentration of amino acids. Polysome profiles derived from livers perfused for 60min. with ten times the normal plasma concentration of amino acids were essentially the same as the polysome profiles of normal non-perfused livers. 3. The ability of ribosome preparations from perfused livers to incorporate amino acids into protein in vitro decreased with increasing time of perfusion when no amino acids were added to the medium, but increased as the concentration of amino acids in the perfusion medium was increased. 4. The ability of cell sap from perfused livers to support protein synthesis in vitro was not influenced by the amino acid concentration of the perfusion medium. 5. Livers were perfused for 60min. with medium containing amino acid mixtures at ten times the normal plasma concentration but deficient in one amino acid. Maximal incorporation of labelled amino acid into liver protein, the stability of the polysome profile and the ability of ribosome preparations to incorporate amino acids into protein were found to depend on the presence of 11 amino acids: arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine. A mixture of these 11 amino acids, at ten times their normal plasma concentration, stimulated the incorporation of labelled amino acid into liver protein, stabilized the polysome profile and increased the ability of ribosome preparations to incorporate amino acids into protein to the same extent as the complete mixture. 6. It is concluded that the availability of certain amino acids plays an important role in the control of protein synthesis, possibly by stimulating the ability of ribosomes to become, and to remain, attached to messenger RNA.

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