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Sample records for acids phenylalanine tyrosine

  1. Zur Biosynthese von Phenylalanin und Tyrosin

    NASA Astrophysics Data System (ADS)

    Lingens, F.; Keller, E.

    1983-03-01

    With the discovery of arogenic acid two new pathways for the biosynthesis of phenylalanine and tyrosine have been revealed. The occurrence of two, three, or four pathways for the biosynthesis of phenylalanine and tyrosine in microorganisms and plants may be a useful tool for taxonomic classifications. Investigations on enterobacteriaceae, pseudomonads, flavobacteria, streptomycetes, archaebacteria, and on Sphaerotilus, Trichococcus and Leptothrix species from bulking sludge are described. The possible role of arogenate in the evolution of the pathways for tyrosine and phenylalanine biosynthesis is discussed.

  2. An Experimental and Computational Investigation into the Gas-Phase Acidities of Tyrosine and Phenylalanine: Three Structures for Deprotonated Tyrosine

    SciTech Connect

    Bokatzian, Samantha S.; Stover, Michele L.; Plummer, Chelsea E.; Dixon, David A.; Cassady, Carolyn J.

    2014-11-06

    Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol.

  3. An experimental and computational investigation into the gas-phase acidities of tyrosine and phenylalanine: three structures for deprotonated tyrosine.

    PubMed

    Bokatzian, Samantha S; Stover, Michele L; Plummer, Chelsea E; Dixon, David A; Cassady, Carolyn J

    2014-11-01

    Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol. PMID:25299802

  4. The cysteine, total sulfur amino acid, tyrosine, phenylalanine + tyrosine, and non-essential amino acid maintenance requirements of broiler breeders.

    PubMed

    Ekmay, R D; Mei, S J; Sakomura, N K; Coon, C N

    2016-06-01

    Two hundred and fifty Cobb-Vantress broiler breeders were used to determine the maintenance requirement and efficiency of utilization of dietary Cys, Tyr, and non-essential amino acids (AA) in a 21-day experiment. The breeders were fed crystalline amino acid diets containing graded levels of Cys or Tyr representing 0, 10, 20, 30, and 40% of their suggested requirement level with all other amino acids maintained at 40% of their suggested requirement level. To determine the non-essential AA maintenance requirement, graded levels of non-essential AA were provided by glutamic acid to represent 12, 19, 26, 33, and 40% of the ideal level of glutamic acid with all other amino acids maintained at their maintenance requirement level. The total sulfur amino acid (TSAA) and Phe + Tyr requirements were calculated by combining Cys and Tyr results, respectively, with previously determined Met and Phe, respectively. The slope of Cys, Tyr, and non-essential AA accretion regression line indicated that 29% Cys, 24% TSAA, 21% Tyr, 20% Phe + Tyr, and 9% non-essential AA of crystalline amino acids were retained. The Cys requirement for zero protein accretion was calculated to be 30.48 mg/d or 17.006 mg/ kgBW(0.75)/d or 75.426 mg/kgCP/d. The TSAA requirement for zero accretion was calculated to be 132.25 mg/b/d, 71.48 mg/kgBW(0.75)/d, and 307.55 mg/kgCP/d. The Tyr requirement for zero protein accretion was calculated to be 65.907 mg/d or 37.233 mg/ kgBW(0.75)/d or 175.566 mg/kgCP/d. The Phe + Tyr requirement for zero protein accretion was calculated to be 352.18 mg/b/d, 190.37 mg/kgBW(0.75)/d, and 749.33 mg/kgCP/d. The non-essential AA requirement for zero protein accretion was calculated to be 3715.194 mg/d or 2003.155 mg/kgBW(0.75)/d or 9452.954 mg/kgCP/d. PMID:26994191

  5. Diurnal variation of phenylalanine and tyrosine concentrations in adult patients with phenylketonuria: subcutaneous microdialysis is no adequate tool for the determination of amino acid concentrations

    PubMed Central

    2013-01-01

    Background Metabolic control and dietary management of patients with phenylketonuria (PKU) are based on single blood samples obtained at variable intervals. Sampling conditions are often not well-specified and intermittent variation of phenylalanine concentrations between two measurements remains unknown. We determined phenylalanine and tyrosine concentrations in blood over 24 hours. Additionally, the impact of food intake and physical exercise on phenylalanine and tyrosine concentrations was examined. Subcutaneous microdialysis was evaluated as a tool for monitoring phenylalanine and tyrosine concentrations in PKU patients. Methods Phenylalanine and tyrosine concentrations of eight adult patients with PKU were determined at 60 minute intervals in serum, dried blood and subcutaneous microdialysate and additionally every 30 minutes postprandially in subcutaneous microdialysate. During the study period of 24 hours individually tailored meals with defined phenylalanine and tyrosine contents were served at fixed times and 20 min bicycle-ergometry was performed. Results Serum phenylalanine concentrations showed only minor variations while tyrosine concentrations varied significantly more over the 24-hour period. Food intake within the patients’ individual diet had no consistent effect on the mean phenylalanine concentration but the tyrosine concentration increased up to 300% individually. Mean phenylalanine concentration remained stable after short-term bicycle-exercise whereas mean tyrosine concentration declined significantly. Phenylalanine and tyrosine concentrations in dried blood were significantly lower than serum concentrations. No close correlation has been found between serum and microdialysis fluid for phenylalanine and tyrosine concentrations. Conclusions Slight diurnal variation of phenylalanine concentrations in serum implicates that a single blood sample does reliably reflect the metabolic control in this group of adult patients. Phenylalanine

  6. Single-channel studies on linear gramicidins with altered amino acid sequences. A comparison of phenylalanine, tryptophane, and tyrosine substitutions at positions 1 and 11.

    PubMed Central

    Mazet, J L; Andersen, O S; Koeppe, R E

    1984-01-01

    The relation between chemical structure and permeability characteristics of transmembrane channels has been investigated with the linear gramicidins (A, B, and C), where the amino acid at position 1 was chemically replaced by phenylalanine, tryptophane or tyrosine. The purity of most of the compounds was estimated to be greater than 99.99%. The modifications resulted in a wide range of conductance changes in NaCl solutions: sixfold from tryptophane gramicidin A to tyrosine gramicidin B. The conductance changes induced by a given amino acid substitution at position 1 are not the same as at position 11. The only important change in the Na+ affinity was observed when the first amino acid was tyrosine. No major conformational changes of the polypeptide backbone structure could be detected on the basis of experiments with mixtures of different analogues and valine gramicidin A (except possibly with tyrosine at position 1), as all the compounds investigated could form hybrid channels with valine gramicidin A. The side chains are not in direct contact with the permeating ions. The results were therefore interpreted in terms of modifications of the energy profile for ion movement through the channel, possibly due to an electrostatic interaction between the dipoles of the side chains and ions in the channel. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:6201199

  7. Vibrational analysis of amino acids and short peptides in hydrated media. VIII. Amino acids with aromatic side chains: L-phenylalanine, L-tyrosine, and L-tryptophan.

    PubMed

    Hernández, Belén; Pflüger, Fernando; Adenier, Alain; Kruglik, Sergei G; Ghomi, Mahmoud

    2010-11-25

    Four out of the 20 natural α-amino acids (α-AAs) contain aromatic rings in their side chains. In a recent paper (J. Phys. Chem. B 2010, 114, 9072-9083), we have analyzed the structural and vibrational features of l-histidine, one of the potent elements of this series. Here, we report on the three remaining members of this family, i.e., l-phenylalanine, l-tyrosine, and l-tryptophan. Their solution (H(2)O and D(2)O) Raman scattering and Fourier transform infrared absorption attenuated total reflection (FT-IR ATR) spectra were measured at room temperature from the species corresponding to those existing at physiological conditions. Because of the very low water solubility of tyrosine, special attention was paid to avoid any artifact concerning the report of the vibrational spectra corresponding to nondissolved powder of this AA in aqueous solution. Finally, we could obtain for the first time the Raman and FT-IR spectra of tyrosine at very low concentration (2.3 mM) upon long accumulation time. To clarify this point, those vibrational spectra of tyrosine recorded either in the solid phase or in a heterogeneous state, where dissolved and nondissolved species of this AA coexist in aqueous solution, are also provided as Supporting Information . To carry out a discussion on the general geometrical and vibrational behavior of these AAs, we resorted to quantum mechanical calculations at the DFT/B3LYP/6-31++G* level, allowing (i) determination of potential energy surfaces of these AAs in a continuum solvent as a function of the torsion angles χ(1) and χ(2), defining the conformation of each aromatic side chain around C(α)-C(β) and C(β)-C(γ) bonds, respectively; (ii) analysis of geometrical features of the AAs surrounded by clusters of n explicit (n = 5-7) water molecules interacting with the backbone and aromatic rings; and (iii) assignment of the observed vibrational modes by means of the theoretical data provided by the lowest energy conformers of explicitly

  8. Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity.

    PubMed

    MacDonald, Marybeth C; Arivalagan, Pugazhendhi; Barre, Douglas E; MacInnis, Judith A; D'Cunha, Godwin B

    2016-01-01

    Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications. PMID:27014206

  9. Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity

    PubMed Central

    MacDonald, Marybeth C.; Arivalagan, Pugazhendhi; Barre, Douglas E.; MacInnis, Judith A.; D’Cunha, Godwin B.

    2016-01-01

    Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 μM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications. PMID:27014206

  10. Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

    PubMed

    Das, Mrinal Kumar; Bishwal, Subasa Chandra; Das, Aleena; Dabral, Deepti; Badireddy, Vinod Kumar; Pandit, Bhaswati; Varghese, George M; Nanda, Ranjan Kumar

    2015-04-01

    Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB. PMID:25693719

  11. First hyperpolarizability of the natural aromatic amino acids tryptophan, tyrosine, and phenylalanine and the tripeptide lysine-tryptophan-lysine determined by hyper-Rayleigh scattering.

    PubMed

    Duboisset, J; Matar, G; Russier-Antoine, I; Benichou, E; Bachelier, G; Jonin, Ch; Ficheux, D; Besson, F; Brevet, P F

    2010-11-01

    We report the first hyperpolarizability of tryptophan (Trp) and tyrosine (Tyr) and an upper limit for that of phenylalanine (Phe), three natural aromatic amino acids. The measurements were performed with hyper-Rayleigh scattering in an aqueous Tris buffer solution at a pH of 8.5 and 150 mM salt concentration with a fundamental wavelength of 780 nm. A value of (4.7 ± 0.7) × 10(-30) esu is found for Trp and (4.1 ± 0.7) × 10(-30) esu for Tyr whereas the upper limit of 1.4 × 10(-30) esu is found for that of Phe due to its limited solubility. The influence of the presence of lysine (Lys) in close vicinity of Trp is investigated with a measurement of the first hyperpolarizabilty of Trp in an excess of Lys and compared to the first hyperpolarizability obtained for the tripeptide Lys-Trp-Lys. The clear decrease of the values measured in these two cases indicates that the first hyperpolarizabilty of Trp is very sensitive to its local environment. PMID:20939548

  12. Study of Interaction Between Tryptophan, Tyrosine, and Phenylalanine Separately with Silver Nanoparticles by Fluorescence Quenching Method

    NASA Astrophysics Data System (ADS)

    Roy, S.; Das, T. K.

    2015-09-01

    Using the spectroscopic method, the individual interaction of the three biochemically important amino acids, which are constituents of protein, namely, tryptophan, tyrosine, and phenylalanine with biologically synthesized silver nanoparticles has been investigated. The obtained UV-Vis spectra show the formation of ground-state complexes between tryptophan, tyrosine, and phenylalanine with silver nanoparticles. Silver nanoparticles possess the ability to quench the intrinsic fluorescence of the aforesaid amino acids by a dynamic quenching process. The binding constant, number of binding sites, and corresponding thermodynamic parameters (Δ H, Δ S, and Δ G) based on the interaction system were calculated for 293, 303, and 313 K. In the case of tryptophan and phenylalanine, with increase in temperature, the binding constant K was found to decrease; conversely, it was found to increase with increase in temperature in the case of tyrosine. The thermodynamic results revealed that the binding process was spontaneous; hydrogen bonding and van der Waals interaction were the predominant forces responsible for the complex stabilization in the case of tryptophan and phenylalanine, respectively, whereas in the case of tyrosine, hydrophobic interaction was the sole force conferring stability. Moreover, the Förster non-radiation energy transfer theory has been applied to calculate the average binding distance among the above amino acids and silver nanoparticles. The results show a binding distance of <7 nm, which ensures that energy transfer does occur between the said amino acids and silver nanoparticles.

  13. Phenylalanine and tyrosine levels in newborn screening blood samples.

    PubMed Central

    Morris, A F; Holton, J B; Burman, D; Colley, J R

    1983-01-01

    A previously described difference in newborn blood phenylalanine concentrations between those living in urban and non-urban areas in the south west of England has been confirmed and shown to be independent of the type of feed. Several factors including the place of abode, type of feed, birthweight, and the accuracy of the test have been found to affect the measured, phenylalanine concentration in the newborn screening blood spot, and the importance of these results to screening practice is considered. Blood tyrosine also varied with the above factors, but severe, neonatal tyrosinaemia was shown to be a rare problem. PMID:6847230

  14. LAT-1 activity of meta-substituted phenylalanine and tyrosine analogs.

    PubMed

    Augustyn, Evan; Finke, Karissa; Zur, Arik A; Hansen, Logan; Heeren, Nathan; Chien, Huan-Chieh; Lin, Lawrence; Giacomini, Kathleen M; Colas, Claire; Schlessinger, Avner; Thomas, Allen A

    2016-06-01

    The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described. PMID:27106710

  15. Dopamine and food reward: effects of acute tyrosine/phenylalanine depletion on appetite.

    PubMed

    Hardman, Charlotte A; Herbert, Vanessa M B; Brunstrom, Jeffrey M; Munafò, Marcus R; Rogers, Peter J

    2012-03-20

    It has been suggested that obese individuals over-eat in order to compensate for deficits in the dopaminergic reward system. The current study used acute tyrosine/phenylalanine depletion (ATPD) to investigate the effect of reduced dopamine function on appetite and the reward value of food in healthy volunteers. The compensatory-eating hypothesis would predict an increase in the reward value and consumption of food following depletion by this method. In a double-blind, counterbalanced, crossover study, 17 male participants (mean age=29.2 (SEM=2.7) years; mean body mass index=24.4 (SEM=0.6) kg/m(2)) were administered with a tyrosine/phenylalanine-free mixture (TYR/PHE-free; depletion condition) and a balanced amino acid mixture (BAL; control). Plasma amino acid levels were measured at baseline and peak depletion (300 min). Appetite, willingness to pay for food, liking, desired portion size and ad libitum food intake were also assessed. The TYR/PHE-free mixture was associated with significant decreases in tyrosine, phenylalanine, and the ratio of tyrosine+phenylalanine to the other large neutral amino acids (all p<.001). There were no effects on our measures of willingness to pay for food or liking. However, in the TYR/PHE-free condition, participants reported significantly lower levels of hunger following a fixed-test meal relative to the BAL condition. In conclusion, we found no evidence for compensatory eating following ATPD. Our results also provide support for the role of dopamine in motivational components of eating. PMID:22230253

  16. New peptide architectures through C–H activation stapling between tryptophan–phenylalanine/tyrosine residues

    PubMed Central

    Mendive-Tapia, Lorena; Preciado, Sara; García, Jesús; Ramón, Rosario; Kielland, Nicola; Albericio, Fernando; Lavilla, Rodolfo

    2015-01-01

    Natural peptides show high degrees of specificity in their biological action. However, their therapeutical profile is severely limited by their conformational freedom and metabolic instability. Stapled peptides constitute a solution to these problems and access to these structures lies on a limited number of reactions involving the use of non-natural amino acids. Here, we describe a synthetic strategy for the preparation of unique constrained peptides featuring a covalent bond between tryptophan and phenylalanine or tyrosine residues. The preparation of such peptides is achieved in solution and on solid phase directly from the corresponding sequences having an iodo-aryl amino acid through an intramolecular palladium-catalysed C–H activation process. Moreover, complex topologies arise from the internal stapling of cyclopeptides and double intramolecular arylations within a linear peptide. Finally, as a proof of principle, we report the application to this new stapling method to relevant biologically active compounds. PMID:25994485

  17. Evaluation of an Adsorbent Based on Agricultural Waste (Corn Cobs) for Removal of Tyrosine and Phenylalanine from Aqueous Solutions

    PubMed Central

    Alves, Cibele C. O.; Franca, Adriana S.; Oliveira, Leandro S.

    2013-01-01

    Adsorption of phenolic amino acids, such as phenylalanine and tyrosine, is quite relevant for the production of protein hydrolysates used as dietary formulations for patients suffering from congenital disorders of amino acid metabolism, such as phenylketonuria. In this study, an adsorbent prepared from corn cobs was evaluated for the removal of tyrosine (Tyr) from both a single component solution and a binary aqueous solution with phenylalanine (Phe). The adsorption behavior of tyrosine was similar to that of phenylalanine in single component solutions, however, with a much lower adsorption capacity (14 mg g−1 for Tyr compared to 109 mg g−1 for Phe). Tyr adsorption kinetics was satisfactorily described by a pseudosecond-order model as it was for Phe. In adsorption equilibrium studies for binary mixtures, the presence of Tyr in Phe solutions favored Phe faster adsorption whereas the opposite behavior was observed for the presence of Phe in Tyr solutions. Such results indicate that, in binary systems, Phe will be adsorbed preferably to Tyr, and this is a welcome feature when employing the prepared adsorbent for the removal of Phe from protein hydrolysates to be used in dietary formulations for phenylketonuria treatment. PMID:23936863

  18. Identification of β-phenylalanine as a non-protein amino acid in cultivated rice, Oryza sativa

    PubMed Central

    Yokoo, Takayuki; Takata, Ryo; Yan, Jian; Matsumoto, Fuka; Teraishi, Masayoshi; Okumoto, Yutaka; Jander, Georg; Mori, Naoki

    2015-01-01

    Non-protein amino acids, often analogs of the standard 20 protein amino acids, have been discovered in many plant species. Recent research with cultivated rice (Oryza sativa) identified (3R)-β-tyrosine, as well as a tyrosine amino mutase that synthesizes (3R)-β-tyrosine from the protein amino acid (2S)-α-tyrosine. Gas chromatography-mass spectrometry (GC-MS) assays and comparison to an authentic standard showed that β-phenylalanine is also a relatively abundant non-protein amino acid in rice leaves and that its biosynthesis occurs independently from that of β-tyrosine. PMID:27066169

  19. Syntheses of halogen derivatives of L-tryptophan, L-tyrosine and L-phenylalanine labeled with hydrogen isotopes.

    PubMed

    Pająk, Małgorzata; Pałka, Katarzyna; Winnicka, Elżbieta; Kańska, Marianna

    2016-01-01

    Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L-tryptophan, i.e. 5'-bromo-[2-(3)H]-, 5'-bromo-[2-(2)H/(3)H]-, 5'-fluoro-[2-(3)H]-5'-fluoro-[2-(2)H/(3)H]-, 6'-fluoro-[2-(3)H]-, 6'-fluoro-[2-(2)H/(3)H]-L-tryptophan, as well as, L-tyrosine, i.e. 3'-fluoro-[2-(3)H]-, 3'-fluoro-[2-(2)H/(3)H]-, 3'-chloro-[2-(3)H]-, and 3'-chloro-[2-(2)H/(3)H]-L-tyrosine, and also L-phenylalanine, i.e. 2'-fluoro-[(3S)-(3)H]-, 2'-fluoro-[(3S)-(2)H/(3) H]-, 2'-chloro-[(3S)-(3)H]-, 2'-chloro-[(3S)-(2)H/(3)H]-, 4'-chloro-[(3S)-(3)H]-, and 4'-chloro-[(3S)-(2)H/(3)H]-L-phenylalanine were synthesized using enzymatic methods. Isotopomers of L-tryptophan were synthesized by coupling of halogenated indoles with S-methyl-L-cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L-tyrosine were obtained by the enzymatically supported exchange between halogenated L-tyrosine and isotopic water. Labeled halogenated isotopologues of L-Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D2O) with addition of HTO were used. PMID:26586485

  20. Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution.

    PubMed

    Song, Yanting; Takatsuki, Katsuya; Sekiguchi, Tetsushi; Funatsu, Takashi; Shoji, Shuichi; Tsunoda, Makoto

    2016-07-01

    This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field. PMID:27209196

  1. No Tryptophan, Tyrosine and Phenylalanine Abnormalities in Children with Attention-Deficit/Hyperactivity Disorder

    PubMed Central

    Bergwerff, Catharina Elisabeth; Luman, Marjolein; Blom, Henk J.; Oosterlaan, Jaap

    2016-01-01

    Background The aim of the current study was to explore the role of aromatic amino acids (AAAs) in blood in relation to attention-deficit/hyperactivity disorder (ADHD). Given their impact on the synthesis of serotonin and dopamine, decreased concentrations of the AAAs tryptophan, tyrosine and phenylalanine in blood may contribute to the expression of ADHD symptoms. Decreased AAA blood concentrations, in turn, may be related to lowered dietary protein intake or to abnormal AAA catabolism, as evidenced by increased urinary AAA concentrations. Methods Eighty-three children with ADHD (75% males) and 72 typically developing (TD) children (51% males), aged 6 to 13 years, participated in the study. AAA concentrations were assessed in blood spots and an 18-hour urinary sample. A nutritional diary was filled out by parents to calculate dietary protein intake. Parent and teacher questionnaires assessed symptoms of ADHD, oppositional defiant disorder, conduct disorder, and autism spectrum disorder. Results Children with ADHD showed normal AAA concentrations in blood spots and urine, as well as normal protein intake compared to controls. No associations between AAA concentrations and symptoms of ADHD or comorbid psychiatric disorders were found. Conclusions This study is the first to explore AAA metabolism in children with ADHD using a well-defined and relatively large sample. We found that AAA deficiencies are not related to ADHD. The results do not support treatment with AAA supplements in children with ADHD. Future studies regarding the cause of serotonin and dopamine alterations in ADHD should focus on other explanations, such as effects of altered transport of AAAs. PMID:26938936

  2. [Simultaneous resolution and determination of tyrosine, tryptophan and phenylalanine by alternating penalty trilinear decomposition algorithm coupled with 3D emission-excitation matrix fluorometry].

    PubMed

    Xiao, Jin; Ren, Feng-lian; Song, Ge; Liao, Lü; Yu, Wen-feng; Zeng, Tao

    2007-10-01

    A new method using alternating penalty trilinear decomposition algorithm coupled with excitation-emission matrix fluorometry has been developed for simultaneous resolution and determination of tyrosine, phenylalanine and tryptophan. Their correlation coefficients were 0.9987, 0.9995 and 0.9993 respectively. The contents of tyrosine, phenylalanine and tryptophan in Hibiscus syriacus L. leaves were also be determined by this method after being extracted by ultrasonic. The coefficients of variation and the recoveries of the three amino acids were 0.84%, 0.36%, 1.59% and 101.0%-92.7%, 106.5%-93.0%, 103.0%-95.0% respectively. All these show that this is a simple, fast and cridible method. PMID:18306802

  3. SERS study of transformation of phenylalanine to tyrosine under particle irradiation

    NASA Astrophysics Data System (ADS)

    Zhang, Jingjing; Huang, Qing; Yao, Guohua; Ke, Zhigang; Zhang, Hong; Lu, Yilin

    2014-08-01

    Surface enhanced Raman scattering or spectroscopy (SERS) is a very powerful analytical tool which has been widely applied in many scientific research and application fields. It is therefore also very intriguing for us to introduce SERS technique in the radiobiological research, where in many cases only a very few of biomolecules are subjected to changes which however can lead to significant biological effects. The radiation induced biochemical reactions are normally very sophisticated with different substances produced in the system, so currently it is still a big challenge for SERS to analyze such a mixture system which contains multiple analytes. In this context, this work aimed to establish and consolidate the feasibility of SERS as an effective tool in radiation chemistry, and this purpose, we employed SERS as a sensitive probe to a known process, namely, the oxidation of phenylalanine (Phe) under particle irradiation, where the energetic particles were obtained from either plasma discharge or electron-beam. During the irradiation, three types of tyrosine (Tyr), namely, p-Tyr, m-Tyr and o-Tyr were produced, and all these tyrosine isomers together with Phe could be identified and measured based on the SERS spectral analysis of the corresponding enhanced characteristic signals, namely, 1002 cm-1 for Phe, 1161 cm-1 for p-Tyr, 990 cm-1 for m-Tyr, and 970 cm-1 for o-Tyr, respectively. The estimation of the quantities of different tyrosine isomers were also given and verified by conventional method such as high performance liquid chromatography (HPLC). As for comparison of different ways of particle irradiation, our results also indicated that electron-beam irradiation was more efficient for converting Phe into Tyr than plasma discharge treatment, confirming the role of hydroxyl radicals in the Phe-Tyr conformation. Therefore, our work has not only demonstrated that SERS can be successfully applied in the radiobiological study, but also given insights into the

  4. [Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases.

    PubMed Central

    Desmarais, S; Friesen, R W; Zamboni, R; Ramachandran, C

    1999-01-01

    Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPbeta, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs. PMID:9882618

  5. Dopamine and Pain Sensitivity: Neither Sulpiride nor Acute Phenylalanine and Tyrosine Depletion Have Effects on Thermal Pain Sensations in Healthy Volunteers

    PubMed Central

    Becker, Susanne; Ceko, Marta; Louis-Foster, Mytsumi; Elfassy, Nathaniel M.; Leyton, Marco; Shir, Yoram; Schweinhardt, Petra

    2013-01-01

    Based on animal studies and some indirect clinical evidence, dopamine has been suggested to have anti-nociceptive effects. Here, we investigated directly the effects of increased and decreased availability of extracellular dopamine on pain perception in healthy volunteers. In Study 1, participants ingested, in separate sessions, a placebo and a low dose of the centrally acting D2-receptor antagonist sulpiride, intended to increase synaptic dopamine via predominant pre-synaptic blockade. No effects were seen on thermal pain thresholds, tolerance, or temporal summation. Study 2 used the acute phenylalanine and tyrosine depletion (APTD) method to transiently decrease dopamine availability. In one session participants ingested a mixture that depletes the dopamine amino acid precursors, phenylalanine and tyrosine. In the other session they ingested a nutritionally balanced control mixture. APTD led to a small mood-lowering response following aversive thermal stimulation, but had no effects on the perception of cold, warm, or pain stimuli. In both studies the experimental manipulation of dopaminergic neurotransmission was successful as indicated by manipulation checks. The results contradict proposals that dopamine has direct anti-nociceptive effects in acute experimental pain. Based on dopamine’s well-known role in reward processing, we hypothesize that also in the context of pain, dopamine acts on stimulus salience and might play a role in the initiation of avoidance behavior rather than having direct antinociceptive effects in acute experimental pain. PMID:24236199

  6. Effect of amino acids on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in creatinine/phenylalanine and creatinine/phenylalanine/4-oxo-2-nonenal reaction mixtures.

    PubMed

    Zamora, Rosario; Alcón, Esmeralda; Hidalgo, Francisco J

    2013-12-15

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) formation in mixtures of creatinine, phenylalanine, amino acids and 4-oxo-2-nonenal was studied, to analyse the role of amino acids on the generation of this heterocyclic aromatic amine. When oxidised lipid was absent, cysteine, serine, aspartic acid, threonine, asparagine, tryptophan, tyrosine, proline, and methionine increased significantly (p < 0.05) the amount of PhIP formed in comparison to the control. When lipid was present, only the addition of methionine, glycine, and serine increased significantly (p < 0.05) the amount of PhIP produced, while histidine, cysteine, lysine, tryptophan, tyrosine, and alanine reduced significantly (p < 0.05) PhIP. These results may be a consequence of the different competitive reactions that occur. Thus, in the absence of lipids, thermal decomposition of the amino acids produced reactive carbonyls that converted phenylalanine into phenylacetaldehyde as a key step in the formation of PhIP. When oxidised lipid was present, amino acids competed with phenylalanine for the lipid, and amino acid degradation products were formed, among which alpha-keto acids seemed to play a role in these reactions. These results suggest that PhIP can be produced by several alternative reaction pathways from all major food components, including amino acids and lipids, in addition to carbohydrates. PMID:23993611

  7. Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension

    PubMed Central

    Manela, Neta; Oliva, Moran; Ovadia, Rinat; Sikron-Persi, Noga; Ayenew, Biruk; Fait, Aaron; Galili, Gad; Perl, Avichai; Weiss, David; Oren-Shamir, Michal

    2015-01-01

    Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as well. PMID:26236327

  8. Grass roots chemistry: meta-Tyrosine, an herbicidal nonprotein amino acid

    PubMed Central

    Bertin, Cécile; Weston, Leslie A.; Huang, Tengfang; Jander, Georg; Owens, Thomas; Meinwald, Jerrold; Schroeder, Frank C.

    2007-01-01

    Fine fescue grasses displace neighboring plants by depositing large quantities of an aqueous phytotoxic root exudate in the soil rhizosphere. Via activity-guided fractionation, we have isolated and identified the nonprotein amino acid m-tyrosine as the major active component. m-Tyrosine is significantly more phytotoxic than its structural isomers o- and p-tyrosine. We show that m-tyrosine exposure results in growth inhibition for a wide range of plant species and propose that the release of this nonprotein amino acid interferes with root development of competing plants. Acid hydrolysis of total root protein from Arabidopsis thaliana showed incorporation of m-tyrosine, suggesting this as a possible mechanism of phytotoxicity. m-Tyrosine inhibition of A. thaliana root growth is counteracted by exogenous addition of protein amino acids, with phenylalanine having the most significant effect. The discovery of m-tyrosine, as well as a further understanding of its mode(s) of action, could lead to the development of biorational approaches to weed control. PMID:17940026

  9. Stability decrease of RNA double helices by phenylalanine-, tyrosine- and tryptophane-amides. Analysis in terms of site binding and relation to melting proteins.

    PubMed Central

    Pörschke, D; Jung, M

    1982-01-01

    The amides of L-phenylalanine, L-tyrosine and L-tryptophane decrease the melting temperatures tm of poly(A)*poly(U) and poly(I)*poly(C) double helices at low concentrations (1 mM), whereas high concentrations finally lead to an increase of tm. This dependence of the tm-values upon the ligand concentration can be represented quantitatively by a simple site binding model, providing binding parameters for the interaction between the amides and the nucleic acids both in the double- and the single-stranded conformation. According to these data the affinity to the single strands is higher than that to the double strands and increases in the series Phe less than Tyr less than Trp. The binding constants decrease with increasing salt concentration as expected for an interaction driven by electrostatic attraction. However, part of the interaction is also due to stacking between the aromatic amides and the nucleic acid bases. The present results indicate a direct correlation between the presence of aromatic amino acids at the binding site of helix destabilising proteins and the properties of simple derivatives of these amino acids. Furthermore the results suggest that very simple peptides containing aromatic amino acids served as a starting point for the evolution of helix destabilising proteins. PMID:7145717

  10. Replacement of tyrosine D with phenylalanine affects the normal proton transfer pathways for the reduction of P680+ in oxygen-evolving photosystem II particles from Chlamydomonas.

    PubMed

    Jeans, C; Schilstra, M J; Ray, N; Husain, S; Minagawa, J; Nugent, J H A; Klug, D R

    2002-12-31

    We have probed the electrostatics of P680(+) reduction in oxygenic photosynthesis using histidine-tagged and histidine-tagged Y(D)-less Photosystem II cores. We make two main observations: (i) that His-tagged Chlamydomonas cores show kinetics which are essentially identical to those of Photosystem II enriched thylakoid membranes from spinach; (ii) that the microsecond kinetics, previously shown to be proton/hydrogen transfer limited [Schilstra et al. (1998) Biochemistry 37, 3974-3981], are significantly different in Y(D)-less Chlamydomonas particles when compared with both the His-tagged Chlamydomonas particles and the spinach membranes. The oscillatory nature of the kinetics in both Chlamydomonas samples is normal, indicating that S-state cycling is unaffected by either the histidine-tagging or the replacement of tyrosine D with phenylalanine. We propose that the effects on the proton-coupled electron transfers of P680(+) reduction in the absence of Y(D) are likely to be due to pK shifts of residues in a hydrogen-bonded network of amino acids in the vicinity of Y(Z). Tyrosine D is 35 A from Y(Z) and yet has a significant influence on proton-coupled electron transfer events in the vicinity of Y(Z). This finding emphasizes the delicacy of the proton balance that Photosystem II has to achieve during the water splitting process. PMID:12501204

  11. Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network.

    PubMed

    Widhalm, Joshua R; Gutensohn, Michael; Yoo, Heejin; Adebesin, Funmilayo; Qian, Yichun; Guo, Longyun; Jaini, Rohit; Lynch, Joseph H; McCoy, Rachel M; Shreve, Jacob T; Thimmapuram, Jyothi; Rhodes, David; Morgan, John A; Dudareva, Natalia

    2015-01-01

    In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network. PMID:26356302

  12. Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network

    PubMed Central

    Widhalm, Joshua R.; Gutensohn, Michael; Yoo, Heejin; Adebesin, Funmilayo; Qian, Yichun; Guo, Longyun; Jaini, Rohit; Lynch, Joseph H.; McCoy, Rachel M.; Shreve, Jacob T.; Thimmapuram, Jyothi; Rhodes, David; Morgan, John A.; Dudareva, Natalia

    2015-01-01

    In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network. PMID:26356302

  13. Amino acid supplementation does not alter whole-body phenylalanine kinetics in Arabian geldings.

    PubMed

    Urschel, Kristine L; Geor, Raymond J; Hanigan, Mark D; Harris, Pat A

    2012-03-01

    Stable isotope infusion methods have not been extensively used in horses to study protein metabolism. The objectives were to develop infusion and sampling methodologies for [1-(13)C] phenylalanine and apply these methods to determine whether the addition of supplemental amino acids to a control diet affected whole-body phenylalanine kinetics in mature horses. Arabian geldings were studied using a 6-h primed (9 μmol/kg), constant (6 μmol · kg(-1) · h(-1)) i.v. infusion of L-[1-(13)C] phenylalanine, with blood and breath sampled every 30 min, to measure whole-body phenylalanine kinetics in response to receiving the control diet (n = 12) or the control diet supplemented with equimolar amounts of glutamate (+Glu; 55 mg · kg(-1) · d(-1); n = 5), leucine (+Leu; 49 mg · kg(-1) · d(-1); n = 5), lysine (+Lys; 55 mg · kg(-1) · d(-1); n = 5), or phenylalanine (+Phe; 62 mg · kg(-1) · d(-1); n = 6). The plasma concentrations of the supplemented amino acid in horses receiving the +Leu, +Lys, and +Phe diets were 58, 53, and 36% greater, respectively, than for the control treatment (P < 0.05). Isotopic plateau was attained in blood [1-(13)C] phenylalanine and breath (13)CO(2) enrichments by 60 and 270 min, respectively. Phenylalanine flux (+20%) and oxidation (+110%) were greater (P < 0.05) in horses receiving the +Phe treatment than in those fed the control diet. There was no effect of treatment diet on nonoxidative phenylalanine disposal or phenylalanine release from protein breakdown. The developed methods are a valuable way to study protein metabolism and assess dietary amino acid adequacy in horses and will provide a useful tool for studying amino acid requirements in the future. PMID:22259192

  14. Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing.

    PubMed

    Nair, U J; Nair, J; Friesen, M D; Bartsch, H; Ohshima, H

    1995-05-01

    The habit of betel quid chewing, common in South-East Asia and the South Pacific islands, is causally associated with an increased risk of oral cancer. Reactive oxygen species formed from polyphenolic betel quid ingredients and lime at alkaline pH have been implicated as the agents responsible for DNA and tissue damage. To determine whether hydroxyl radical (HO.) is generated in the human oral cavity during chewing of betel quid, the formation of o- and m-tyrosine from L-phenylalanine was measured. Both o- and m-tyrosine were formed in vitro in the presence of extracts of areca nut and/or catechu, transition metal ions such as Cu2+ and Fe2+ and lime or sodium carbonate (alkaline pH). Omission of any of these ingredients from the reaction mixture significantly reduced the yield of tyrosines. Hydroxyl radical scavengers such as ethanol, D-mannitol and dimethylsulfoxide inhibited the phenylalanine oxidation in a dose-dependent fashion. Five volunteers chewed betel quid consisting of betel leaf, areca nut, catechu and slaked lime (without tobacco). Their saliva, collected after chewing betel quid, contained high concentrations of p-tyrosine, but no appreciable amounts of o- or m-tyrosine. Saliva samples from the same subjects after chewing betel quid to which 20 mg phenylalanine had been added contained o- and m-tyrosine at concentrations ranging from 1010 to 3000 nM and from 1110 to 3140 nM respectively. These levels were significantly higher (P < 0.005) than those of subjects who kept phenylalanine in the oral cavity without betel quid, which ranged from 14 to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine. These studies clearly demonstrate that the HO. radical is formed in the human oral cavity during betel quid chewing and is probably implicated in the genetic damage that has been observed in oral epithelial cells of chewers. PMID:7767985

  15. Light-switched inhibitors of protein tyrosine phosphatase PTP1B based on phosphonocarbonyl phenylalanine as photoactive phosphotyrosine mimetic.

    PubMed

    Wagner, Stefan; Schütz, Anja; Rademann, Jörg

    2015-06-15

    Phosphopeptide mimetics containing the 4-phosphonocarbonyl phenylalanine (pcF) as a photo-active phosphotyrosine isoster are developed as potent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. The photo-active inhibitors 6-10 are derived from phosphopeptide substrates and are prepared from the suitably protected pcF building block 12 by Fmoc-based solid phase peptide synthesis. All pcF-containing peptides are moderate inhibitors of PTP1B with KI values between 10 and 50μM. Irradiation of the inhibitors at 365nm in the presence of the protein PTP1B amplify the inhibitory activity of pcF-peptides up to 120-fold, switching the KI values of the best inhibitors to the sub-micromolar range. Photo-activation of the inhibitors results in the formation of triplet intermediates of the benzoylphosphonate moiety, which deactivate PTP1B following an oxidative radical mechanism. Deactivation of PTP1B proceeds without covalent crosslinking of the protein target with the photo-switched inhibitors and can be reverted by subsequent addition of reducing agent dithiothreitol (DTT). PMID:25907367

  16. Serum phenylalanine screening

    MedlinePlus

    ... The test detects abnormally high levels of an amino acid called phenylalanine. ... body lacks a substance needed to breakdown the amino acid phenylalanine. If PKU is not detected early, increasing ...

  17. Remnants of an ancient pathway to L-phenylalanine and L-tyrosine in enteric bacteria: evolutionary implications and biotechnological impact.

    PubMed Central

    Bonner, C A; Fischer, R S; Ahmad, S; Jensen, R A

    1990-01-01

    The pathway construction for biosynthesis of aromatic amino acids in Escherichia coli is atypical of the phylogenetic subdivision of gram-negative bacteria to which it belongs (R. A. Jensen, Mol. Biol. Evol. 2:92-108, 1985). Related organisms possess second pathways to phenylalanine and tyrosine which depend upon the expression of a monofunctional chorismate mutase (CM-F) and cyclohexadienyl dehydratase (CDT). Some enteric bacteria, unlike E. coli, possess either CM-F or CDT. These essentially cryptic remnants of an ancestral pathway can be a latent source of biochemical potential under certain conditions. As one example of advantageous biochemical potential, the presence of CM-F in Salmonella typhimurium increases the capacity for prephenate accumulation in a tyrA auxotroph. We report the finding that a significant fraction of the latter prephenate is transaminated to L-arogenate. The tyrA19 mutant is now the organism of choice for isolation of L-arogenate, uncomplicated by the presence of other cyclohexadienyl products coaccumulated by a Neurospora crassa mutant that had previously served as the prime biological source of L-arogenate. Prephenate aminotransferase activity was not conferred by a discrete enzyme, but rather was found to be synonymous with the combined activities of aspartate aminotransferase (aspC), aromatic aminotransferase (tyrB), and branched-chain aminotransferase (ilvE). This conclusion was confirmed by results obtained with combinations of aspC-, tyrB-, and ilvE-deficient mutations in E. coli. An example of disadvantageous biochemical potential is the presence of a cryptic CDT in Klebsiella pneumoniae, where a mutant carrying multiple enzyme blocks is the standard organism used for accumulation and isolation of chorismate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2082822

  18. An unnatural amino acid based fluorescent probe for phenylalanine ammonia lyase.

    PubMed

    Tian, Zhenlin; Zhu, Weiping; Xu, Yufang; Qian, Xuhong

    2014-08-21

    A fluorescent probe (2a-LP) based on an unnatural amino acid (UAA) is developed for the detection of phenylalanine ammonia lyase (PAL). In the presence of PAL, 2a-LP is catalytically deaminated to ortho-amino-transcinnamic acid (o-a-CA), which shows a remarkable “off–on” fluorescence signal. Thus, the probe 2a-LP enables direct visualization of the PAL activity in tomato under UV illumination and has potential in vitro assays. PMID:24971756

  19. NON-PHYSIOLOGICAL AMINO ACID (NPAA) THERAPY TARGETING BRAIN PHENYLALANINE REDUCTION: PILOT STUDIES IN PAHENU2 MICE

    PubMed Central

    Vogel, Kara R.; Arning, Erland; Wasek, Brandi L.; Bottiglieri, Teodoro; Gibson, K. Michael

    2012-01-01

    Transport of large neutral amino acids (LNAA) across the blood brain barrier (BBB) is facilitated by the L-type amino acid transporter, LAT1. Peripheral accumulation of one LNAA (e.g., phenylalanine (phe) in PKU) is predicted to increase uptake of the offending amino acid to the detriment of others, resulting in disruption of brain amino acid homeostasis. We hypothesized that selected non-physiological amino acids (NPAAs) such as DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), 2-aminoisobutyrate (AIB), and N-methyl-aminoisobutyrate (MAIB), acting as competitive inhibitors of various brain amino acid transporters, could reduce brain phe in Pahenu2 mice, a relevant murine model of PKU. Oral feeding of 5% NL, 5% AIB, 0.5% NB and 3% MAIB reduced brain phe by 56% (p<0.01), −1% (p=NS), 27% (p<0.05) and 14% (p<0.01), respectively, compared to untreated subjects. Significant effects on other LNAAs (tyrosine, methionine, branched chain amino acids) were also observed, however, with MAIB displaying the mildest effects. Of interest, MAIB represents an inhibitor of the system A (alanine) transporter that primarily traffics small amino acids and not LNAAs. Our studies represent the first in vivo use of these NPAAs in Pahenu2 mice, and provide proof-of-principle for their further preclinical development, with the long-term objective of identifying NPAA combinations and concentrations that selectively restrict brain phe transport while minimally impacting other LNAAs and downstream intermediates. PMID:22976763

  20. Performance of piglets in response to the standardized ileal digestible phenylalanine and tyrosine supply in low-protein diets.

    PubMed

    Gloaguen, M; Le Floc'h, N; Primot, Y; Corrent, E; van Milgen, J

    2014-09-01

    Reducing the CP level of the diet allows for a reduction in N excretion without limiting performance as long as the amino acid (AA) requirements are covered. The availability of crystalline AA has permitted for a considerable reduction in the CP level of diets, practically used in pig nutrition. The adoption of low CP diets and the extent to which the CP content can be reduced further depends on the knowledge of the minimum levels of indispensable AA that maximize growth. The standardized ileal digestible (SID) Phe : Lys and Tyr : Lys requirements and the possibility to substitute Tyr by Phe have never been studied in piglets. The objectives of this study were to estimate these requirements in 10 to 20 kg pig as well as to determine the extent to which Phe can be used to cover the Tyr requirement. In three dose-response studies, six pigs within each of 14 blocks were assigned to six low CP diets (14.5% CP) sub-limiting in Lys at 1.00% SID. In experiment 1, the SID Phe : Lys requirement estimate was assessed by supplementing a Phe-deficient diet with different levels of l-Phe to attain 33%, 39%, 46%, 52%, 58%, and 65% SID Phe : Lys. Because Phe can be used for Tyr synthesis, the diets provided a sufficient Tyr supply. A similar approach was used in experiment 2 with six levels of l-Tyr supplementation to attain 21%, 27%, 33%, 39%, 45% and 52% SID Tyr : Lys. Phenylalanine was supplied at a level sufficient to sustain maximum growth (estimated in experiment 1). The SID Phe : Lys and SID Tyr : Lys requirements for maximizing daily gain were 54% and 40% using a curvilinear-plateau model, respectively. A 10% deficiency in Phe and Tyr reduced daily gain by 3.0% and 0.7%, respectively. In experiment 3, the effect of the equimolar substitution of dietary SID Tyr by Phe to obtain 50%, 57%, and 64% SID Phe : (Phe+Tyr) was studied at two limiting levels of Phe+Tyr. From 57% to 64% SID Phe : (Phe+Tyr), performance was slightly reduced. In conclusion, it is recommended not to

  1. Heterologous production of caffeic acid from tyrosine in Escherichia coli.

    PubMed

    Rodrigues, J L; Araújo, R G; Prather, K L J; Kluskens, L D; Rodrigues, L R

    2015-04-01

    Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62mM (472mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56mM (280mg/L) and 1mM (180mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids. PMID:25765308

  2. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

    PubMed Central

    2016-01-01

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

  3. Diseases of Phenylalanine Metabolism

    PubMed Central

    Parker, Charles E.

    1979-01-01

    Continuing investigation of the system that hydroxylates phenylalanine to tyrosine has led to new insights into diseases associated with the malfunction of this system. Good evidence has confirmed that phenylketonuria (PKU) is not caused by a simple lack of phenylalanine hydroxylase. Dihydropteridine reductase deficiency as well as defects in biopterin metabolism may also cause the clinical features of phenylketonuria. Furthermore, these diseases do not respond to the standard treatment for phenylketonuria. PMID:388868

  4. Phenylalanine sensitive K562-D cells for the analysis of the biochemical impact of excess amino acid

    PubMed Central

    Sanayama, Yoshitami; Matsumoto, Akio; Shimojo, Naoki; Kohno, Yoichi; Nakaya, Haruaki

    2014-01-01

    Although it is recognized that the abnormal accumulation of amino acid is a cause of the symptoms in metabolic disease such as phenylketonuria (PKU), the relationship between disease severity and serum amino acid levels is not well understood due to the lack of experimental model. Here, we present a novel in vitro cellular model using K562-D cells that proliferate slowly in the presence of excessive amount of phenylalanine within the clinically observed range, but not phenylpyruvate. The increased expression of the L-type amino acid transporter (LAT2) and its adapter protein 4F2 heavy chain appeared to be responsible for the higher sensitivity to phenylalanine in K562-D cells. Supplementation with valine over phenylalanine effectively restored cell proliferation, although other amino acids did not improve K562-D cell proliferation over phenylalanine. Biochemical analysis revealed mammalian target of rapamycin complex (mTORC) as a terminal target of phenylalanine in K562-D cell proliferation, and supplementation of valine restored mTORC1 activity. Our results show that K562-D cell can be a potent tool for the investigation of PKU at the molecular level and to explore new therapeutic approaches to the disease. PMID:25373594

  5. Non-ionic surfactant modified ligand exchange chromatography using copper (II) complex of N,N-dimethyl-L-phenylalanine as the chiral additive for enantioselective amino acids separation.

    PubMed

    Dimitrova, Pepa; Bart, Hans-Jörg

    2010-03-17

    The influence of non-ionic surfactants on the selectivity and retention in the ligand exchange chromatography for the enantioselective separation of racemic mixtures of the amino acids dl-methionine, dl-leucine, dl-valine and dl-tyrosine applying chiral mobile phases was investigated, whereas five different surfactants were tested as modifiers. The experiments were carried out using a commercially available non-chiral RP-C8 column and the copper (II) complex of N,N-dimethyl-l-phenylalanine as the chiral additive. Varying the surfactant concentrations the retention factors and the selectivity could be controlled and in general no negative influence on the separation (due to surfactant adsorption on the non-chiral stationary phase) occurred. Changing the temperature the van't Hoff plots were obtained and the thermodynamic parameters calculated. Temperature had influence on the selectivity for each surfactant and lowered the retention times as expected. PMID:20172105

  6. Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps

    NASA Technical Reports Server (NTRS)

    Guerra, D.; Anderson, A. J.; Salisbury, F. B.

    1985-01-01

    Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

  7. Self-assembled phenylalanine-α,β-dehydrophenylalanine nanotubes for sustained intravitreal delivery of a multi-targeted tyrosine kinase inhibitor.

    PubMed

    Panda, Jiban J; Yandrapu, Sarath; Kadam, Rajendra S; Chauhan, Virander S; Kompella, Uday B

    2013-12-28

    Current standard of care for sustained back of the eye drug delivery is surgical placement or injection of large, slow release implants using a relatively large 22 gauge needle. We designed novel dipeptide (phenylalanine-α,β-dehydrophenylalanine; Phe-∆Phe) based nanotubes with a diameter of ~15-30 nm and a length of ~1500 nm that could be injected with a 33 gauge needle for sustained intravitreal delivery of pazopanib, a multi-targeted tyrosine kinase inhibitor. The drug could be loaded during nanotube assembly or post-loaded after nanotube formation, with the former being more efficient at 25% w/w pazopanib loading and ~55% loading efficiency. Plain and peptide loaded nanotube were non-cytotoxic to retinal pigment epithelial cells even at a concentration of 200 μg/ml. Following intravitreal injection of fluorescently labeled nanotubes using a 33 gauge needle in a rat model, the nanotube persistence and drug delivery were monitored using noninvasive fluorophotometry, electron microscopy and mass spectrometry analysis. Nanotubes persisted in the vitreous humor during the 15 days study and pazopanib levels in the vitreous humor, retina, and choroid-RPE at the end of the study were 4.5, 5, and 2.5-folds higher, respectively, compared to the plain drug. Thus, Phe-∆Phe nanotubes allow intravitreal injections with a small gauge needle and sustain drug delivery. PMID:24075925

  8. Effect of aspartame and protein, administered in phenylalanine-equivalent doses, on plasma neutral amino acids, aspartate, insulin and glucose in man.

    PubMed

    Møller, S E

    1991-05-01

    Six human males each received 0.56 g phenylalanine (Phe) in the form of 1.0 g aspartame or 12.2 g bovine albumin in 200 ml water or water alone. Venous blood samples collected before consumption and during the following 4 hr were assayed for plasma levels of large, neutral amino acids (LNAA), aspartate, insulin and glucose. The area under the curve for plasma Phe was 40% greater, although not significant, after aspartame compared with albumin intake. The indicated increased clearance rate of plasma Phe after albumin may be caused by the significant increase of insulin, on which aspartame had no effect. There was a significant main effect of aspartame for plasma tyrosine but not for tryptophan, valine, isoleucine or leucine. Plasma aspartate was significantly increased at 0.25 hr after the aspartame intake. The percentage Phe/LNAA decreased slightly in response to albumin but increased 55% after aspartame and remained significantly increased for 2 hr. Tyrosine/LNAA increased and tryptophan/LNAA decreased modestly after aspartame intake. The study showed that the intake of aspartame in a not unrealistically high dose produced a marked and persistent increase of the availability of Phe to the brain, which was not observed after protein intake. The study indicated, furthermore, that Phe was cleared faster from the plasma after consumption of protein compared with aspartame. PMID:1946186

  9. Phenylketonuria: brain phenylalanine concentrations relate inversely to cerebral protein synthesis

    PubMed Central

    de Groot, Martijn J; Sijens, Paul E; Reijngoud, Dirk-Jan; Paans, Anne M; van Spronsen, Francjan J

    2015-01-01

    In phenylketonuria, elevated plasma phenylalanine concentrations may disturb blood-to-brain large neutral amino acid (LNAA) transport and cerebral protein synthesis (CPS). We investigated the associations between these processes, using data obtained by positron emission tomography with l-[1-11C]-tyrosine (11C-Tyr) as a tracer. Blood-to-brain transport of non-Phe LNAAs was modeled by the rate constant for 11C-Tyr transport from arterial plasma to brain tissue (K1), while CPS was modeled by the rate constant for 11C-Tyr incorporation into cerebral protein (k3). Brain phenylalanine concentrations were measured by magnetic resonance spectroscopy in three volumes of interest (VOIs): supraventricular brain tissue (VOI 1), ventricular brain tissue (VOI 2), and fluid-containing ventricular voxels (VOI 3). The associations between k3 and each predictor variable were analyzed by multiple linear regression. The rate constant k3 was inversely associated with brain phenylalanine concentrations in VOIs 2 and 3 (adjusted R2=0.826, F=19.936, P=0.021). Since brain phenylalanine concentrations in these VOIs highly correlated with each other, the specific associations of each predictor with k3 could not be determined. The associations between k3 and plasma phenylalanine concentration, K1, and brain phenylalanine concentrations in VOI 1 were nonsignificant. In conclusion, our study shows an inverse association between k3 and increased brain phenylalanine concentrations. PMID:25352046

  10. Food products made with glycomacropeptide, a low-phenylalanine whey protein, provide a new alternative to amino Acid-based medical foods for nutrition management of phenylketonuria.

    PubMed

    van Calcar, Sandra C; Ney, Denise M

    2012-08-01

    Phenylketonuria (PKU), an inborn error in phenylalanine metabolism, requires lifelong nutrition management with a low-phenylalanine diet, which includes a phenylalanine-free amino acid-based medical formula to provide the majority of an individual's protein needs. Compliance with this diet is often difficult for older children, adolescents, and adults with PKU. The whey protein glycomacropeptide (GMP) is ideally suited for the PKU diet because it is naturally low in phenylalanine. Nutritionally complete, acceptable medical foods and beverages can be made with GMP to increase the variety of protein sources for the PKU diet. As an intact protein, GMP improves protein use and increases satiety compared with amino acids. Thus, GMP provides a new, more physiologic source of low-phenylalanine dietary protein for people with PKU. PMID:22818728

  11. Defects of tyrosine289phenylalanine mutation on binding and functional properties of the human tachykinin NK2 receptor stably expressed in chinese hamster ovary cells.

    PubMed

    Renzetti, A R; Catalioto, R M; Carloni, C; Criscuoli, M; Cucchi, P; Giolitti, A; Zappitelli, S; Rotondaro, L; Maggi, C A

    1999-04-15

    A point mutation was made at position 289 in the transmembrane segment 7 of the human tachykinin NK2 receptor to yield a tyrosine/phenylalanine (Tyr/Phe) substitution. Chinese hamster ovary cells stably transfected with the wild-type or Tyr289Phe mutant NK2 receptor both bound neurokinin A (NKA) and the synthetic NK2 receptor-selective agonists, GR 64349 and [betaAla8]NKA(4-10), with high and even affinities. Neurokinin B (NKB) and substance P (SP) also displayed sizeable binding affinities, albeit with lower affinity as compared to NKA. In a functional assay (production of inositol-1,4,5-trisphosphate, IP3), NKA, GR 64349, and [betaAla8]INKA(4-10) stimulated IP3 accumulation via the wild-type and mutant receptors with similar potencies. On the other hand, NKB and SP exhibited a dramatic reduction in their agonist efficacies at the mutant receptor, NKB acting as a partial agonist (maximum effect = 50% of the response to NKA) and SP being totally inactive. The results obtained with phenoxybenzamine inactivation experiments indicated that a large and similar receptor reserve existed for both the wild-type and the mutant receptor. SP, which displayed sizeable binding affinity for the mutant receptor but did not stimulate IP3 accumulation, antagonized the agonist effect of NKA. The antagonist action of SP at the mutant NK2 receptor cannot be ascribed to receptor internalization. The Tyr/Phe replacement at position 289 markedly reduced the binding affinity and antagonist potency of the non-peptide ligand, SR 48968, without affecting the binding affinity and antagonist potency of the bicyclic peptide antagonist MEN 11420. The results indicate that the hydroxyl radical function of Tyr289 in transmembrane segment 7 of the human NK2 receptor is, directly or indirectly, involved in stimulus transduction when the NK2 receptor is occupied by NKB or SP, but not when using NKA or NK2 receptor-selective agonists. PMID:10086323

  12. The global gene expression response of Escherichia coli to L-phenylalanine.

    PubMed

    Polen, T; Krämer, M; Bongaerts, J; Wubbolts, M; Wendisch, V F

    2005-02-01

    We investigated the global gene expression changes of Escherichia coli due to the presence of different concentrations of phenylalanine or shikimate in the growth medium. The response to 0.5 g l(-1) phenylalanine primarily reflected a perturbed aromatic amino acid metabolism, in particular due to TyrR-mediated regulation. The addition of 5g l(-1) phenylalanine reduced the growth rate by half and elicited a great number of likely indirect effects on genes regulated in response to changed pH, nitrogen or carbon availability. Consistent with the observed gene expression changes, supplementation with shikimate, tyrosine and tryptophan relieved growth inhibition by phenylalanine. In contrast to the wild-type, a tyrR disruption strain showed increased expression of pckA and of tktB in the presence of phenylalanine, but its growth was not affected by phenylalanine at the concentrations tested. The absence of growth inhibition by phenylalanine suggested that at high phenylalanine concentrations TyrR-defective strains might perform better in phenylalanine production. PMID:15639085

  13. A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

    PubMed Central

    2015-01-01

    The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of

  14. Aspartame and sucrose produce a similar increase in the plasma phenylalanine to large neutral amino acid ratio in healthy subjects.

    PubMed

    Burns, T S; Stargel, W W; Tschanz, C; Kotsonis, F N; Hurwitz, A

    1991-01-01

    Aspartame (L-aspartyl-L-phenylalanine methyl ester) consumption has been postulated to increase brain phenylalanine levels by increasing the molar ratio of the plasma phenylalanine concentration to the sum of the plasma concentrations of the other large neutral amino acids (Phe/LNAA). Dietary manipulations with carbohydrate or protein can also produce changes in the Phe/LNAA value. To compare the effects of aspartame and carbohydrate on Phe/LNAA, beverages sweetened with aspartame, sucrose, and aspartame plus sucrose, and unsweetened beverage were ingested by 8 healthy, fasted subjects in a randomized, four-way crossover design. The beverages were sweetened with an amount of aspartame (500 mg) and/or sucrose (100 g) approximately equivalent to that used to sweeten 1 liter of soft drink. The baseline-corrected plasma Phe/LNAA values did not differ significantly following ingestion of aspartame or sucrose. Following aspartame alone, the high mean ratio increased 26% over baseline 1 h after ingestion. Following sucrose alone, the high mean ratio increased 19% at 2.5 h. Sucrose increased the Phe/LNAA value due to an insulin-mediated decrease in the plasma LNAA, while aspartame increased the ratio by increasing the plasma Phe concentration. These findings indicate that similar increases in plasma Phe/LNAA occur when healthy, fasting subjects ingest amounts of equivalent sweetness of sucrose or aspartame. PMID:1771173

  15. Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum) 1

    PubMed Central

    Cheng, Christina K.-C.; Marsh, H. V.

    1968-01-01

    The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10−6-10−4 m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10−4 m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification. PMID:16656968

  16. Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum).

    PubMed

    Cheng, C K; Marsh, H V

    1968-11-01

    The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10(-6)-10(-4)m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10(-4)m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification. PMID:16656968

  17. Study of the effects of proline, phenylalanine, and urea foliar application to Tempranillo vineyards on grape amino acid content. Comparison with commercial nitrogen fertilisers.

    PubMed

    Garde-Cerdán, T; López, R; Portu, J; González-Arenzana, L; López-Alfaro, I; Santamaría, P

    2014-11-15

    The aim of this work was to study the influence of foliar application of different nitrogen sources on grape amino acid content. The nitrogen sources applied to Tempranillo grapevines were proline, phenylalanine, urea, and two commercial nitrogen fertilisers, both without and with amino acids in their formulations. All treatments were applied at veraison and one week later. Proline treatment did not affect the must nitrogen composition. However, phenylalanine and urea foliar application enhanced the plants' synthesis of most of the amino acids, producing similar effects. In addition, the spray of commercial nitrogen fertilisers over leaves also induced a rise in grape amino acid concentrations regardless of the presence or absence of amino acids in their formulation. The most effective treatments were phenylalanine and urea followed by nitrogen fertilisers. This finding is of oenological interest for improved must nitrogen composition, ensuring better fermentation kinetics and most likely enhancing wine quality. PMID:24912708

  18. Expression, purification, crystallization, data collection and preliminary biochemical characterization of methicillin-resistant Staphylococcus aureus Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase

    SciTech Connect

    Seetharamappa, Jaldappagari; Oke, Muse; Liu, Huanting; McMahon, Stephen A.; Johnson, Kenneth A.; Carter, Lester; Dorward, Mark; Zawadzki, Michal; Overton, Ian M.; Niekirk, C. A. Johannes van; Graham, Shirley; Botting, Catherine H.; Taylor, Garry L.; White, Malcolm F.; Barton, Geoffrey J.; Coote, Peter J.; Naismith, James H.

    2007-05-01

    As part of work on S. aureus, the crystallization of Sar2028, a protein that is upregulated in MRSA, is reported. Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5′-phosphate-dependent aminotransferase with a molecular weight of 48 168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 Å, α = β = γ = 90°. Analysis of the systematic absences along the three principal axes indicated the space group to be P2{sub 1}2{sub 1}2{sub 1}. A complete data set was collected to 2.5 Å resolution.

  19. Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase.

    PubMed

    Gannavaram, Swathi; Sirin, Sarah; Sherman, Woody; Gadda, Giovanni

    2014-10-21

    The flavin-mediated enzymatic oxidation of a CN bond in amino acids can occur through hydride transfer, carbanion, or polar nucleophilic mechanisms. Previous results with D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) using multiple deuterium kinetic isotope effects (KIEs) and computational studies established preferred binding of the substrate protonated on the α-amino group, with cleavages of the NH and CH bonds occurring in asynchronous fashion, consistent with the three possible mechanisms. The hydroxyl groups of Y53 and Y249 are ≤4 Å from the imino and carboxylate groups of the reaction product iminoarginine, suggesting participation in binding and catalysis. In this study, we have investigated the reductive half-reactions of the Y53F and Y249F variants of PaDADH using substrate and solvent deuterium KIEs, solvent viscosity and pH effects, and quantum mechanical/molecular mechanical computational approaches to gain insights into the catalytic roles of the tyrosines and evaluate whether their mutations affect the transition state for substrate oxidation. Both Y53F and Y249F enzymes oxidized D-arginine with steady-state kinetic parameters similar to those of the wild-type enzyme. Rate constants for flavin reduction (k(red)) with D-leucine, a slow substrate amenable to rapid kinetics, were 3-fold smaller than the wild-type value with similar pKa values for an unprotonated group of ∼10.0. Similar pKa values were observed for (app)Kd in the variant and wild-type enzymes. However, cleavage of the substrate NH and CH bonds in the enzyme variants occurred in synchronous fashion, as suggested by multiple deuterium KIEs on k(red). These data can be reconciled with a hydride transfer mechanism, but not with carbanion and polar nucleophilic mechanisms. PMID:25243743

  20. Spectroscopic analyses on interaction of o-Vanillin- D-Phenylalanine, o-Vanillin- L-Tyrosine and o-Vanillin- L-Levodopa Schiff Bases with bovine serum albumin (BSA)

    NASA Astrophysics Data System (ADS)

    Gao, Jingqun; Guo, Yuwei; Wang, Jun; Wang, Zhiqiu; Jin, Xudong; Cheng, Chunping; Li, Ying; Li, Kai

    2011-04-01

    In this work, three o-Vanillin Schiff Bases (o-VSB: o-Vanillin- D-Phenylalanine (o-VDP), o-Vanillin- L-Tyrosine (o-VLT) and o-Vanillin- L-Levodopa (o-VLL)) with alanine constituent were synthesized by direct reflux method in ethanol solution, and then were used to study the interaction to bovine serum albumin (BSA) molecules by fluorescence spectroscopy. Based on the fluorescence quenching calculation, the bimolecular quenching constant ( Kq), apparent quenching constant ( Ksv), effective binding constant ( KA) and corresponding dissociation constant ( KD) as well as binding site number ( n) were obtained. In addition, the binding distance ( r) was also calculated according to Foster's non-radioactive energy transfer theory. The results show that these three o-VSB can efficiently bind to BSA molecules, but the binding array order is o-VDP-BSA > o-VLT-BSA > o-VLL-BSA. Synchronous fluorescence spectroscopy indicates that the o-VDP is more accessibility to tryptophan (Trp) residues of BSA molecules than to tyrosine (Tyr) residues. Nevertheless, the o-VLT and o-VLL are more accessibility to Tyr residues than to Trp residues.

  1. Utilization of [14C]phenylalanine derived from arylphorin or free amino acid in Manduca sexta pharate adults

    NASA Technical Reports Server (NTRS)

    Wu, M.; Tischler, M. E.

    1995-01-01

    The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.

  2. A comparative study of the vibrational spectra of the anticancer drug melphalan and its fundamental molecules 3-phenylpropionic acid and L-phenylalanine

    NASA Astrophysics Data System (ADS)

    Badawi, Hassan M.; Khan, Ibrahim

    2016-04-01

    The structural stability and the vibrational spectra of the anticancer drug melphalan and its parent compounds 3-phenylpropionic acid and L-phenylalanine were investigated by the DFT B3LYP/6-311G** calculations. Melphalan and its fundamental compounds were predicted to exist predominantly in non-planar structures. The vibrational frequencies of the low energy structures of melphalan, 3-phenylpropionic acid, and phenylalanine were computed at the DFT B3LYP level of theory. Complete vibrational assignments of the normal modes of melphalan, 3-phenylpropionic acid, and phenylalanine were provided by combined theoretical and experimental data of the molecules. The experimental infrared spectra of phenylalanine and melphalan show a significantly different pattern of the Cdbnd O stretching mode as compared to those of normal carboxylic acids. A comparison of the 3700-2000 cm-1 infrared spectral region of the three molecules suggests the presence of similar intermolecular H-bonding in their condensed phases. The observed infrared and Raman spectra are consistent with the presence of one predominant melphalan conformation at room temperature.

  3. Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor alpha.

    PubMed

    Mailfait, S; Belaiche, D; Kouach, M; Dallery, N; Chavatte, P; Formstecher, P; Sablonnière, B

    2000-03-01

    Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha mutants: Y208A, Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly

  4. Fungal and Plant Phenylalanine Ammonia-lyase

    PubMed Central

    Hyun, Min Woo; Yun, Yeo Hong; Kim, Jun Young

    2011-01-01

    L-Phenylalanine is one of the essential amino acids that cannot be synthesized in mammals in adequate amounts to meet the requirements for protein synthesis. Fungi and plants are able to synthesize phenylalanine via the shikimic acid pathway. L-Phenylalanine, derived from the shikimic acid pathway, is used directly for protein synthesis in plants or metabolized through the phenylpropanoid pathway. This phenylpropanoid metabolism leads to the biosynthesis of a wide array of phenylpropanoid secondary products. The first step in this metabolic sequence involves the action of phenylalanine ammonia-lyase (PAL). The discovery of PAL enzyme in fungi and the detection of 14CO2 production from 14C-ring-labeled phenylalanine and cinnamic acid demonstrated that certain fungi can degrade phenylalanine by a pathway involving an initial deamination to cinnamic acid, as happens in plants. In this review, we provide background information on PAL and a recent update on the presence of PAL genes in fungi. PMID:22783113

  5. The relationship between low leucocyte ascorbic-acid levels and tyrosine metabolism in the elderly.

    PubMed

    Windsor, A C; Hobbs, C B; Treby, D A; Gupta, C B

    1975-11-01

    Twenty-seven elderly men aged 69-94 years had tyrosine tolerance tests measured before and after receiving ascorbic acid 1 g daily for four days. There was a significant rise in the fasting and half-hourly mean plasma tyrosine levels after ascorbic acid administration in those subjects with an initial leucocyte ascorbic-acid level (LAA) below 15 micrograms/10(8) w.c.c. The peak of the tyrosine tolerance curve was also advanced following administration of ascorbic acid in those subjects with an initial LAA below 15 micrograms/10(8) w.c.c. A further ten elderly men aged 73-89 years had simultaneous measurements of the circadian rhythms of LAA, plasma cortisol and plasma tyrosine levels before and after receiving ascorbic acid 1 g daily for four days. All ten subjects showed a peak LAA level at 5 p.m. when saturated with vitamin C but there was no significant correlation between plasma cortisol changes and plasma tyrosine changes. Possible explanations for the results are discussed. PMID:803133

  6. Molecular aggregation in selected crystalline 1:1 complexes of hydrophobic D- and L-amino acids. IV. The L-phenylalanine series.

    PubMed

    Görbitz, Carl Henrik; Rissanen, Kari; Valkonen, Arto; Husabø, Asmund

    2009-06-01

    The amino acid L-phenylalanine has been cocrystallized with D-2-aminobutyric acid, C(9)H(11)NO(2).C(4)H(9)NO(2), D-norvaline, C(9)H(11)NO(2).C(5)H(11)NO(2), and D-methionine, C(9)H(11)NO(2).C(5)H(11)NO(2)S, with linear side chains, as well as with D-leucine, C(9)H(11)NO(2).C(6)H(13)NO(2), D-isoleucine, C(9)H(11)NO(2).C(6)H(13)NO(2), and D-allo-isoleucine, C(9)H(11)NO(2).C(6)H(13)NO(2), with branched side chains. The structures of these 1:1 complexes fall into two classes based on the observed hydrogen-bonding pattern. From a comparison with other L:D complexes involving hydrophobic amino acids and regular racemates, it is shown that the structure-directing properties of phenylalanine closely parallel those of valine and isoleucine but not those of leucine, which shares side-chain branching at C(gamma) with phenylalanine and is normally considered to be the most closely related non-aromatic amino acid. PMID:19498234

  7. Enantioselective acylation of β-phenylalanine acid and its derivatives catalyzed by penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Li, Dengchao; Ji, Lilian; Wang, Xinfeng; Wei, Dongzhi

    2013-01-01

    This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however, increased to 42.9% as the pH value and the molar ratio of phenylacetamide to BPA were elevated to 11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conversion efficiencies of BPA derivatives all reached about 50% in a relatively short reaction time (45-90 min). The enantiomeric excess value of product (ee(p)) and enantiomeric excess value of substrate (ee(s)) were all above 98% and 95%, respectively. These results suggest that the method established in this study is practical, effective, and environmentally benign and may be applied to industrial production of enantiomerically pure BPA and its derivatives. PMID:23302108

  8. Urine homogentisic acid and tyrosine: simultaneous analysis by liquid chromatography tandem mass spectrometry.

    PubMed

    Hughes, A T; Milan, A M; Christensen, P; Ross, G; Davison, A S; Gallagher, J A; Dutton, J J; Ranganath, L R

    2014-07-15

    Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid (HGA) which although excreted in gram quantities in the urine, is deposited as an ochronotic pigment in connective tissues, especially cartilage. Ochronosis leads to a severe, early-onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, Nitisinone, an inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of HGA, resulting in accumulation of the upstream pre-cursor, tyrosine. Using reverse phase LC-MS/MS, a method has been developed to simultaneously quantify urinary HGA and tyrosine. Using matrix-matched calibration standards, two product ion transitions were identified for each compound and their appropriate isotopically labelled internal standards. Validation was performed across the AKU and post-treatment concentrations expected. Intrabatch accuracy for acidified urine was 96-109% for tyrosine and 94-107% for HGA; interbatch accuracy (n=20 across ten assays) was 95-110% for tyrosine and 91-109% for HGA. Precision, both intra- and interbatch was <10% for tyrosine and <5% for HGA. Matrix effects observed with acidified urine (12% decrease, CV 5.6%) were normalised by the internal standard. Tyrosine and HGA were proved stable under various storage conditions and no carryover, was observed. Overall the method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of patients with AKU, pre and post-nitisinone therapy. PMID:24952314

  9. Role of Tyrosine Isomers in Acute and Chronic Diseases Leading to Oxidative Stress - A Review

    PubMed Central

    Molnár, Gergő A.; Kun, Szilárd; Sélley, Eszter; Kertész, Melinda; Szélig, Lívia; Csontos, Csaba; Böddi, Katalin; Bogár, Lajos; Miseta, Attila; Wittmann, István

    2016-01-01

    Oxidative stress plays a major role in the pathogenesis of a variety of acute and chronic diseases. Measurement of the oxidative stress-related end products may be performed, e.g. that of structural isomers of the physiological para-tyrosine, namely meta- and ortho-tyrosine, that are oxidized derivatives of phenylalanine. Recent data suggest that in sepsis, serum level of meta-tyrosine increases, which peaks on the 2nd and 3rd days (p<0.05 vs. controls), and the kinetics follows the intensity of the systemic inflammation correlating with serum procalcitonin levels. In a similar study subset, urinary meta-tyrosine excretion correlated with both need of daily insulin dose and the insulin-glucose product in non-diabetic septic cases (p<0.01 for both). Using linear regression model, meta-tyrosine excretion, urinary meta-tyrosine/para-tyrosine, urinary ortho-tyrosine/para-tyrosine and urinary (meta- + ortho-tyrosine)/para-tyrosine proved to be markers of carbohydrate homeostasis. In a chronic rodent model, we tried to compensate the abnormal tyrosine isomers using para-tyrosine, the physiological amino acid. Rats were fed a standard high cholesterol-diet, and were given para-tyrosine or vehicle orally. High-cholesterol feeding lead to a significant increase in aortic wall meta-tyrosine content and a decreased vasorelaxation of the aorta to insulin and the glucagon-like peptide-1 analogue, liraglutide, that both could be prevented by administration of para-tyrosine. Concluding, these data suggest that meta- and ortho-tyrosine are potential markers of oxidative stress in acute diseases related to oxidative stress, and may also interfere with insulin action in septic humans. Competition of meta- and ortho-tyrosine by supplementation of para-tyrosine may exert a protective role in oxidative stress-related diseases. PMID:26785996

  10. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  11. Replacement of a phenylalanine by a tyrosine in the active site confers fructose-6-phosphate aldolase activity to the transaldolase of Escherichia coli and human origin.

    PubMed

    Schneider, Sarah; Sandalova, Tatyana; Schneider, Gunter; Sprenger, Georg A; Samland, Anne K

    2008-10-31

    Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe(178) to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. PMID:18687684

  12. Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin*S⃞

    PubMed Central

    Schneider, Sarah; Sandalova, Tatyana; Schneider, Gunter; Sprenger, Georg A.; Samland, Anne K.

    2008-01-01

    Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe178 to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody. PMID:18687684

  13. Role of Tyrosine Isomers in Acute and Chronic Diseases Leading to Oxidative Stress - A Review.

    PubMed

    Molnár, Gergő A; Kun, Szilárd; Sélley, Eszter; Kertész, Melinda; Szélig, Lívia; Csontos, Csaba; Böddi, Katalin; Bogár, Lajos; Miseta, Attila; Wittmann, István

    2016-01-01

    Oxidative stress plays a major role in the pathogenesis of a variety of acute and chronic diseases. Measurement of the oxidative stress-related end products may be performed, e.g. that of structural isomers of the physiological para-tyrosine, namely meta- and ortho-tyrosine, that are oxidized derivatives of phenylalanine. Recent data suggest that in sepsis, serum level of meta-tyrosine increases, which peaks on the 2(nd) and 3(rd) days (p<0.05 vs. controls), and the kinetics follows the intensity of the systemic inflammation correlating with serum procalcitonin levels. In a similar study subset, urinary meta-tyrosine excretion correlated with both need of daily insulin dose and the insulin-glucose product in non-diabetic septic cases (p<0.01 for both). Using linear regression model, meta-tyrosine excretion, urinary meta-tyrosine/para-tyrosine, urinary ortho-tyrosine/para-tyrosine and urinary (meta- + orthotyrosine)/ para-tyrosine proved to be markers of carbohydrate homeostasis. In a chronic rodent model, we tried to compensate the abnormal tyrosine isomers using para-tyrosine, the physiological amino acid. Rats were fed a standard high cholesterol-diet, and were given para-tyrosine or vehicle orally. High-cholesterol feeding lead to a significant increase in aortic wall meta-tyrosine content and a decreased vasorelaxation of the aorta to insulin and the glucagon-like peptide-1 analogue, liraglutide, that both could be prevented by administration of para-tyrosine. Concluding, these data suggest that meta- and ortho-tyrosine are potential markers of oxidative stress in acute diseases related to oxidative stress, and may also interfere with insulin action in septic humans. Competition of meta- and ortho-tyrosine by supplementation of para-tyrosine may exert a protective role in oxidative stress-related diseases. PMID:26785996

  14. Shikimate and Phenylalanine Biosynthesis in the Green Lineage

    PubMed Central

    Tohge, Takayuki; Watanabe, Mutsumi; Hoefgen, Rainer; Fernie, Alisdair R.

    2013-01-01

    The shikimate pathway provides carbon skeletons for the aromatic amino acids l-tryptophan, l-phenylalanine, and l-tyrosine. It is a high flux bearing pathway and it has been estimated that greater than 30% of all fixed carbon is directed through this pathway. These combined pathways have been subjected to considerable research attention due to the fact that mammals are unable to synthesize these amino acids and the fact that one of the enzymes of the shikimate pathway is a very effective herbicide target. However, in addition to these characteristics these pathways additionally provide important precursors for a wide range of important secondary metabolites including chlorogenic acid, alkaloids, glucosinolates, auxin, tannins, suberin, lignin and lignan, tocopherols, and betalains. Here we review the shikimate pathway of the green lineage and compare and contrast its evolution and ubiquity with that of the more specialized phenylpropanoid metabolism which this essential pathway fuels. PMID:23543266

  15. Phenylalanine Metabolism Regulates Reproduction and Parasite Melanization in the Malaria Mosquito

    PubMed Central

    Fuchs, Silke; Behrends, Volker; Bundy, Jacob G.; Crisanti, Andrea; Nolan, Tony

    2014-01-01

    The blood meal of the female malaria mosquito is a pre-requisite to egg production and also represents the transmission route for the malaria parasite. The proper and rapid assimilation of proteins and nutrients in the blood meal creates a significant metabolic challenge for the mosquito. To better understand this process we generated a global profile of metabolite changes in response to blood meal of Anopheles gambiae, using Gas Chromatography-Mass Spectrometry (GC-MS). To disrupt a key pathway of amino acid metabolism we silenced the gene phenylalanine hydroxylase (PAH) involved in the conversion of the amino acid phenylalanine into tyrosine. We observed increased levels of phenylalanine and the potentially toxic metabolites phenylpyruvate and phenyllactate as well as a reduction in the amount of tyrosine available for melanin synthesis. This in turn resulted in a significant impairment of the melanotic encapsulation response against the rodent malaria parasite Plasmodium berghei. Furthermore silencing of PAH resulted in a significant impairment of mosquito fertility associated with reduction of laid eggs, retarded vitellogenesis and impaired melanisation of the chorion. Carbidopa, an inhibitor of the downstream enzyme DOPA decarboxylase that coverts DOPA into dopamine, produced similar effects on egg melanization and hatching rate suggesting that egg chorion maturation is mainly regulated via dopamine. This study sheds new light on the role of amino acid metabolism in regulating reproduction and immunity. PMID:24409310

  16. Specificity of Deoxyribonucleic Acid Intercalating Compounds in the Control of Phenylalanine Ammonia Lyase and Pisatin Levels 1

    PubMed Central

    Hadwiger, Lee A.; Schwochau, Martin E.

    1971-01-01

    Compounds with planar triple ring systems such as acridine orange, 9-amino acridine, 9-amino-1,2,3,4-tetrahydroacridine (tacrine), 6,9-diamino-2-ethoxyacridine lactate monohydrate (DE-acridine), 6-chloro-9-(3′-diethylamino-2′-hydroxypropylamino) -2-methoxyacridine·2 HCl (CDM-acridine), quinacrine, 6-chloro-9-(4′-diethylamino-1′-methylbutylamino) -2-methoxy-1,10-diazaanthracene (CDM 1,10-diazaanthracene), thionine, azure A, methylene blue, and pyronine Y when applied to excised pea pods were potent inducers of phenylalanine ammonia lyase or of pisatin, or of both. Compounds with an array of structural variation around the planar three-ring system were tested for their ability to induce these responses in pea tissue. In general, dimethylamino, diethylamino, or amino substitutions at position 2 and 6 or an amino (with or without an aliphatic side chain) substitution at position 9 of the three-ring system augmented induction potential. Methyl green, methylene blue, 2,7-diaminofluorene, nile blue, neutral red, pyrogallol red, ethidium bromide, nogalamycin, quinine, chloroquine, spermine, 8-azaguanine, gliotoxin, chromomycin A3, actinomycin D, and mitomycin C were also potent inducers. The inhibition of phenylalanine ammonia lyase induction by the application of actinomycin D (300 micrograms per milliliter) or 6-methylpurine (1 milligram per milliliter) within 1 hour after inducer application indicated that newly synthesized RNA is necessary for induction. Phenylalanine ammonia lyase induction was also inhibited by cycloheximide (150 micrograms per milliliter). PMID:16657620

  17. Abscisic Acid Suppression of Phenylalanine Ammonia-Lyase Activity and mRNA, and Resistance of Soybeans to Phytophthora megasperma f.sp. glycinea1

    PubMed Central

    Ward, Edmund W. B.; Cahill, David M.; Bhattacharyya, Madan K.

    1989-01-01

    Etiolated hypocotyls of the resistant soybean (Glycine max [L.] Merr.) cultivar Harosoy 63 became susceptible to Phytophthora megasperma (Drechs.) f.sp. glycinea (Hildeb.) Kuan and Erwin race 1 after treatment with abscisic acid. Susceptibility was expressed by increases in lesion size and a major decrease in accumulation of the isoflavonoid phytoalexin, glyceollin. In untreated hypocotyls, activity of phenylalanine ammonia-lyase and accumulation of mRNA for this enzyme increased rapidly after infection, but these increases were suppressed in abscisic acid-treated hypocotyls. The results suggest the possibility that biosynthesis of glyceollin in the resistance response of soybeans may be controlled at the transcriptional level by changes in abscisic acid concentrations caused by infection. Images Figure 2 PMID:16667002

  18. Phenylketonuria: Direct and indirect effects of phenylalanine.

    PubMed

    Schlegel, Gudrun; Scholz, Ralf; Ullrich, Kurt; Santer, René; Rune, Gabriele M

    2016-07-01

    High phenylalanine concentrations in the brain due to dysfunctional phenylalanine hydroxylase (Pah) are considered to account for mental retardation in phenylketonuria (PKU). In this study, we treated hippocampal cultures with the amino acid in order to determine the role of elevated levels of phenylalanine in PKU-related mental retardation. Synapse density and dendritic length were dramatically reduced in hippocampal cultures treated with phenylalanine. Changes in cofilin expression and phosphorylation status, which were restored by NMDA, as well as reduced activation of the small GTPase Rac1, likely underlie these structural alterations. In the Pah(enu2) mouse, which carries a mutated Pah gene, we previously found higher synaptic density due to delayed synaptic pruning in response to insufficient microglia function. Microglia activity and C3 complement expression, both of which were reduced in the Pah(enu2) mouse, however, were unaffected in hippocampal cultures treated with phenylalanine. The lack of a direct effect of phenylalanine on microglia is the key to the opposite effects regarding synapse stability in vitro and in the Pah(enu2) mouse. Judging from our data, it appears that another player is required for the inactivation of microglia in the Pah(enu2) mouse, rather than high concentrations of phenylalanine alone. Altogether, the data underscore the necessity of a lifelong phenylalanine-restricted diet. PMID:27091224

  19. Disturbed Amino Acid Metabolism in HIV: Association with Neuropsychiatric Symptoms

    PubMed Central

    Gostner, Johanna M.; Becker, Kathrin; Kurz, Katharina; Fuchs, Dietmar

    2015-01-01

    Blood levels of the amino acid phenylalanine, as well as of the tryptophan breakdown product kynurenine, are found to be elevated in human immunodeficiency virus type 1 (HIV-1)-infected patients. Both essential amino acids, tryptophan and phenylalanine, are important precursor molecules for neurotransmitter biosynthesis. Thus, dysregulated amino acid metabolism may be related to disease-associated neuropsychiatric symptoms, such as development of depression, fatigue, and cognitive impairment. Increased phenylalanine/tyrosine and kynurenine/tryptophan ratios are associated with immune activation in patients with HIV-1 infection and decrease upon effective antiretroviral therapy. Recent large-scale metabolic studies have confirmed the crucial involvement of tryptophan and phenylalanine metabolism in HIV-associated disease. Herein, we summarize the current status of the role of tryptophan and phenylalanine metabolism in HIV disease and discuss how inflammatory stress-associated dysregulation of amino acid metabolism may be part of the pathophysiology of common HIV-associated neuropsychiatric conditions. PMID:26236243

  20. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

    PubMed

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

    2015-10-01

    Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids. PMID:26109004

  1. Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1.

    PubMed

    Zhang, Jinhua; Shi, Junling; Liu, Yanlin

    2013-12-01

    To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously. PMID:24068334

  2. Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate.

    PubMed

    Rodrigues, Joana L; Araújo, Rafael G; Prather, Kristala L J; Kluskens, Leon D; Rodrigues, Ligia R

    2015-04-01

    Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we report an engineered artificial pathway in Escherichia coli to produce natural curcuminoids through caffeic acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used to produce curcuminoids and 70 mg/L of curcumin was obtained from ferulic acid. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used. Caffeoyl-CoA 3-O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 mg/L of curcumin were produced, respectively. This is the first time DCS and CURS1 were used in vivo to produce curcuminoids and that curcumin was produced by feeding tyrosine. We have shown that curcumin can be produced using a pathway involvoing caffeic acid. This alternative pathway represents a step forward in the heterologous production of curcumin using E. coli. PMID:25641677

  3. Regulation of the Tyrosine Biosynthetic Enzymes in Salmonella typhimurium: Analysis of the Involvement of Tyrosyl-Transfer Ribonucleic Acid and Tyrosyl-Transfer Ribonucleic Acid Synthetase1

    PubMed Central

    Heinonen, J.; Artz, S. W.; Zalkin, H.

    1972-01-01

    Mutants of Salmonella typhimurium were isolated that require tyrosine for growth because of an altered tyrosyl-transfer ribonucleic acid (tRNA) synthetase. Extracts of one strain (JK10) contain a labile enzyme with decreased ability to transfer tyrosine to tRNATyr and a higher Km for tyrosine than the wild-type enzyme. Strain JK10 maintains repressed levels of the tyrosine biosynthetic enzymes when the growth rate is restricted due to limitation of charged tRNATyr. Several second-site revertants of strain JK10 exhibit temperature-sensitive growth due to partially repaired, heat-labile tyrosyl-tRNA synthetase. The tyrosine biosynthetic enzymes are not derepressed in thermosensitive strains grown at the restrictive temperature. A class of tyrosine regulatory mutants, designated tyrR, contains normal levels of tyrosyl-tRNA synthetase and tRNATyr. These results suggest that charging of tRNATyr is not necessary for repression. This conclusion is substantiated by the finding that 4-aminophenylalanine, a tyrosine analogue which causes repression of the tyrosine biosynthetic enzymes, is not attached to tRNATyr in vivo, nor does it inhibit the attachment reaction in vitro. A combined regulatory effect due to the simultaneous presence of tyrS and tyrR mutations in the same strain was detected. The possibility of direct participation of tyrosyl-tRNA synthetase in tyrosine regulation is discussed. PMID:4404819

  4. Characterization of 2-(methylamino)alkanoic acid capacity to restrict blood–brain phenylalanine transport in Pahenu2 mice: Preliminary findings✩

    PubMed Central

    Vogel, Kara R.; Arning, Erland; Wasek, Brandi L.; Bottiglieri, Teodoro; Gibson, K. Michael

    2014-01-01

    Background Our laboratory seeks a pharmacotherapeutic intervention for PKU that utilizes non-physiological amino acids (NPAAs) to block the accumulation of phenylalanine (Phe) in the brain. In previous studies (Vogel et al. 2013), methylation of the amino group of 2-aminoisobutyrate (AIB) provided an enhanced degree of selectivity for Phe restriction into the brain of Pahenu2 mice in comparison to unmethylated AIB, leading to the hypothesis that 2-(methylamino)alkanoic acid analogs of AIB might represent targeted inhibitors of Phe accretion into the brain. Methods Pahenu2 and control mice were intraperitoneally administered (500–750 mg/kg body weight, once daily; standard 19% protein diet) AIB, methyl AIB (MAIB), isovaline, and two MAIB analogs, 2-methyl-2-(methylamino)butanoic (MeVal) and 3-methyl-2-(methylamino)pentanoic (MePent) acids for one week, followed by brain and blood isolation for amino acid analyses using UPLC. Results In the brain, AIB significantly reduced Phe accretion in Pahenu2 mice, while MeVal significantly improved glutamine and aspartic acids. Four of five test compounds improved brain threonine and arginine levels. AIB, MAIB and IsoVal significantly reduced blood Phe, with no effect of any drug intervention on other sera amino acids. Conclusions Further evaluation of AIB and the 2-(methylamino)alkanoic acids as inhibitors of brain Phe accumulation in Pahenu2 mice is warranted, with more detailed evaluations of route of administration, combinatorial intervention, and detailed toxicity studies. PMID:23999161

  5. Simultaneous and selective decarboxylation of L-serine and deamination of L-phenylalanine in an amino acid mixture--a means of separating amino acids for synthesizing biobased chemicals.

    PubMed

    Teng, Yinglai; Scott, Elinor L; Witte-van Dijk, Susan C M; Sanders, Johan P M

    2016-01-25

    Amino acids (AAs) obtained from the hydrolysis of biomass-derived proteins are interesting feedstocks for the chemical industry. They can be prepared from the byproduct of biofuel production and agricultural wastes. They are rich in functionalities needed in petrochemicals, providing the opportunity to save energy, reagents, and process steps. However, their separation is required before they can be applied for further applications. Electrodialysis (ED) is a promising separation method, but its efficiency needs to be improved when separating AAs with similar isoelectric points. Thus, specific conversions are required to form product with different charges. Here we studied the enzymatic conversions which can be used as a means to aid the ED separation of neutral AAs. A model mixture containing L-serine, L-phenylalanine and L-methionine was used. The reactions of L-serine decarboxylase and L-phenylalanine ammonia-lyase were employed to specifically convert serine and phenylalanine into ethanolamine and trans-cinnamic acid. At the isoelectric point of methionine (pH 5.74), the charge of ethanolamine and trans-cinnamic acid are +1 and -1, therefore facilitating potential separation into three different streams by electrodialysis. Here the enzyme kinetics, specificity, inhibition and the operational stabilities were studied, showing that both enzymes can be applied simultaneously to aid the ED separation of neutral AAs. PMID:25976628

  6. Siglec-5 (CD170) can mediate inhibitory signaling in the absence of immunoreceptor tyrosine-based inhibitory motif phosphorylation.

    PubMed

    Avril, Tony; Freeman, Sylvie D; Attrill, Helen; Clarke, Rosemary G; Crocker, Paul R

    2005-05-20

    Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit FcepsilonRI-mediated calcium fluxing and serotonin release after co-cross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation. PMID:15769739

  7. Antibodies to Yeast Phenylalanine Transfer Ribonucleic Acid Are Specific for the Odd Nucleoside Y in the Anticodon Loop

    PubMed Central

    Fuchs, Sara; Aharonov, Aharon; Sela, Michael; Von Der Haar, Friedrich; Cramer, Friedrich

    1974-01-01

    Antibodies with specificity to a single species of tRNA were elicited in a goat by immunization with a glutaraldehyde conjugate of yeast phenylalanine transfer RNA with bovine gamma globulin. The specificity of the antibodies was studied by a radioimmunoassay measuring the direct binding of [3H]tRNAPhe or the inhibition of the binding. The antibodies formed are predominantly directed towards the characteristic highly modified nucleoside Y, which is located right next to the anticodon. The antibodies bind specifically to tRNAPhe, to oligonucleotides derived by enzymatic digestion from the anticodon loop of tRNAPhe, and to the Y nucleoside itself. tRNA species which do not contain Y in their sequences, or tRNAPhe from which the Y base has been excised, do not bind to the antibodies. Yeast tRNAPhe can be separated from other tRNA species with an immunoadsorbent of antibodies to tRNAPhe. PMID:4527666

  8. Inhibition of acid, alkaline, and tyrosine (PTP1B) phosphatases by novel vanadium complexes.

    PubMed

    McLauchlan, Craig C; Hooker, Jaqueline D; Jones, Marjorie A; Dymon, Zaneta; Backhus, Emily A; Greiner, Bradley A; Dorner, Nicole A; Youkhana, Mary A; Manus, Lisa M

    2010-03-01

    In the course of our investigations of vanadium-containing complexes for use as insulin-enhancing agents, we have generated a series of novel vanadium coordination complexes with bidentate ligands. Specifically we have focused on two ligands: anthranilate (anc(-)), a natural metabolite of tryptophan, and imidizole-4-carboxylate (imc(-)), meant to mimic naturally occurring N-donor ligands. For each ligand, we have generated a series of complexes containing the V(III), V(IV), and V(V) oxidation states. Each complex was investigated using phosphatase inhibition studies of three different phosphatases (acid, alkaline, and tyrosine (PTP1B) phosphatase) as prima facia evidence for potential use as an insulin-enhancing agent. Using p-nitrophenyl phosphate as an artificial phosphatase substrate, the levels of inhibition were determined by measuring the absorbance of the product at 405nm using UV/vis spectroscopy. Under our experimental conditions, for instance, V(imc)(3) appears to be as potent an inhibitor of alkaline phosphatase as sodium orthovanadate when comparing the K(cat)/K(m) term. VO(anc)(2) is as potent an inhibitor of acid phosphatase and tyrosine phosphatase as the Na(3)VO(4). Thus, use of these complexes can increase our mechanistic understanding of the effects of vanadium in vivo. PMID:20071031

  9. Mechanistic, Mutational, and Structural Evaluation of a Taxus Phenylalanine Aminomutase

    SciTech Connect

    Feng, Lei; Wanninayake, Udayanga; Strom, Susan; Geiger, James; Walker, Kevin D.

    2014-10-02

    The structure of a phenylalanine aminomutase (TcPAM) from Taxus canadensis has been determined at 2.4 {angstrom} resolution. The active site of the TcPAM contains the signature 4-methylidene-1H-imidazol-5(4H)-one prosthesis, observed in all catalysts of the class I lyase-like family. This catalyst isomerizes (S)-{alpha}-phenylalanine to the (R)-{beta}-isomer by exchange of the NH{sub 2}/H pair. The stereochemistry of the TcPAM reaction product is opposite of the (S)-{beta}-tyrosine made by the mechanistically related tyrosine aminomutase (SgTAM) from Streptomyces globisporus. Since TcPAM and SgTAM share similar tertiary- and quaternary-structures and have several highly conserved aliphatic residues positioned analogously in their active sites for substrate recognition, the divergent product stereochemistries of these catalysts likely cannot be explained by differences in active site architecture. The active site of the TcPAM structure also is in complex with (E)-cinnamate; the latter functions as both a substrate and an intermediate. To account for the distinct (3R)-{beta}-amino acid stereochemistry catalyzed by TcPAM, the cinnamate skeleton must rotate the C{sub 1}-C{sub {alpha}} and C{sub ipso}-C{sub {beta}} bonds 180{sup o} in the active site prior to exchange and rebinding of the NH{sub 2}/H pair to the cinnamate, an event that is not required for the corresponding acrylate intermediate in the SgTAM reaction. Moreover, the aromatic ring of the intermediate makes only one direct hydrophobic interaction with Leu-104. A L104A mutant of TcPAM demonstrated an 1.5-fold increase in k{sub cat} and a decrease in K{sub M} values for sterically demanding 3'-methyl-{alpha}-phenylalanine and styryl-{alpha}-alanine substrates, compared to the kinetic parameters for TcPAM. These parameters did not change significantly for the mutant with 4'-methyl-{alpha}-phenylalanine compared to those for TcPAM.

  10. Tyrosine depletion lowers in vivo DOPA synthesis in ventral hippocampus.

    PubMed

    Bongiovanni, Rodolfo; Kyser, Abby N; Jaskiw, George E

    2012-12-01

    In vivo dopamine synthesis in the medial prefrontal cortex of the rat is sensitive to the availability of tyrosine. Whether other limbic cortical dopamine terminal regions are similarly tyrosine-dependent is not known. In this study we examined the effects of tyrosine depletion on dopamine synthesis and catecholamine levels in the ventral hippocampus. A tyrosine- and phenylalanine-free neutral amino acid mixture was used to lower brain tyrosine levels in rats undergoing in vivo microdialysis. In one group, NSD-1015 was included in perfusate to permit measurement of DOPA levels. In a second group, NSD-1015 was not included in perfusate so that catecholamine levels could be assayed. Tyrosine depletion significantly lowered DOPA levels in the NSD-1015 treated group and lowered DOPAC but not dopamine or noradrenaline levels in the group not exposed to NSD-1015. We conclude that while catecholamine synthesis in the ventral hippocampus declines when tyrosine availability is lowered, under basal conditions, compensatory mechanisms are able to maintain stable extracellular catecholamine levels. PMID:23022716

  11. Heterologous expression and characterization of tyrosine decarboxylase from Enterococcus faecalis R612Z1 and Enterococcus faecium R615Z1.

    PubMed

    Liu, Fang; Xu, Wenjuan; Du, Lihui; Wang, Daoying; Zhu, Yongzhi; Geng, Zhiming; Zhang, Muhan; Xu, Weimin

    2014-04-01

    Tyrosine decarboxylase (TDC) is responsible for tyramine production and can catalyze phenylalanine to produce β-phenylethylamine. Enterococcus strains are a group of bacteria predominantly producing tyramine and β-phenylethylamine in water-boiled salted duck. In this study, the heterologous expression and characterization of two TDCs from Enterococcus faecalis R612Z1 (612TDC) and Enterococcus faecium R615Z1 (615TDC) were studied. The recombinant putative proteins of 612TDC and 615TDC were heterologously expressed in Escherichia coli. 612TDC is a 620-amino-acid protein with a molecular mass of 70.0 kDa, whereas 615TDC is a 625-amino-acid protein with a molecular mass of 70.3 kDa. Both 612TDC and 615TDC showed an optimum temperature of 25 °C for the tyrosine and phenylalanine substrates. However, 612TDC revealed maximal activity at pH 5.5, whereas 615TDC revealed maximal activity at pH 6.0. Kinetic studies showed that 612TDC and 615TDC exhibited higher specificity for tyrosine than for phenylalanine. The catalysis abilities of both 612TDC and 615TDC for phenylalanine were restrained significantly with the increase in NaCl concentration, but this was not the case for tyrosine. This study revealed that the enzyme properties of the purified recombinant 612TDC and 615TDC were similar, although their amino acid sequences had 84% identity. PMID:24680070

  12. Synthesis and antimicrobial evaluation of L-phenylalanine-derived C5-substituted rhodanine and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone.

    PubMed

    Jin, Xin; Zheng, Chang-Ji; Song, Ming-Xia; Wu, Yan; Sun, Liang-Peng; Li, Yin-Jing; Yu, Li-Jun; Piao, Hu-Ri

    2012-10-01

    Four novel series of compounds, including the l-phenylalanine-derived C5-substituted rhodanine (6a-q, 7a-j) and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone (9a-e, 11a-e) have been designed, synthesized, characterized, and evaluated for their antibacterial activity. Some of these compounds showed significant antibacterial activity against Gram-positive bacterias, especially against the strains of multidrug-resistant clinical isolates, among which compounds 6c-e, 6g, 6i, 6j and 6q exhibiting high levels of antimicrobial activity against Staphylococcus aureus RN4220 with minimum inhibitory concentration (MIC) values of 2 μg/mL. Compound 6q showed the most potent activity of all of the compounds against all of the test multidrug-resistant clinical isolates tested. Unfortunately, however, none of the compounds were active against Gram-negative bacteria at 64 μg/mL. PMID:22982124

  13. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  14. Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering.

    PubMed

    Meisburger, Steve P; Taylor, Alexander B; Khan, Crystal A; Zhang, Shengnan; Fitzpatrick, Paul F; Ando, Nozomi

    2016-05-25

    Mammalian phenylalanine hydroxylase (PheH) is an allosteric enzyme that catalyzes the first step in the catabolism of the amino acid phenylalanine. Following allosteric activation by high phenylalanine levels, the enzyme catalyzes the pterin-dependent conversion of phenylalanine to tyrosine. Inability to control elevated phenylalanine levels in the blood leads to increased risk of mental disabilities commonly associated with the inherited metabolic disorder, phenylketonuria. Although extensively studied, structural changes associated with allosteric activation in mammalian PheH have been elusive. Here, we examine the complex allosteric mechanisms of rat PheH using X-ray crystallography, isothermal titration calorimetry (ITC), and small-angle X-ray scattering (SAXS). We describe crystal structures of the preactivated state of the PheH tetramer depicting the regulatory domains docked against the catalytic domains and preventing substrate binding. Using SAXS, we further describe the domain movements involved in allosteric activation of PheH in solution and present the first demonstration of chromatography-coupled SAXS with Evolving Factor Analysis (EFA), a powerful method for separating scattering components in a model-independent way. Together, these results support a model for allostery in PheH in which phenylalanine stabilizes the dimerization of the regulatory domains and exposes the active site for substrate binding and other structural changes needed for activity. PMID:27145334

  15. Resolving phenylalanine metabolism sheds light on natural synthesis of penicillin G in Penicillium chrysogenum.

    PubMed

    Veiga, Tânia; Solis-Escalante, Daniel; Romagnoli, Gabriele; ten Pierick, Angela; Hanemaaijer, Mark; Deshmukh, Amit T; Deshmuhk, Amit; Wahl, Aljoscha; Pronk, Jack T; Daran, Jean-Marc

    2012-02-01

    The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-(13)C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH(4))(2)SO(4) as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate. PMID:22158714

  16. Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

    PubMed Central

    Veiga, Tânia; Solis-Escalante, Daniel; Romagnoli, Gabriele; ten Pierick, Angela; Hanemaaijer, Mark; Deshmuhk, Amit; Wahl, Aljoscha; Pronk, Jack T.

    2012-01-01

    The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-13C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH4)2SO4 as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate. PMID:22158714

  17. An additional substrate binding site in a bacterial phenylalanine hydroxylase

    PubMed Central

    Ronau, Judith A.; Paul, Lake N.; Fuchs, Julian E.; Corn, Isaac R.; Wagner, Kyle T.; Liedl, Klaus R.; Abu-Omar, Mahdi M.; Das, Chittaranjan

    2014-01-01

    Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

  18. Production of l-Phenylalanine from Starch by Analog-Resistant Mutants of Bacillus polymyxa†

    PubMed Central

    Shetty, Kalidas; Crawford, Don L.; Pometto, Anthony L.

    1986-01-01

    p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated by ethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts of l-phenylalanine (l-phe) from starch. A β-2-thienylalanine-resistant mutant (BTR-7) derived from p-fluorophenylalanine mutant (C-4000 FPR-4) and resistant to both p-fluorophenylalanine and β-2-thienylalanine produced 0.5 g of l-phe and 0.15 g of l-tyrosine per liter from 10 g of starch per liter when growing in a minimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility of l-phenylalanine ammonia-lyase-induced deamination of l-phe to CA. The amount of l-phe-derived CA detected in BTR-7 was less compared with mutant C-4000 FPR-4. CA production was induced in the parent only when l-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayed by addition of other nitrogen sources, an indication of possible l-phenylalanine ammonia-lyase inhibition or repression. The presence of l-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FPR-4 was confirmed by assays of cell-free extracts from cells grown in starch minimal medium containing l-phe as the sole nitrogen source. Preliminary studies of the regulation of deoxy-d-arabino-heptulosonate-7-phosphate synthase and prephenate dehydratase in the wild-type strain showed that deoxy-d-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by l-phe, l-tyrosine, and l-tryptophan. Inhibition by each amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid. Prephenate dehydratase was feedback inhibited by l-phe, but not by l-tyrosine or l-tryptophan or both. In the double analog-resistant mutant BTR-7, deoxy-d-arabino-heptulosonate-7-phosphate synthase had specific activity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However

  19. Conformational Structure of Tyrosine, Tyrosyl-Glycine, and Tyrosyl-Glycyl-Glycine by Double Resonance Spectroscopy

    NASA Technical Reports Server (NTRS)

    Abo-Riziq, Ali; Grace, Louis; Crews, Bridgit; Callahan, Michael P,; van Mourik, Tanja; de Vries, Mattanjah S,

    2011-01-01

    We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YC) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine glycine (FGG) and tryptophan-glycine-glycine (WGG)j however) the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

  20. Nordihydroguaiaretic acid inhibits IFN-gamma-induced STAT tyrosine phosphorylation in rat brain astrocytes.

    PubMed

    Jeon, Sae-Bom; Ji, Kyung-Ae; You, Hye-Jin; Kim, Jae-Hong; Jou, Ilo; Joe, Eun-Hye

    2005-03-11

    The Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signal cascades are major pathways that mediate the inflammatory functions of interferon-gamma (IFN-gamma), an important pro-inflammatory cytokine. Therefore, regulation of JAK/STAT signaling should modulate IFN-gamma-mediated inflammation. In this study, we found that nordihydroguaiaretic acid (NDGA), a well-known lipoxygenase (LO) inhibitor, suppressed IFN-gamma-induced inflammatory responses in brain astrocytes. In the presence of NDGA, interferon regulatory factor-1 expression was significantly reduced. Expression of monocyte chemotactic protein-1 and interferon-gamma inducible protein-10 mRNA in response to IFN-gamma was significantly suppressed in the presence of NDGA, as was tyrosine-phosphorylation of JAK and STAT. However, the 5-LO products, leukotriene B(4) (LTB(4)) and leukotriene C(4), were not detected in cells treated with IFN-gamma, indicating that the effect of NDGA seemed to be independent of 5-LO inhibition. In addition, two other 5-LO inhibitors (Rev5901 and AA861) did not mimic the effect of NDGA, and the 5-LO metabolites, 5-hydroxyeicosatetraenoic acid and LTB(4), were unable to reverse NDGA-driven suppression of STAT activation or affect basal STAT phosphorylation. Taken together, these results suggest that NDGA regulates IFN-gamma-mediated inflammation through mechanisms unrelated to the inhibition of 5-LO. PMID:15694390

  1. D-Amino acids influence ultrasonic calling in mice pups: effects of D-phenylalanine and D-leucine.

    PubMed

    Albonetti, M E; D'Udine, B; Oliverio, A

    1985-06-24

    6-day-old mice pups were injected with D-amino acids (D-phenyl-alanine + D-leucine), and their ultrasonic distress vocalizations were measured. D-Amino acids, which exert opioid-like effects, reduce the number of ultrasonic calls without affecting the activity of the pups. This effect is reversed by naloxone, an opioid antagonist. The role of endogenous opioids in modulating early attachment is discussed. PMID:4041020

  2. Dual role for the tyrosine decarboxylation pathway in Enterococcus faecium E17: response to an acid challenge and generation of a proton motive force.

    PubMed

    Pereira, C I; Matos, D; San Romão, M V; Crespo, M T Barreto

    2009-01-01

    In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products. PMID:19011061

  3. Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica.

    PubMed Central

    Mauch-Mani, B.; Slusarenko, A. J.

    1996-01-01

    Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)-[beta]-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific for the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility. PMID:12239383

  4. New Method for Determining Isotopic Values of Glutamic Acid and Phenylalanine for Estimation of Precise Trophic Position in Food Web Studies

    NASA Astrophysics Data System (ADS)

    Kamath, T.; Broek, T.; McCarthy, M.

    2012-12-01

    Compound Specific Isotope Analysis of Amino Acids (CSI-AA) has emerged as a highly precise new method of determining trophic levels of both aquatic and terrestrial organisms. Multiple studies have now shown that δ15N values for glutamic acid (Glu) and phenylalanine (Phe) can be coupled to provide extremely precise estimates of trophic position in diverse food web studies. The standard gas chromatography—isotope ratio mass spectrometer (GC-IRMS) approach is presently limited to a select number of labs since necessary equipment is both expensive and not widely accessible. Furthermore, typical GC-IRMS δ15N precision (±1‰) is significantly lower than usual bulk δ15N values (±0.1‰), thus presenting a considerable setback for precise trophic level calculations. In this study, we develop a new dual-column method to purify Glu and Phe using high performance liquid chromatography (HPLC). Phe is purified using an analytical scale reverse phase column embedded with anionic ion-pairing reagents and collected using automated fraction collection. Glu is separated from the non-polar amino acids using the same column and further purified using a hydrophilic interaction liquid chromatography (HILIC) cation and anion-exchange column and collected via automated fraction collection. Isotopic analysis of the purified AAs is then conducted on an elemental analyzer—isotope ratio mass spectrometer (EA-IRMS). As a test of this method, we present and compare the trophic position of five marine organisms—cyanobacteria, deep-sea bamboo coral, juvenile and adult white sea bass, and harbor seal, calculated using Glu and Phe δ15N values produced by both GC-IRMS and our HPLC-EA-IRMS approach. The preliminary results of this study suggest that the HPLC-EA-IRMS method is a viable alternative to GC-IRMS, which should allow accurate trophic position estimates to be made by more researchers using more readily available instrumentation.

  5. L-leucine, L-methionine, and L-phenylalanine share a Na(+)/K (+)-dependent amino acid transporter in shrimp hepatopancreas.

    PubMed

    Duka, Ada; Ahearn, Gregory A

    2013-08-01

    Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J

  6. Synthesis of D- and L-phenylalanine derivatives by phenylalanine ammonia lyases: a multienzymatic cascade process.

    PubMed

    Parmeggiani, Fabio; Lovelock, Sarah L; Weise, Nicholas J; Ahmed, Syed T; Turner, Nicholas J

    2015-04-01

    The synthesis of substituted D-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural D-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the D-configured product. Furthermore, the system was extended to the preparation of those L-phenylalanines which are obtained with a low ee value using PAL amination. PMID:25728350

  7. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius.

    PubMed

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-01-01

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT-PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress. PMID:24865400

  8. Differential inductions of phenylalanine ammonia-lyase and chalcone synthase during wounding, salicylic acid treatment, and salinity stress in safflower, Carthamus tinctorius

    PubMed Central

    Dehghan, Sara; Sadeghi, Mahnaz; Pöppel, Anne; Fischer, Rainer; Lakes-Harlan, Reinhard; Kavousi, Hamid Reza; Vilcinskas, Andreas; Rahnamaeian, Mohammad

    2014-01-01

    Safflower (Carthamus tinctorius L.) serves as a reference dicot for investigation of defence mechanisms in Asteraceae due to abundant secondary metabolites and high resistance/tolerance to environmental stresses. In plants, phenylpropanoid and flavonoid pathways are considered as two central defence signalling cascades in stress conditions. Here, we describe the isolation of two major genes in these pathways, CtPAL (phenylalanine ammonia-lyase) and CtCHS (chalcone synthase) in safflower along with monitoring their expression profiles in different stress circumstances. The aa (amino acid) sequence of isolated region of CtPAL possesses the maximum identity up to 96% to its orthologue in Cynara scolymus, while that of CtCHS retains the highest identity to its orthologue in Callistephus chinensis up to 96%. Experiments for gene expression profiling of CtPAL and CtCHS were performed after the treatment of seedlings with 0.1 and 1 mM SA (salicylic acid), wounding and salinity stress. The results of semi-quantitative RT–PCR revealed that both CtPAL and CtCHS genes are further responsive to higher concentration of SA with dissimilar patterns. Regarding wounding stress, CtPAL gets slightly induced upon injury at 3 hat (hours after treatment) (hat), whereas CtCHS gets greatly induced at 3 hat and levels off gradually afterward. Upon salinity stress, CtPAL displays a similar expression pattern by getting slightly induced at 3 hat, but CtCHS exhibits a biphasic expression profile with two prominent peaks at 3 and 24 hat. These results substantiate the involvement of phenylpropanoid and particularly flavonoid pathways in safflower during wounding and especially salinity stress. PMID:24865400

  9. GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

    PubMed

    Liu, Changlu; Bonaventure, Pascal; Lee, Grace; Nepomuceno, Diane; Kuei, Chester; Wu, Jiejun; Li, Qingqin; Joseph, Victory; Sutton, Steven W; Eckert, William; Yao, Xiang; Yieh, Lynn; Dvorak, Curt; Carruthers, Nicholas; Coate, Heather; Yun, Sujin; Dugovic, Christine; Harrington, Anthony; Lovenberg, Timothy W

    2015-11-01

    GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain. PMID:26349500

  10. Tyrosine-rich acidic matrix protein (TRAMP) accelerates collagen fibril formation in vitro.

    PubMed

    MacBeath, J R; Shackleton, D R; Hulmes, D J

    1993-09-15

    Tyrosine-rich acidic matrix protein (TRAMP) is a recently discovered protein that co-purifies with porcine skin lysyl oxidase and is equivalent to the M(r) 22,000 extracellular matrix protein from bovine skin that co-purifies with dermatan sulfate proteoglycans (Cronshaw, A. D., MacBeath, J. R. E., Shackleton, D. R., Collins, J. F., Fothergill-Gilmore, L. A., and Hulmes, D. J. S. (1993) Matrix 13, 255-266; Neame, P. J., Choi, H. U., and Rosenberg, L. C. (1989) J. Biol. Chem. 264, 5474-5479). The effect of TRAMP on collagen fibril formation was studied in vitro by reconstitution of fibrils from lathyritic rat skin collagen I. Fibril formation was initiated by the warm start procedure, in which acidic collagen solutions and double strength neutral buffer, both preincubated separately at 34 degrees C, were mixed. When monitored by turbidimetry, TRAMP was found to accelerate collagen fibril formation. Acceleration occurred at sub-stoichiometric molar ratios of TRAMP collagen, and the presence of TRAMP stabilized the fibrils against low temperature dissociation. It was confirmed by centrifugation that the amount of fibrillar collagen formed in the presence of TRAMP was greater than in its absence. By SDS-polyacrylamide gel electrophoresis and scanning densitometry, binding of TRAMP to collagen was detected that approached saturation with a molar ratio of TRAMP to collagen of approximately 1:2. Fibrils formed in the presence of TRAMP were normal when observed by electron microscopy, although fibril diameters were smaller than the controls. TRAMP was found to partially reverse the inhibitory effects of urea and increased ionic strength on the kinetics of fibril formation, although inhibition by glucose was unaffected. TRAMP also accelerated the assembly of pepsin-treated collagen, where the non-helical, telopeptide regions were partially removed. Acceleration of collagen fibril formation by TRAMP is discussed in the light of the known effects of other extracellular matrix

  11. Synthesis of hydrophobic photoluminescent carbon nanodots by using L-tyrosine and citric acid through a thermal oxidation route.

    PubMed

    Gude, Venkatesh

    2014-01-01

    Hydrophobic photoluminescent carbon nanodots (CNDs) were fabricated by using citric acid and L-tyrosine precursor molecules through a simple, facile thermal oxidation process in air. These CNDs (less than 4 nm in size) exhibited a characteristic excitation wavelength dependent emission and upconversion emission properties and are insoluble in water, but soluble in organic solvents. FTIR and (1)H NMR analyses showed a selective participation of L-tyrosine molecule during the carbonization process at 220 °C without a disturbance of its benzylic protons and aromatic phenyl ring bearing hydroxy group. TEM and XRD studies revealed a quasi-spherical morphology and poor-crystalline nature of CNDs. Because the presence of the hydroxy group of L-tyrosine is dominating at the surface, these CNDs are also soluble in water under basic conditions. The effects of base and silver nanoparticles on the luminescence properties of CNDs were studied and a quenching of fluorescence was observed. These tyrosine-passivated CNDs are applicable for both biologically and commercially. PMID:25247134

  12. Does oral administration of the amino acid tyrosine affect oestradiol-17β concentration and sexual behaviour in the bitch?

    PubMed

    Spankowsky, S; Heuwieser, W; Arlt, S P

    2013-02-23

    The oral administration of the amino acid, tyrosine, has been for years recommended in order to improve fertility, especially to improve copulation behaviour in female dogs. However, evidence is comparatively poor. The objective of our study was to determine whether oral administration of tyrosine has an effect on oestradiol-17β concentrations and the oestrous behaviour in the bitch. Fifty bitches were randomly allocated to one of two treatment groups in which each dog received 100mg/kg/day of either tyrosine or milk sugar orally between Day 3 and Day 9 of heat. Every two to three days, a gynaecological examination was performed and blood samples were taken to determine oestradiol-17β and progesterone concentrations. The day of ovulation was estimated by clinical findings, and according to the specifications of the laboratory, once progesterone values exceeded 12.7nmol/l. The observed copulation behaviour was not different between the groups. No differences in volume and visual nature of vaginal discharge were observed. At the day of ovulation, mean oestradiol-17β concentration in the treated group was 163.4pmol/l and 162.2pmol/l in the placebo group, respectively. In conclusion, feeding tyrosine to female dogs between Day 3 and Day 9 of heat did not alter visual signs of heat or copulation behaviour, and did not alter oestradiol-17β concentration. PMID:23315766

  13. Synthesis of Hydroxylated Bicyclic Amino Acids from l-Tyrosine: Octahydro-1H-indole Carboxylates

    PubMed Central

    Pierce, Joshua G.; Kasi, Dhanalakshmi; Fushimi, Makoto; Cuzzupe, Anthony; Wipf, Peter

    2009-01-01

    A stereoselective approach to polyhydroxylated l-Choi derivatives has been developed. The oxidative cyclization of l-tyrosine was optimized to avoid partial racemization and to allow a more efficient scale-up. PMID:18767800

  14. Brain catechol synthesis - Control by brain tyrosine concentration

    NASA Technical Reports Server (NTRS)

    Wurtman, R. J.; Larin, F.; Mostafapour, S.; Fernstrom, J. D.

    1974-01-01

    Brain catechol synthesis was estimated by measuring the rate at which brain dopa levels rose following decarboxylase inhibition. Dopa accumulation was accelerated by tyrosine administration, and decreased by treatments that lowered brain tyrosine concentrations (for example, intraperitoneal tryptophan, leucine, or parachlorophenylalanine). A low dose of phenylalanine elevated brain tyrosine without accelerating dopa synthesis. Our findings raise the possibility that nutritional and endocrine factors might influence brain catecholamine synthesis by controlling the availability of tyrosine.

  15. In vivo amino acid transport of subacute and chronic cerebral infarction evaluated by 12-18F-phenylalanine

    SciTech Connect

    Shimosegawa, E.; Miura, S.; Murakami, M.

    1994-05-01

    On the basis of previous validation of kinetic two-compartment model and the determination of normal values of three parameters (k{sub 1}:influx rate constant, k{sub 2}:outflux rate constant, Vd:distribution volume), PET measurements of in vivo amino acid transport from blood to brain using L-(2-18F)-fluorophenylalanine ({sup 18}F-Phe) were undergone in the patients with cerebral infarction. The purposes of this study are to evaluate the alteration of amino acid transport in subacute and chronic stage of cerebral infarction and to compare with cerebral blood flow (CBF) and oxygen metabolism. Dynamic {sup 18}F-Phe PET studies for 50 minutes were performed in 7 patients with cerebral infarction. The input function was obtained by 27 points of arterial sampling. In all patients, measurements of CBF, cerebral blood volume (CBV), cerebral metabolic rate of oxygen (CMRO{sub 2}), and oxygen extraction fraction (OEF) were made on the same day of {sup 18}F-Phe PET measurement. Each patient was studied twice, within 2 weeks of the onset and 3 months later. Weighted integration technique with table look-up method was applied for the reconstruction of parametric images of {sup 18}F-Phe and ROI analysis of k{sub 1}, k{sub 2}, and Vd. In subacute stage, significant reduction of k{sub 2} value in infarct area was observed when compared to that in periinfarct area (p<0.05) and in normal cortices (p<0.001). k{sub 1} value in this stage showed only slightly decrease in infarct area, therefore, Vd value in infarct area increased significantly compared to normal cortices (p<0.001). In chronic stage, both k{sub 1} and k{sub 2} values in infarct area were significantly lower than that in normal cortices (p<0.001), and corresponding Vd value reduced to normal level. Correlativity between kinetic parameters of {sup 18}F-Phe and CBF or oxygen metabolism was not observed both in subacute and chronic stage of infarction.

  16. Competition between pi and non-pi cation-binding sites in aromatic amino acids: a theoretical study of alkali metal cation (Li+, Na+, K+)-phenylalanine complexes.

    PubMed

    Siu, Fung Ming; Ma, Ngai Ling; Tsang, Chun Wai

    2004-04-19

    To understand the cation-pi interaction in aromatic amino acids and peptides, the binding of M(+) (where M(+) = Li(+), Na(+), and K(+)) to phenylalanine (Phe) is studied at the best level of density functional theory reported so far. The different modes of M(+) binding show the same order of binding affinity (Li(+)>Na(+)>K(+)), in the approximate ratio of 2.2:1.5:1.0. The most stable binding mode is one in which the M(+) is stabilized by a tridentate interaction between the cation and the carbonyl oxygen (O[double bond]C), amino nitrogen (--NH(2)), and aromatic pi ring; the absolute Li(+), Na(+), and K(+) affinities are estimated theoretically to be 275, 201, and 141 kJ mol(-1), respectively. Factors affecting the relative stabilities of various M(+)-Phe binding modes and conformers have been identified, with ion-dipole interaction playing an important role. We found that the trend of pi and non-pi cation bonding distances (Na(+)-pi>Na(+)-N>Na(+)-O and K(+)-pi>K(+)-N>K(+)-O) in our theoretical Na(+)/K(+)-Phe structures are in agreement with the reported X-ray crystal structures of model synthetic receptors (sodium and potassium bound lariat ether complexes), even though the average alkali metal cation-pi distance found in the crystal structures is longer. This difference between the solid and the gas-phase structures can be reconciled by taking the higher coordination number of the cations in the lariat ether complexes into account. PMID:15079836

  17. A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna[C][W

    PubMed Central

    Bedewitz, Matthew A.; Góngora-Castillo, Elsa; Uebler, Joseph B.; Gonzales-Vigil, Eliana; Wiegert-Rininger, Krystle E.; Childs, Kevin L.; Hamilton, John P.; Vaillancourt, Brieanne; Yeo, Yun-Soo; Chappell, Joseph; DellaPenna, Dean; Jones, A. Daniel; Buell, C. Robin; Barry, Cornelius S.

    2014-01-01

    The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine. PMID:25228340

  18. 21 CFR 201.21 - Declaration of presence of phenylalanine as a component of aspartame in over-the-counter and...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... methylester of a dipeptide composed of two amino acids, phenylalanine and aspartic acid. When these two amino acids are so combined to form aspartame (1-methyl N-L-α-aspartyl-L-phenylalanine), they produce...

  19. 21 CFR 201.21 - Declaration of presence of phenylalanine as a component of aspartame in over-the-counter and...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... methylester of a dipeptide composed of two amino acids, phenylalanine and aspartic acid. When these two amino acids are so combined to form aspartame (1-methyl N-L-α-aspartyl-L-phenylalanine), they produce...

  20. 21 CFR 201.21 - Declaration of presence of phenylalanine as a component of aspartame in over-the-counter and...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... methylester of a dipeptide composed of two amino acids, phenylalanine and aspartic acid. When these two amino acids are so combined to form aspartame (1-methyl N-L-α-aspartyl-L-phenylalanine), they produce...

  1. Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

    PubMed Central

    Pandey, Manoj K.; Sung, Bokyung; Aggarwal, Bharat B.

    2009-01-01

    STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents. PMID:19937797

  2. Aromatic/heterocyclic amino acids and the simulated sunlight-ultraviolet inactivation of the Heliothis/Helicoverpa baculovirus

    SciTech Connect

    Ignoffo, C.M.; Garcia, C.

    1995-04-01

    Tryptophan, of five aromatic/heterocyclic amino acids (tyrosine, phenylalanine, proline, histidine) provided significant protection of the Heliothis baculovirus (HzSNPV) from inactivation by simulated ultraviolet (SUV). Fifty percent of SUV protection of HzSNPV with tryptophan or tyrosine was obtained at 0.03 mg/ml and 0.5 mg/ml, respectively. Rates as high as 100.0 mg/ml of phenylalanine, histidine, or proline provided <50% protection. The extent of tryptophan protection was correlated with its absorption in the sunlight UV-B spectra. 16 refs., 2 tabs.

  3. Molecular Docking Study of Catecholamines and [4-(Propan-2-yl) Phenyl]Carbamic acid with Tyrosine Hydroxylase

    PubMed Central

    Parveen, Zahida; Nawaz, Muhammad Sulaman; Shakil, Shazi; Greig, Nigel H.; Kamal, Mohammad A.

    2016-01-01

    Parkinson’s disease is a major age-related neurodegenerative disorder. As the classical disease-related motor symptoms are associated with the loss of dopamine-generating cells within the substantia nigra, tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines has become an important target in the development of Parkinson’s disease drug candidates, with the focus to augment TH levels or its activity. By contrast, TH inhibitors are of relevance in the treatment of conditions associated with catecholamine over-production, as occurs in pheochromocytomas. To aid characterizing new drug candidates, a molecular docking study of catecholamines and a novel hypothetical compound [4-(propan-2-yl) phenyl]carbamic acid (PPCA) with TH is described. Docking was performed using Autodock4.2 and results were analyzed using Chimera1.5.2. All the studied ligands were found to bind within a deep narrow groove lined with polar aromatic and acidic residues within TH. Our results corroborated a ‘hexa interacting amino acids unit’ located in this deep narrow groove crucial to the interaction of PPCA and the studied catecholamines with TH, whereby the ‘His361-His336 dyad’ was found to be even more crucial to these binding interactions. PPCA displayed a binding interaction with human TH that was comparable to the original TH substrate, L-tyrosine. Hence PPCA may warrant in vitro and in vivo characterization with TH to assess its potential as a candidate therapeutic. PMID:22583429

  4. Poly(carbonate–amide)s Derived from Bio-Based Resources: Poly(ferulic acid-co-tyrosine)

    PubMed Central

    2015-01-01

    Ferulic acid (FA), a bio-based resource found in fruits and vegetables, was coupled with a hydroxyl-amino acid to generate a new class of monomers to afford poly(carbonate–amide)s with potential to degrade into natural products. l-Serine was first selected as the hydroxyl-amino partner for FA, from which the activated p-nitrophenyl carbonate monomer was synthesized. Unfortunately, polymerizations were unsuccessful, and the elimination product was systematically obtained. To avoid elimination, we revised our strategy and used l-tyrosine ethyl ester, which lacks an acidic proton on the α position of the ethyl ester. Four new monomers were synthesized and converted into the corresponding poly(carbonate–amide)s with specific regioselectivities. The polymers were fully characterized through thermal and spectroscopic analyses. Preliminary fluorescent studies revealed interesting photophysical properties for the monomers and their corresponding poly(carbonate–amide)s, beyond the fluorescence characteristics of l-tyrosine and FA, making these materials potentially viable for sensing and/or imaging applications, in addition to their attractiveness as engineering materials derived from renewable resources. PMID:24839309

  5. A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.

    PubMed

    Sarkissian, C N; Shao, Z; Blain, F; Peevers, R; Su, H; Heft, R; Chang, T M; Scriver, C R

    1999-03-01

    Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease. PMID:10051643

  6. Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.

    PubMed

    Masuda, Katsuyoshi; Koizumi, Ayako; Misaka, Takumi; Hatanaka, Yasumaru; Abe, Keiko; Tanaka, Takaharu; Ishiguro, Masaji; Hashimoto, Makoto

    2010-02-01

    Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor. PMID:20031409

  7. Interaction of bacterial fatty-acid-displaced regulators with DNA is interrupted by tyrosine phosphorylation in the helix-turn-helix domain

    PubMed Central

    Derouiche, Abderahmane; Bidnenko, Vladimir; Grenha, Rosa; Pigonneau, Nathalie; Ventroux, Magali; Franz-Wachtel, Mirita; Nessler, Sylvie; Noirot-Gros, Marie-Françoise; Mijakovic, Ivan

    2013-01-01

    Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators. PMID:23939619

  8. Protein tyrosine kinase regulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons.

    PubMed

    Wang, H; Yu, L C; Li, Y C

    2016-01-01

    This study was performed to investigate the modulation effect of protein tyrosine kinase on postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons. The cultured neurons were exposed to 1% O2 and the expression of AMPA receptor subunit GluR2 on the cell surface was significantly increased, while total GluR2 was not markedly changed. Furthermore, the hypoxia-induced increase in GluR2 expression on the cell surface was partially blocked by the protein tyrosine kinase membrane-permeable inhibitor genistein. In contrast, both the protein tyrosine kinase agonist nerve growth factor and protein tyrosine phosphatase inhibitor vanadate promoted the hypoxia-induced increase of GluR2 expression on cell surface. Moreover, GluR2 could be phosphorylated by tyrosine under normoxia and hypoxia conditions in vitro on brainstem neurons, and tyrosine phosphorylation of GluR2 was significantly stronger under hypoxia conditions. Our results indicate that acute hypoxia induces the AMPA receptor subunit GluR2 to rapidly migrate to the cell membrane to modify the strength of the synapse. This study indicates that tyrosine phosphorylation of the receptor is an important pathway regulating the rapid migration of GluR2 in the postsynaptic domain induced by hypoxia. PMID:27525851

  9. Identification of a potential general acid/base in the reversible phosphoryl transfer reactions catalyzed by tyrosine recombinases: Flp H305

    PubMed Central

    Whiteson, Katrine L.; Chen, Yu; Chopra, Neeraj; Raymond, Amy C.; Rice, Phoebe A.

    2007-01-01

    Summary Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate, similar to the mechanism of type Ib topoisomerases [1]. Unlike most related enzymes, Flp’s nucleophilic tyrosine derives from a different protomer than the remainder of its active site [2, 3]. Because the tyrosine can be supplied exogenously, non-natural synthetic analogs can be used. Here we examine the catalytic role of Flp’s conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa≅10), or 3-fluoro-tyrosine (pKa≅8.4). Religation was studied using DNA substrates with 3’-phospho-cresol (pKa≅10) or 3’-para-nitro-phenol (pKa≅7.1) mimicking the covalent protein-DNA intermediate. In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide the first experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases. PMID:17317566

  10. A strategy for the study of cerebral amino acid transport using iodine-123-labeled amino acid radiopharmaceutical: 3-iodo-alpha-methyl-L-tyrosine

    SciTech Connect

    Kawai, K.; Fujibayashi, Y.; Saji, H.; Yonekura, Y.; Konishi, J.; Kubodera, A.; Yokoyama, A. )

    1991-05-01

    We examined the brain accumulation of iodine-123-iodo-alpha-methyl-L-tyrosine ({sup 123}I-L-AMT) in mice and rats. I-L-AMT showed high brain accumulation in mice, and in rats; rat brain uptake index exceeded that of {sup 14}C-L-tyrosine. The brain uptake index and the brain slice studies indicated the affinity of I-L-AMT for carrier-mediated and stereoselective active transport systems, respectively; both operating across the blood-brain barrier and cell membranes of the brain. The tissue homogenate analysis revealed that most of the accumulated radioactivity belonged to intact I-L-AMT, an indication of its stability. Thus, {sup 123}I-L-AMT appears to be a useful radiopharmaceutical for the selective measurement of cerebral amino acid transport.

  11. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

    PubMed Central

    Stahlhut, Steen Gustav; Li, Mingji; Gaspar, Paula; Siedler, Solvej; Förster, Jochen; Maury, Jérôme; Borodina, Irina

    2015-01-01

    Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 μM p-coumaric acid OD600 unit−1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases. PMID:25911487

  12. 13C-phenylalanine breath test detects altered phenylalanine kinetics in schizophrenia patients.

    PubMed

    Teraishi, T; Ozeki, Y; Hori, H; Sasayama, D; Chiba, S; Yamamoto, N; Tanaka, H; Iijima, Y; Matsuo, J; Kawamoto, Y; Kinoshita, Y; Hattori, K; Ota, M; Kajiwara, M; Terada, S; Higuchi, T; Kunugi, H

    2012-01-01

    Phenylalanine is an essential amino acid required for the synthesis of catecholamines including dopamine. Altered levels of phenylalanine and its metabolites in blood and cerebrospinal fluid have been reported in schizophrenia patients. This study attempted to examine for the first time whether phenylalanine kinetics is altered in schizophrenia using L-[1-(13)C]phenylalanine breath test ((13)C-PBT). The subjects were 20 chronically medicated schizophrenia patients (DSM-IV) and the same number of age- and sex-matched controls. (13)C-phenylalanine (99 atom% (13)C; 100 mg) was administered orally and the breath (13)CO(2) /(12)CO(2) ratio was monitored for 120 min. The possible effect of antipsychotic medication (risperidone (RPD) or haloperidol (HPD) treatment for 21 days) on (13)C-PBT was examined in rats. Body weight (BW), age and diagnostic status were significant predictors of the area under the curve of the time course of Δ(13)CO(2) (‰) and the cumulative recovery rate (CRR) at 120 min. A repeated measures analysis of covariance controlled for age and BW revealed that the patterns of CRR change over time differed between the patients and controls and that Δ(13)CO(2) was lower in the patients than in the controls at all sampling time points during the 120 min test, with an overall significant difference between the two groups. Chronic administration of RPD or HPD had no significant effect on (13)C-PBT indices in rats. Our results suggest that (13)C-PBT is a novel laboratory test that can detect altered phenylalanine kinetics in chronic schizophrenia patients. Animal experiments suggest that the observed changes are unlikely to be attributable to antipsychotic medication. PMID:22832963

  13. Effects of totally synthetic, low phenylalanine diet on adolescent phenylketonuric patients

    PubMed Central

    McKean, Charles M.

    1971-01-01

    The long-term responses of 5 adolescent phenylketonuric patients to chemically-defined, synthetic diets with normal and low phenylalanine content were determined. The synthetic preparations were found capable of sustaining good health and rapid growth in this group of profoundly retarded, behaviourally disturbed patients over a 3½-year period without clinical or biochemical evidence of nutritional inadequacy. 4 of these patients who were treated for 6 months on a comparable diet, in which 80% of the phenylalanine was replaced by tyrosine, continued to show weight maintenance and height increases. There was no evidence of poor acceptability of the imbalanced diet, whether the blood phenylalanine concentrations were at phenylketonuric or treatment levels. The phenylalanine intake required to maintain blood phenylalanine concentrations of 3-5 mg/100 ml in these 4 patients was well below normal requirements, and ranged between 6·8 and 20·1 mg/kg per day. Predictably, the phenylalanine requirement varied with individual growth rates. All 4 treated patients had objective signs of improved central nervous system function during the six-month period on the phenylalanine-restricted diet. These electrophysiological and behavioural improvements were manifest after blood phenylalanine concentrations fell below 12 mg/100 ml in 3 cases and below 5 mg/100 ml in the fourth. PMID:5118048

  14. A Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in Formation of the Oxygen-Reactive Complex

    PubMed Central

    Krzyaniak, Matthew D.; Eser, Bekir E.; Ellis, Holly R.; Fitzpatrick, Paul F.; McCracken, John

    2013-01-01

    Tyrosine hydroxylase is a non-heme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin and molecular oxygen. We have used nitric oxide as an O2 surrogate to poise Fe(II) at the catalytic site in an S=3/2, {FeNO}7 form amenable to EPR spectroscopy. 2H-Electron Spin Echo Envelope Modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}7 paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}7 center to the closest deuteron on 6,7-2H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-2H-tyrosine resulted in reorientation of the magnetic axes of the S=3/2 {FeNO}7 center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase. PMID:24168553

  15. Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles.

    PubMed

    Weiser, Diána; Bencze, László Csaba; Bánóczi, Gergely; Ender, Ferenc; Kiss, Róbert; Kókai, Eszter; Szilágyi, András; Vértessy, Beáta G; Farkas, Ödön; Paizs, Csaba; Poppe, László

    2015-11-01

    Phenylalanine ammonia-lyase (PAL), found in many organisms, catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in-line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)-pent-2-ene-4-ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel-Crafts-type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)-pent-2-ene-4-ynoate yielding enantiopure l-PG, contradicts the proposed highly exothermic single-step mechanism. Computations with the QM/MM models of the N-MIO intermediates from L-PG and L-Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N-MIO intermediate. PMID:26345352

  16. Studies on a novel approach for the separation of L-phenylalanine amino acid from fermentation broth using a newly developed charged ultrafiltration membrane. I. Single solute system

    SciTech Connect

    Agarwal, A.K.; Huang, R.Y.M. )

    1994-01-01

    A newly developed thin-film composite (TFC) ultrafiltration membrane made of sulfonated poly(phenylene oxide) (SPPO) was used to establish the feasibility of separating L-phenylalanine from the fermentation broth containing a number of dissolved inorganic and organic solutes as an alternative approach to the currently used complex and uneconomical conventional ion-exchange schemes. It was found that the rejection of inorganic salts in a single component system was highly dependent on the feed solution concentration and varied inversely with it. The pH of the feed solution was found to have a strong effect on the rejection of L-phenylalanine, changing it from - 10 to 90%. This rejection behavior was identical for the two TFC-SPPO membrane samples which had molecular weight cutoff ratings of 10,000 and 20,000, respectively, although the permeate flux of the latter sample was almost twice that of the former sample. It was found that glucose molecules were not rejected by the membrane. 11 refs., 18 figs., 2 tab.

  17. Molecular cloning and characterization of tyrosine aminotransferase and hydroxyphenylpyruvate reductase, and rosmarinic acid accumulation in Scutellaria baicalensis.

    PubMed

    Kim, Yeon Bok; Uddina, Md Romij; Kim, YeJi; Park, Chun Geon; Park, Sang Un

    2014-09-01

    Rosmarinic acid (a-O-caffeoyl-3,4-dihydroxyphenylacetic acid, RA) is a caffeoyl ester widely distributed in plants. cDNA clones encoding tyrosine aminotransferase (TAT1 and 2) and hydroxyphenylpyruvate reductase (HPPR) have been isolated from Scutellaria baicalensis. The open reading frames (ORFs) of SbTAT1 and 2 were 1230 and 1272 bp long and encoded 409 and 423 amino acid residues, respectively. HPPR corresponded to a 942-bp ORF and 313 amino acid residues of translated protein. To study the molecular mechanisms of TAT and HPPR and investigate RA accumulation in S. baicalensis, we examined the transcript levels of TAT isoforms and HPPR with quantitative real-time PCR and analyzed the RA content in different organs by using high-performance liquid chromatography. The transcript levels of SbTATI SbTAT2, and SbHPPR in the flowers were higher than those in other organs. RA was also highly accumulated in the flowers and with a trace amount in the roots. No RA was detected in the leaves and stems of S. baicalensis. The amount of accumulated RA in the flowers was 28.7 times higher than that in the roots. Our results will be helpful in elucidating the mechanisms of RA biosynthesis in S. baicalensis. PMID:25918800

  18. Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis.

    PubMed Central

    Marusich, W C; Jensen, R A; Zamir, L O

    1981-01-01

    Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than a complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-Phenylalanine will also induce lyase synthesis during exponential growth in minimal in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared from cultures induced with doubly labeled (U-14C; ring-4-3H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate. PMID:7195398

  19. Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis

    SciTech Connect

    Marusich, W.C.; Jensen, R.A.; Zamir, L.O.

    1981-06-01

    Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than in complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-phenylalanine will also induce lyase synthesis during exponential growth in minimal medium in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared fom cultures induced with doubly labeled (U-/sup 14/C; ring-4-/sup 3/H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate.

  20. Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein.

    PubMed

    Bergt, Constanze; Fu, Xiaoyun; Huq, Nabiha P; Kao, Jeff; Heinecke, Jay W

    2004-02-27

    Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation. PMID:14660678

  1. Treating phenylketonuria by a phenylalanine-free diet.

    PubMed

    Start, K

    1998-01-01

    The phenylalanine-free diet is needed for the treatment of phenylketonuria. Phenylketonuria is an inherited metabolic condition in which there is a deficiency of the enzyme phenylalanine hydroxylase. Lack of this enzyme means the body cannot metabolise the essential amino acid phenylalanine, which then builds up in the blood and causes mental retardation and other abnormalities. Retardation can be prevented if phenylketonuria is diagnosed in the first three weeks of infancy and dietary treatment started straightaway. There is a universal screening test in the UK (the Guthrie test). Heel-prick blood samples are taken from all babies between 6-14 days old and analysed at a regional screening centre. For infants, a phenylalanine-free formula is needed, either as a supplement before breast feeds or following a formula feed. The diet must continue during weaning and childhood, with a low protein diet. Foods such as meat, fish, eggs, milk, cheese, nuts and pulses are excluded as they contain high levels of phenylalanine. Vegetables and fruit are allowed in measured amounts only! Special low protein bread, pasta, biscuits and flour are used to supplement the diet and ensure adequate calorie intake. Whether the diet can be stopped at the end of adolescence is debatable. If stopped, it should be re-started at conception and maintained during pregnancy, as high levels of phenylalanine in the mother can affect the fetus. PMID:9814366

  2. Tyrosine sulfation of human trypsin steers S2' subsite selectivity towards basic amino acids.

    PubMed

    Szabó, András; Salameh, Moh'd A; Ludwig, Maren; Radisky, Evette S; Sahin-Tóth, Miklós

    2014-01-01

    Human cationic and anionic trypsins are sulfated on Tyr154, a residue which helps to shape the prime side substrate-binding subsites. Here, we used phage display technology to assess the significance of tyrosine sulfation for the specificity of human trypsins. The prime side residues P1'-P4' in the binding loop of bovine pancreatic trypsin inhibitor (BPTI) were fully randomized and tight binding inhibitor phages were selected against non-sulfated and sulfated human cationic trypsin. The selection pattern for the two targets differed mostly at the P2' position, where variants selected against non-sulfated trypsin contained primarily aliphatic residues (Leu, Ile, Met), while variants selected against sulfated trypsin were enriched also for Arg. BPTI variants carrying Arg, Lys, Ile, Leu or Ala at the P2' position of the binding loop were purified and equilibrium dissociation constants were determined against non-sulfated and sulfated cationic and anionic human trypsins. BPTI variants harboring apolar residues at P2' exhibited 3-12-fold lower affinity to sulfated trypsin relative to the non-sulfated enzyme, whereas BPTI variants containing basic residues at P2' had comparable affinity to both trypsin forms. Taken together, the observations demonstrate that the tyrosyl sulfate in human trypsins interacts with the P2' position of the substrate-like inhibitor and this modification increases P2' selectivity towards basic side chains. PMID:25010489

  3. New tyrosinase inhibitory decapeptide: Molecular insights into the role of tyrosine residues.

    PubMed

    Ochiai, Akihito; Tanaka, Seiya; Imai, Yuta; Yoshida, Hisashi; Kanaoka, Takumi; Tanaka, Takaaki; Taniguchi, Masayuki

    2016-06-01

    Tyrosinase, a rate-limiting enzyme in melanin biosynthesis, catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxy-l-phenylalanine (l-dopa) (monophenolase reaction) and the subsequent oxidation of l-dopa to l-dopaquinone (diphenolase reaction). Thus, tyrosinase inhibitors have been proposed as skin-lightening agents; however, many of the existing inhibitors cannot be widely used in the cosmetic industry due to their high cytotoxicity and instability. On the other hand, some tyrosinase inhibitory peptides have been reported as safe. In this study, we found that the peptide TH10, which has a similar sequence to the characterized inhibitory peptide P4, strongly inhibits the monophenolase reaction with a half-maximal inhibitory concentration of 102 μM. Seven of the ten amino acid residues in TH10 were identical to P4; however, TH10 possesses one N-terminal tyrosine, whereas P4 contains three tyrosine residues located at its N-terminus, center, and C-terminus. Subsequent analysis using sequence-shuffled variants indicated that the tyrosine residues located at the N-terminus and center of P4 have little to no contribution to its inhibitory activity. Furthermore, docking simulation analysis of these peptides with mushroom tyrosinase demonstrated that the active tyrosine residue was positioned close to copper ions, suggesting that TH10 and P4 bind to tyrosinase as a substrate analogue. PMID:26589783

  4. eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

    PubMed Central

    Fazioli, F; Minichiello, L; Matoskova, B; Wong, W T; Di Fiore, P P

    1993-01-01

    An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals. Images PMID:7689153

  5. Systems engineering of tyrosine 195, tyrosine 260, and glutamine 265 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance maltodextrin specificity for 2-O-(D)-glucopyranosyl-(L)-ascorbic acid synthesis.

    PubMed

    Han, Ruizhi; Liu, Long; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    In this work, the site saturation mutagenesis of tyrosine 195, tyrosine 260 and glutamine 265 in the cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase for maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G). Specifically, the site-saturation mutagenesis of three sites-tyrosine 195, tyrosine 260, and glutamine 265-was performed, and it was found that the resulting mutants (containing the mutations Y195S [tyrosine → serine], Y260R [tyrosine → arginine], and Q265K [glutamine → lysine]) produced higher AA-2G yields than the wild type and the other mutant CGTases when maltodextrin was used as the glycosyl donor. Furthermore, double and triple mutations were introduced, and four mutants (containing Y195S/Y260R, Y195S/Q265K, Y260R/Q265K, and Y260R/Q265K/Y195S) were obtained and evaluated for the capacity to produce AA-2G. The Y260R/Q265K/Y195S triple mutant produced the highest titer of AA-2G at 1.92 g/liter, which was 60% higher than that (1.20 g/liter) produced by the wild-type CGTase. The kinetics analysis of AA-2G synthesis by the mutant CGTases confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, all seven mutants had lower cyclization activities and higher hydrolysis and disproportionation activities. Finally, the mechanism responsible for the enhanced substrate specificity was explored by structure modeling, which indicated that the enhancement of maltodextrin specificity may be related to the changes of hydrogen bonding interactions between the side chain of residue at the three positions (195, 260, and 265) and the substrate sugars. This work adds to our understanding of the synthesis of AA-2G and makes the Y260R/Q265K/Y195S mutant a good starting point for further development by protein engineering. PMID:23160123

  6. Phenylalanine transfer across the isolated perfused human placenta: an experimental and modeling investigation

    PubMed Central

    Lofthouse, E. M.; Perazzolo, S.; Brooks, S.; Crocker, I. P.; Glazier, J. D.; Johnstone, E. D.; Panitchob, N.; Sibley, C. P.; Widdows, K. L.; Sengers, B. G.

    2015-01-01

    Membrane transporters are considered essential for placental amino acid transfer, but the contribution of other factors, such as blood flow and metabolism, is poorly defined. In this study we combine experimental and modeling approaches to understand the determinants of [14C]phenylalanine transfer across the isolated perfused human placenta. Transfer of [14C]phenylalanine across the isolated perfused human placenta was determined at different maternal and fetal flow rates. Maternal flow rate was set at 10, 14, and 18 ml/min for 1 h each. At each maternal flow rate, fetal flow rates were set at 3, 6, and 9 ml/min for 20 min each. Appearance of [14C]phenylalanine was measured in the maternal and fetal venous exudates. Computational modeling of phenylalanine transfer was undertaken to allow comparison of the experimental data with predicted phenylalanine uptake and transfer under different initial assumptions. Placental uptake (mol/min) of [14C]phenylalanine increased with maternal, but not fetal, flow. Delivery (mol/min) of [14C]phenylalanine to the fetal circulation was not associated with fetal or maternal flow. The absence of a relationship between placental phenylalanine uptake and net flux of phenylalanine to the fetal circulation suggests that factors other than flow or transporter-mediated uptake are important determinants of phenylalanine transfer. These observations could be explained by tight regulation of free amino acid levels within the placenta or properties of the facilitated transporters mediating phenylalanine transport. We suggest that amino acid metabolism, primarily incorporation into protein, is controlling free amino acid levels and, thus, placental transfer. PMID:26676251

  7. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues.

    PubMed

    Cook, Naomi L; Moeke, Cassidy H; Fantoni, Luca I; Pattison, David I; Davies, Michael J

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN(-)) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces cellular dysfunction in cultured macrophage-like cells. The present study extends this previous work and shows that physiologically-relevant concentrations of HOSCN alter the activity and structure of other members of the wider PTP family (including leukocyte antigen-related PTP, PTP-LAR; T-cell PTP, TC-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 µM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids or sulfenyl-thiocyanates (detected by Western blotting, and LC-MS as dimedone adducts), and structural changes. LC-MS/MS peptide mass-mapping has provided data on the modified Cys residues in PTP-LAR. This study indicates that inflammation-induced oxidants, and particularly myeloperoxidase-derived species, can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation. PMID:26616646

  8. Cytoplasmic Tyrosine Phosphatase Shp2 Coordinates Hepatic Regulation of Bile Acid and FGF15/19 Signaling to Repress Bile Acid Synthesis

    PubMed Central

    Li, Shuangwei; Hsu, Diane D.F.; Li, Bing; Luo, Xiaolin; Alderson, Nazilla; Qiao, Liping; Ma, Lina; Zhu, Helen H.; He, Zhao; Suino-Powell, Kelly; Ji, Kaihong; Li, Jiefu; Shao, Jianhua; Xu, H. Eric; Li, Tiangang; Feng, Gen-Sheng

    2015-01-01

    Summary Bile acid (BA) biosynthesis is tightly controlled by intrahepatic negative feedback signaling elicited by BA binding to farnesoid X receptor (FXR), and also by enterohepatic communication involving ileal BA reabsorption and FGF15/19 secretion. However, how these pathways are coordinated is poorly understood. We show here that non-receptor tyrosine phosphatase Shp2 is a critical player that couples and regulates the intrahepatic and enterohepatic signals for repression of BA synthesis. Ablating Shp2 in hepatocytes suppressed signal relay from FGFR4, receptor for FGF15/19, and attenuated BA activation of FXR signaling, resulting in elevation of systemic BA levels and chronic hepatobiliary disorders in mice. Acting immediately downstream of FGFR4, Shp2 associates with FRS2α and promotes the receptor activation and signal relay to several pathways. These results elucidate a molecular mechanism for the control of BA homeostasis by Shp2 through orchestration of multiple signals in hepatocytes. PMID:24981838

  9. Association between insulin resistance and plasma amino acid profile in non-diabetic Japanese subjects

    PubMed Central

    Yamada, Chizumi; Kondo, Masumi; Kishimoto, Noriaki; Shibata, Takeo; Nagai, Yoko; Imanishi, Tadashi; Oroguchi, Takashige; Ishii, Naoaki; Nishizaki, Yasuhiro

    2015-01-01

    Aims/Introduction Elevation of the branched-chain amino acids (BCAAs), valine, leucine and isoleucine; and the aromatic amino acids, tyrosine and phenylalanine, has been observed in obesity-related insulin resistance. However, there have been few studies on Asians, who are generally less obese and less insulin-resistant than Caucasian or African-Americans. In the present study, we investigated the relationship between homeostasis model assessment of insulin resistance (HOMA-IR) and plasma amino acid concentration in non-diabetic Japanese participants. Materials and Methods A total of 94 healthy men and women were enrolled, and plasma amino acid concentration was measured by liquid chromatography/mass spectrometry after overnight fasting. The associations between HOMA-IR and 20 amino acid concentrations, and anthropometric and clinical parameters of lifestyle-related diseases were evaluated. Results The mean age and body mass index were 40.1 ± 9.6 years and 22.7 ± 3.9, respectively. Significantly positive correlations were observed between HOMA-IR and valine, isoleucine, leucine, tyrosine, phenylalanine and total BCAA concentration. Compared with the HOMA-IR ≤ 1.6 group, the HOMA-IR > 1.6 group showed significantly exacerbated anthropometric and clinical parameters, and significantly elevated levels of valine, isoleucine, leucine, tyrosine, phenylalanine and BCAA. Conclusions The present study shows that the insulin resistance-related change in amino acid profile is also observed in non-diabetic Japanese subjects. These amino acids include BCAAs (valine, isoleucine and leucine) and aromatic amino acids (tyrosine and phenylalanine), in agreement with previous studies carried out using different ethnic groups with different degrees of obesity and insulin resistance. PMID:26221519

  10. Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib.

    PubMed

    Beckmann, Svenja; Long, Thavy; Scheld, Christina; Geyer, Rudolf; Caffrey, Conor R; Grevelding, Christoph G

    2014-12-01

    In the search for new drugs and drug targets to treat the flatworm disease schistosomiasis, protein kinases (PKs) have come under particular scrutiny because of their essential roles in developmental and physiological processes in schistosome parasites. In this context the application of the anti-cancer Abl tyrosine kinase (TK) inhibitor Imatinib (Gleevec/Glivec; STI-571) to adult Schistosoma mansoni in vitro has indicated negative effects on diverse physiological processes including survival. Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA) and alpha-1 acid glycoprotein (AGP or orosomucoid) negated Imatinib's deleterious effects on adult S. mansoni and schistosomula (post-infective larvae) in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6-8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments. PMID:25516839

  11. Characterization of all possible single-nucleotide change caused amino acid substitutions in the kinase domain of Bruton tyrosine kinase.

    PubMed

    Väliaho, Jouni; Faisal, Imrul; Ortutay, Csaba; Smith, C I Edvard; Vihinen, Mauno

    2015-06-01

    Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease-causing variations among the human protein kinases, still it is just 10% of all the possible single-nucleotide substitution-caused amino acid variations (SNAVs). In the BTK kinase domain (BTK-KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease-causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty-seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms. PMID:25777788

  12. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  13. Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib

    PubMed Central

    Beckmann, Svenja; Long, Thavy; Scheld, Christina; Geyer, Rudolf; Caffrey, Conor R.; Grevelding, Christoph G.

    2014-01-01

    In the search for new drugs and drug targets to treat the flatworm disease schistosomiasis, protein kinases (PKs) have come under particular scrutiny because of their essential roles in developmental and physiological processes in schistosome parasites. In this context the application of the anti-cancer Abl tyrosine kinase (TK) inhibitor Imatinib (Gleevec/Glivec; STI-571) to adult Schistosoma mansoni in vitro has indicated negative effects on diverse physiological processes including survival. Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA) and alpha-1 acid glycoprotein (AGP or orosomucoid) negated Imatinib’s deleterious effects on adult S. mansoni and schistosomula (post-infective larvae) in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6–8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments. PMID:25516839

  14. Transformation of phenol into phenylalanine by a methanogenic consortium

    SciTech Connect

    Lepine, F.; Milot, S.; Beaudet, R.; Villemur, R.

    1996-03-01

    Phenol is a widely used chemical found in many wastewaters of industrial origin. The degradation of phenol by methanogenic bacterial consortia has been reported by many investigators. To better characterise the metabolism of this consortium, a new metabolic pathway of benzoic acid, an intermediary in the degradation of phenol, is reported. This study describes the transformations of benzoic acid into 3-phenylpropionic acid and phenylalanine. 25 refs., 5 figs.

  15. Au-Pd/reduced graphene oxide composite as a new sensing layer for electrochemical determination of ascorbic acid, acetaminophen and tyrosine.

    PubMed

    Tadayon, Fariba; Vahed, Saba; Bagheri, Hasan

    2016-11-01

    An Au-Pd/reduced graphene oxide composite was employed as a novel electrode material for the sensitive and simultaneous determination of ascorbic acid, acetaminophen and tyrosine. The electrochemical response characteristics of the modified electrode toward the analytes were investigated by differential pulse voltammetry and cyclic voltammetry. The responses of the electrochemical sensor for the target analytes were found to be improved significantly in comparison with those obtained using a conventional carbon paste electrode (CPE) and reduced graphene oxide/CPE. The experimental conditions for simultaneous determination of these species have been established. Ternary mixtures of analytes can be determined in the ranges of 0.03-9.50μM. Under optimal conditions, the limits of detection were 15.7, 7.6 and 11.1nM for ascorbic acid, acetaminophen, and tyrosine, respectively. The method was applied successfully to determine the analytes in urine, serum and pharmaceutical samples simultaneously. PMID:27524083

  16. Gi-mediated tyrosine phosphorylation of Grb2 (growth-factor-receptor-bound protein 2)-bound dynamin-II by lysophosphatidic acid.

    PubMed Central

    Kranenburg, O; Verlaan, I; Moolenaar, W H

    1999-01-01

    Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of growth-factor-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. 16, 3097-3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in fibroblasts. PMID:10085221

  17. Biomolecular interactions of emerging two-dimensional materials with aromatic amino acids

    NASA Astrophysics Data System (ADS)

    Mallineni, Sai Sunil Kumar; Karakaya, Mehmet; Podila, Ramakrishna; Rao, Apparao

    The present work experimentally investigates the interaction of aromatic amino acids, viz., tyrosine, tryptophan, and phenylalanine with novel two-dimensional (2D) materials including graphene (G), graphene oxide (GO), and boron nitride (BN). Photoluminescence, micro-Raman spectroscopy and cyclic voltammetry were employed to investigate the nature of interactions and possible charge transfer between 2D materials and amino acids. Consistent with previous theoretical studies, graphene and BN were observed to interact with amino acids through π- π interactions. Furthermore, we found that GO exhibits strong interactions with tryptophan and tyrosine as compared to graphene and BN, which we attribute to the formation of H-bonds between tryptophan and GO as shown theoretically in Ref. 2. On the other hand, phenylalanine did not exhibit much difference in interactions with G, GO, and BN. Clemson Nanomaterials Center, Clemson University, Clemson, SC, USA.

  18. Prediction of Intramolecular Polarization of Aromatic Amino Acids Using Kriging Machine Learning.

    PubMed

    Fletcher, Timothy L; Davie, Stuart J; Popelier, Paul L A

    2014-09-01

    Present computing power enables novel ways of modeling polarization. Here we show that the machine learning method kriging accurately captures the way the electron density of a topological atom responds to a change in the positions of the surrounding atoms. The success of this method is demonstrated on the four aromatic amino acids histidine, phenylalanine, tryptophan, and tyrosine. A new technique of varying training set sizes to vastly reduce training times while maintaining accuracy is described and applied to each amino acid. Each amino acid has its geometry distorted via normal modes of vibration over all local energy minima in the Ramachandran map. These geometries are then used to train the kriging models. Total electrostatic energies predicted by the kriging models for previously unseen geometries are compared to the true energies, yielding mean absolute errors of 2.9, 5.1, 4.2, and 2.8 kJ mol(-1) for histidine, phenylalanine, tryptophan, and tyrosine, respectively. PMID:26588516

  19. A Unique Dual Activity Amino Acid Hydroxylase in Toxoplasma gondii

    PubMed Central

    Gaskell, Elizabeth A.; Smith, Judith E.; Pinney, John W.; Westhead, Dave R.; McConkey, Glenn A.

    2009-01-01

    The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces l-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to l-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed. PMID:19277211

  20. Tyrosine Aminotransferase: Biochemical and Structural Properties and Molecular Dynamics Simulations

    SciTech Connect

    P Mehere; Q Han; J Lemkul; C Vavricka; H Robinson; D Bevan; J Li

    2011-12-31

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  1. Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations

    SciTech Connect

    Mehere, P.; Robinson, H.; Han, Q.; Lemkul, J. A.; Vavricka, C. J.; Bevan, D. R.; Li, J.

    2010-11-01

    Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using {alpha}-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 {angstrom} resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

  2. Synthesis, uptake mechanism characterization and biological evaluation of 18F labeled fluoroalkyl phenylalanine analogs as potential PET imaging agents

    PubMed Central

    Wang, Limin; Qu, Wenchao; Lieberman, Brian P.; Plössl, Karl; Kung, Hank F.

    2010-01-01

    Introduction Amino acids based tracers represent a promising class of tumor metabolic imaging agents with successful clinical applications. Two new phenylalanine derivatives, p-(2-[18F]fluoroethyl)-L-phenylalanine (FEP, [18F]2) and p-(3-[18F]fluoropropyl)-L-phenylalanine (FPP, [18F]3) were synthesized and evaluated in comparison to clinically utilized O-(2-[18F]fluoroethyl)-L-tyrosine (FET, [18F]1). Methods FEP ([18F]2) and FPP ([18F]3) were successfully synthesized by a rapid and efficient two-step nucleophilic fluorination of tosylate precursors and deprotection reaction. In vitro cell uptake studies were carried out in 9L glioma cells. In vivo studies, 9L tumor xenografts were implanted in Fisher 344 rats. Results FEP ([18F]2) and FPP ([18F]3) could be efficiently labeled within 90 min with good enantiomeric purity (>95%), good yield (11–37%) and high specific activity (21–69 GBq/μmol). Cell uptake studies showed FEP had higher uptake than FPP as well as reference ligand FET ([18F]1). Uptake mechanism studies suggested that FEP is a selective substrate for system L and prefers its subtype LAT1. In vivo biodistribution studies demonstrated FEP had specific accumulation in tumor cells and tumor to background ratio reached 1.45 at 60 min. Small animal PET imaging studies showed FEP was comparable to FET for imaging rats bearing 9L tumor model. FEP had high uptake in 9L tumor compared to surrounding tissue and was quickly excreted through urinary tract. Conclusion Biological evaluations indicate that FEP ([18F]2) is a potential useful tracer for tumor imaging with PET. PMID:21220129

  3. Effects of aspartame and glucose administration on brain and plasma levels of large neutral amino acids and brain 5-hydroxyindoles.

    PubMed

    Yokogoshi, H; Roberts, C H; Caballero, B; Wurtman, R J

    1984-07-01

    Administration of the artificial sweetener aspartame (L-aspartylphenylalanylmethyl ester; 200 mg/kg) by gavage to rats caused large increments in brain and plasma levels of phenylalanine and its product tyrosine. Glucose administration (3 g/kg, by gavage, a dose sufficient to cause insulin-mediated reductions in plasma levels of the large neutral amino acids leucine, isoleucine, and valine) also elevated brain phenylalanine and tyrosine, and enhanced the increments caused by the aspartame, nearly doubling the rise in brain phenylalanine. Each animal's brain phenylalanine or tyrosine levels were highly correlated (r = 0.97 and 0.99, respectively) with its plasma phenylalanine or tyrosine ratios, affirming that aspartame's effects on the brain amino acids result from the changes it produces in plasma composition. As described previously, glucose consumption increased brain tryptophan levels, and consequently, brain levels of the 5-hydroxyindoles serotonin and 5-hydroxyindoleacetic acid. Aspartame alone had no effect on these compounds but completely blocked the changes in 5-hydroxyindoles caused by glucose. Each animal's brain level of tryptophan (r = 0.89) and 5-hydroxyindoles (r = 0.74) was also significantly correlated with its plasma tryptophan ratio, affirming that the effects of glucose or aspartame on these brain constituents also result from the changes they produce in plasma composition. The aspartame-glucose combination also reduced brain levels of leucine, isoleucine, and valine to a significantly greater extent than aspartame or glucose alone. These observations indicate that high aspartame doses can generate major neurochemical changes in rats, especially when consumed along with carbohydrate-containing foods. However, they should not in any way be interpreted as demonstrating that aspartame significantly affects the human brain. PMID:6204522

  4. Critical Role for an Acidic Amino Acid Region in Platelet Signaling by the HemITAM (Hemi-immunoreceptor Tyrosine-based Activation Motif) Containing Receptor CLEC-2 (C-type Lectin Receptor-2)*

    PubMed Central

    Hughes, Craig E.; Sinha, Uma; Pandey, Anjali; Eble, Johannes A.; O'Callaghan, Christopher A.; Watson, Steve P.

    2013-01-01

    CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors. PMID:23264619

  5. L-DOPA Reverses the Increased Free Amino Acids Tissue Levels Induced by Dopamine Depletion and Rises GABA and Tyrosine in the Striatum.

    PubMed

    Solís, Oscar; García-Sanz, Patricia; Herranz, Antonio S; Asensio, María-José; Moratalla, Rosario

    2016-07-01

    Perturbations in the cerebral levels of various amino acids are associated with neurological disorders, and previous studies have suggested that such alterations have a role in the motor and non-motor symptoms of Parkinson's disease. However, the direct effects of chronic L-DOPA treatment, that produces dyskinesia, on neural tissue amino acid concentrations have not been explored in detail. To evaluate whether striatal amino acid concentrations are altered in peak dose dyskinesia, 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian mice were treated chronically with L-DOPA and tissue amino acid concentrations were assessed by HPLC analysis. These experiments revealed that neither 6-OHDA nor L-DOPA treatment are able to alter glutamate in the striatum. However, glutamine increases after 6-OHDA and returns back to normal levels with L-DOPA treatment, suggesting increased striatal glutamatergic transmission with lack of dopamine. In addition, glycine and taurine levels are increased following dopamine denervation and restored to normal levels by L-DOPA. Interestingly, dyskinetic animals showed increased levels of GABA and tyrosine, while aspartate striatal tissue levels are not altered. Overall, our results indicate that chronic L-DOPA treatment, besides normalizing the altered levels of some amino acids after 6-OHDA, robustly increases striatal GABA and tyrosine levels which may in turn contribute to the development of L-DOPA-induced dyskinesia. PMID:26966009

  6. A Possible Role of Divalent Manganese Ions in the Photoinduction of Phenylalanine Ammonia-Lyase

    PubMed Central

    Engelsma, G.

    1972-01-01

    Divalent Mn ions cause an increase in the level of phenylalanine ammonia-lyase in gherkin hypocotyls. With the exception of Mg ions, which had a small effect, no other metal ion has so far been found which could replace the Mn ion in this respect. Invertase and peroxidase were not significantly affected by the Mn treatment. The increase in phenylalanine ammonialyase activity is explained by the removal, under the influence of Mn ions, of hydroxycinnamic acids, which cause repression of phenylalanine ammonia-lyase synthesis and/or inactivation of phenylalanine ammonia-lyase. Arguments are advanced for the hypothesis that photochemical transformations of Mn complexes are involved in the photoinduction of phenylalanine ammonia-lyase in dark-grown gherkin seedlings. PMID:16658225

  7. Splanchnic extraction of phenylalanine in mature mares was not affected by threonine supplementation.

    PubMed

    Mastellar, S L; Barnes, T; Cybulak, K; Urschel, K L

    2016-01-01

    This study determined splanchnic extraction of phenylalanine at two intakes of threonine. Six Thoroughbred mares were supplemented with isonitrogenous amounts of either threonine or glutamate. Dietary threonine intakes were 119 (+Thr) and 58 (Basal) mg/kg/day, respectively. Each horse received each diet twice and each was studied once with an oral and once with an intravenous (IV) infusion of [1-(13)C]phenylalanine. A 2-h primed, constant IV infusion of [(13)C]sodium bicarbonate and a 4-h primed, constant infusion of [1-(13)C]phenylalanine, either orally or IV, were used to measure isotopic enrichments. Phenylalanine kinetics were not affected by diet (P > 0.05). Values for the splanchnic extraction of phenylalanine were 26 ± 5% and 27 ± 3% for the +Thr and Basal supplemented diets, respectively. These values will improve the accuracy of future equine indicator amino acid oxidation studies. PMID:26639820

  8. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

    PubMed Central

    Wiedłocha, A; Falnes, P O; Rapak, A; Muñoz, R; Klingenberg, O; Olsnes, S

    1996-01-01

    U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell. PMID:8524304

  9. Effect of methyl jasmonate application to grapevine leaves on grape amino acid content.

    PubMed

    Garde-Cerdán, Teresa; Portu, Javier; López, Rosa; Santamaría, Pilar

    2016-07-15

    Over the last few years, considerable attention has been paid to the application of elicitors to vineyard. However, research about the effect of elicitors on grape amino acid content is scarce. Therefore, the aim of this study was to evaluate the influence of foliar application of methyl jasmonate on must amino acid content. Results revealed that total amino acid content was not modified by the application of methyl jasmonate. However, the individual content of certain amino acids was increased as consequence of methyl jasmonate foliar application, i.e., histidine, serine, tryptophan, phenylalanine, tyrosine, asparagine, methionine, and lysine. Among them, phenylalanine content was considerably increased; this amino acid is precursor of phenolic and aromatic compounds. In conclusion, foliar application of methyl jasmonate improved must nitrogen composition. This finding suggests that methyl jasmonate treatment might be conducive to obtain wines of higher quality since must amino acid composition could affect the wine volatile composition and the fermentation kinetics. PMID:26948648

  10. Analysis of cyclic pyrolysis products formed from amino acid monomer.

    PubMed

    Choi, Sung-Seen; Ko, Ji-Eun

    2011-11-18

    Amino acid was mixed with silica and tetramethylammonium hydroxide (TMAH) to favor pyrolysis of amino acid monomer. The pyrolysis products formed from amino acid monomer were using GC/MS and GC. 20 amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were analyzed. The pyrolysis products were divided into cyclic and non-cyclic products. Among the 20 amino acids, arginine, asparagine, glutamic acid, glutamine, histidine, lysine, and phenylalanine generated cyclic pyrolysis products of the monomer. New cyclic pyrolysis products were formed by isolation of amino acid monomers. They commonly had polar side functional groups to 5-, 6-, or 7-membered ring structure. Arginine, asparagine, glutamic acid, glutamine, histidine, and phenylalanine generated only 5- or 6-membered ring products. However, lysine generated both 6- and 7-membered ring compounds. Variations of the relative intensities of the cyclic pyrolysis products with the pyrolysis temperature and amino acid concentration were also investigated. PMID:21993510

  11. Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases*

    PubMed Central

    Torrens-Spence, Michael P.; Liu, Pingyang; Ding, Haizhen; Harich, Kim; Gillaspy, Glenda; Li, Jianyong

    2013-01-01

    Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site

  12. Phenylalanine hydroxylase deficiency: diagnosis and management guideline.

    PubMed

    Vockley, Jerry; Andersson, Hans C; Antshel, Kevin M; Braverman, Nancy E; Burton, Barbara K; Frazier, Dianne M; Mitchell, John; Smith, Wendy E; Thompson, Barry H; Berry, Susan A

    2014-02-01

    Phenylalanine hydroxylase deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals and was the first inborn error of metabolism to be identified through population screening. Early identification and treatment prevent the most dramatic clinical sequelae of the disorder, but new neurodevelopmental and psychological problems have emerged in individuals treated from birth. The additional unanticipated recognition of a toxic effect of elevated maternal phenylalanine on fetal development has added to a general call in the field for treatment for life. Two major conferences sponsored by the National Institutes of Health held >10 years apart reviewed the state of knowledge in the field of phenylalanine hydroxylase deficiency, but there are no generally accepted recommendations for therapy. The purpose of this guideline is to review the strength of the medical literature relative to the treatment of phenylalanine hydroxylase deficiency and to develop recommendations for diagnosis and therapy of this disorder. Evidence review from the original National Institutes of Health consensus conference and a recent update by the Agency for Healthcare Research and Quality was used to address key questions in the diagnosis and treatment of phenylalanine hydroxylase deficiency by a working group established by the American College of Medical Genetics and Genomics. The group met by phone and in person over the course of a year to review these reports, develop recommendations, and identify key gaps in our knowledge of this disorder. Above all, treatment of phenylalanine hydroxylase deficiency must be life long, with a goal of maintaining blood phenylalanine in the range of 120-360 µmol/l. Treatment has predominantly been dietary manipulation, and use of low protein and phenylalanine medical foods is likely to remain a major component of therapy for the immediate future. Pharmacotherapy for phenylalanine

  13. The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold.

    PubMed

    Hackel, Benjamin J; Wittrup, K Dane

    2010-04-01

    The design of combinatorial libraries for molecular recognition requires extensive diversity to provide high affinity binding to myriad epitopes while maintaining a high degree of functionality to enable inclusion of binders in the limited screenable library size. In the current work, we directly compare minimal and maximal amino acid diversity libraries in the context of the 10th type III domain of human fibronectin. Libraries with either serine/tyrosine or full 20 amino acid diversity were created, pooled and screened for binding to rabbit and goat immunoglobulin G (IgG), and affinity matured by directed evolution. Multiple picomolar binders to rabbit IgG and nanomolar binders to goat IgG were engineered with peak affinities of 51 +/- 4 pM and 1.2 +/- 0.4 nM, respectively. Sequence analysis reveals that 93% of the selected BC and FG loops, including those from the highest affinity clones, originate from the full diversity library. Thus, with a modest initial library size (approximately 1 x 10(8)) and an efficient affinity maturation scheme, more extensive diversity is superior to a binary serine/tyrosine code for the generation of picomolar to low nanomolar binders in the fibronectin domain. The highest affinity binders demonstrated utility in affinity purification of IgG from serum and as detection reagents in flow cytometry. PMID:20067921

  14. Glycoproteomic Analysis of Prostate Cancer Tissues by SWATH Mass Spectrometry Discovers N-acylethanolamine Acid Amidase and Protein Tyrosine Kinase 7 as Signatures for Tumor Aggressiveness*

    PubMed Central

    Liu, Yansheng; Chen, Jing; Sethi, Atul; Li, Qing K.; Chen, Lijun; Collins, Ben; Gillet, Ludovic C. J.; Wollscheid, Bernd; Zhang, Hui; Aebersold, Ruedi

    2014-01-01

    The identification of biomarkers indicating the level of aggressiveness of prostate cancer (PCa) will address the urgent clinical need to minimize the general overtreatment of patients with non-aggressive PCa, who account for the majority of PCa cases. Here, we isolated formerly N-linked glycopeptides from normal prostate (n = 10) and from non-aggressive (n = 24), aggressive (n = 16), and metastatic (n = 25) PCa tumor tissues and analyzed the samples using SWATH mass spectrometry, an emerging data-independent acquisition method that generates a single file containing fragment ion spectra of all ionized species of a sample. The resulting datasets were searched using a targeted data analysis strategy in which an a priori spectral reference library representing known N-glycosites of the human proteome was used to identify groups of signals in the SWATH mass spectrometry data. On average we identified 1430 N-glycosites from each sample. Out of those, 220 glycoproteins showed significant quantitative changes associated with diverse biological processes involved in PCa aggressiveness and metastasis and indicated functional relationships. Two glycoproteins, N-acylethanolamine acid amidase and protein tyrosine kinase 7, that were significantly associated with aggressive PCa in the initial sample cohort were further validated in an independent set of patient tissues using tissue microarray analysis. The results suggest that N-acylethanolamine acid amidase and protein tyrosine kinase 7 may be used as potential tissue biomarkers to avoid overtreatment of non-aggressive PCa. PMID:24741114

  15. Hybride magnetic nanostructure based on amino acids functionalized polypyrrole

    SciTech Connect

    Nan, Alexandrina Bunge, Alexander; Turcu, Rodica

    2015-12-23

    Conducting polypyrrole is especially promising for many commercial applications because of its unique optical, electric, thermal and mechanical properties. We report the synthesis and characterization of novel pyrrole functionalized monomers and core-shell hybrid nanostructures, consisting of a conjugated polymer layer (amino acids functionalized pyrrole copolymers) and a magnetic nanoparticle core. For functionalization of the pyrrole monomer we used several amino acids: tryptophan, leucine, phenylalanine, serine and tyrosine. These amino acids were linked via different types of hydrophobic linkers to the nitrogen atom of the pyrrole monomer. The magnetic core-shell hybrid nanostructures are characterized by various methods such as FTIR spectroscopy, transmission electron microscopy (TEM) and magnetic measurements.

  16. Hybride magnetic nanostructure based on amino acids functionalized polypyrrole

    NASA Astrophysics Data System (ADS)

    Nan, Alexandrina; Bunge, Alexander; Turcu, Rodica

    2015-12-01

    Conducting polypyrrole is especially promising for many commercial applications because of its unique optical, electric, thermal and mechanical properties. We report the synthesis and characterization of novel pyrrole functionalized monomers and core-shell hybrid nanostructures, consisting of a conjugated polymer layer (amino acids functionalized pyrrole copolymers) and a magnetic nanoparticle core. For functionalization of the pyrrole monomer we used several amino acids: tryptophan, leucine, phenylalanine, serine and tyrosine. These amino acids were linked via different types of hydrophobic linkers to the nitrogen atom of the pyrrole monomer. The magnetic core-shell hybrid nanostructures are characterized by various methods such as FTIR spectroscopy, transmission electron microscopy (TEM) and magnetic measurements.

  17. Highest Plasma Phenylalanine Levels in (Very) Premature Infants on Intravenous Feeding; A Need for Concern

    PubMed Central

    Cortés-Castell, Ernesto; Sánchez-González, Pablo; Palazón-Bru, Antonio; Bosch-Giménez, Vicente; Manero-Soler, Herminia; Juste-Ruiz, Mercedes; Rizo-Baeza, María Mercedes; Gil-Guillén, Vicente Francisco

    2015-01-01

    Objective To analyse the association in newborns between blood levels of phenylalanine and feeding method and gestational age. Study Design This observational, cross-sectional study included a sample of 11,829 infants between 2008 and 2013 in a Spanish region. Data were recorded on phenylalanine values, feeding method [breast, formula, mixed (breast plus formula), or partial or fully intravenous feeding], gestational age in weeks (<32, 32–37, ≥37), gender and days since birth at the moment of blood collection. Outcomes were [phenylalanine] and [phenylalanine] ≥95th percentile. Associations were analysed using multivariate models [linear (means difference) and logistic regression (adjusted odds ratios)]. Results Higher phenylalanine values were associated with lower gestational age (p<0.001) and with intravenous feeding (p<0.001). Conclusion The degree of prematurity and intravenous feeding influenced the plasma concentration of phenylalanine in the newborn. Caution should be taken in [phenylalanine] for newborns with intravenous feeding, monitoring them carefully. Very preterm infants given the recommended amount of amino acids should also be strictly monitored. These findings should be taken into consideration and call for adapting the amounts to the needs of the infant. PMID:26389596

  18. Photochemical Tyrosine Oxidation with a Hydrogen-Bonded Proton Acceptor by Bidirectional Proton-Coupled Electron Transfer

    PubMed Central

    Pizano, Arturo A.; Yang, Jay L.

    2012-01-01

    Amino acid radical generation and transport are fundamentally important to numerous essential biological processes to which small molecule models lend valuable mechanistic insights. Pyridyl-amino acid-methyl esters are appended to a rhenium(I) tricarbonyl 1,10-phenanthroline core to yield rhenium–amino acid complexes with tyrosine ([Re]–Y–OH) and phenylalanine ([Re]–F). The emission from the [Re] center is more significantly quenched for [Re]–Y–OH upon addition of base. Time-resolved studies establish that excited-state quenching occurs by a combination of static and dynamic mechanisms. The degree of quenching depends on the strength of the base, consistent with a proton-coupled electron transfer (PCET) quenching mechanism. Comparative studies of [Re]–Y–OH and [Re]–F enable a detailed mechanistic analysis of a bidirectional PCET process. PMID:23495362

  19. Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

    PubMed Central

    Wang, Hongchao; Hao, Guangfei; Yang, Bo; Feng, Yun; Wang, Yu; Feng, Lu; Zhao, Jianxin; Song, Yuanda; Zhang, Hao; Chen, Yong Q.; Wang, Lei

    2013-01-01

    Mortierella alpina is a filamentous fungus commonly found in soil that is able to produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight. Analysis of the M. alpina genome suggests that there is a phenylalanine-hydroxylating system for the catabolism of phenylalanine, which has never been found in fungi before. We characterized the phenylalanine-hydroxylating system in M. alpina to explore its role in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant changes were found in the profile of fatty acids in M. alpina grown on medium containing an inhibitor of the phenylalanine-hydroxylating system compared to M. alpina grown on medium without inhibitor. Genes encoding enzymes involved in the phenylalanine-hydroxylating system (phenylalanine hydroxylase [PAH], pterin-4α-carbinolamine dehydratase, and dihydropteridine reductase) were expressed heterologously in Escherichia coli, and the resulting proteins were purified to homogeneity. Their enzymatic activity was investigated by high-performance liquid chromatography (HPLC) or visible (Vis)-UV spectroscopy. Two functional PAH enzymes were observed, encoded by distinct gene copies. A novel role for tetrahydrobiopterin in fungi as a cofactor for PAH, which is similar to its function in higher life forms, is suggested. This study establishes a novel scheme for the fungal degradation of an aromatic substance (phenylalanine) and suggests that the phenylalanine-hydroxylating system is functionally significant in lipid metabolism. PMID:23503309

  20. Blood-brain barrier permeability to leucine-enkephalin, D-alanine2-D-leucine5-enkephalin and their N-terminal amino acid (tyrosine).

    PubMed

    Zlokovic, B V; Begley, D J; Chain-Eliash, D G

    1985-06-10

    The permeability of the blood-brain barrier to [tyrosyl-3,5-3H]enkephalin-(5-L-leucine) (abbreviated to Leu-Enk) and of its synthetic analogue D-alanine2-[tyrosyl-3,5-3H]enkephalin-(5-D-leucine) (abbreviated to D-Ala2-D-Leu5-Enk) was studied, in the adult rat, by means of Oldendorf's27 intracarotid injection technique. The brain uptake index (BUI) corrected for residual vascular radioactivity was about the same for both peptides, indicating a low extraction from the blood during a 5- or 15-s period of exposure to the peptides. Transport of Leu-Enk was not saturated by unlabelled Enk at a concentration as high as 5 mM but was completely abolished by 5mM tyrosine and by the inhibitor of aminopeptidase activity, bacitracin (2 mM). Also the typical L-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2 carboxylic acid (BCH)9 at 10 mM concentration markedly reduced (by 80%) Leu-Enk uptake by the brain. In contrast, brain uptake of D-Ala2-D-Leu5-Enk was reduced only to about one-half of its control value by bacitracin or by 25% by BCH. Brain uptake for L-tyrosine was typically large and markedly inhibited by BCH but not inhibited by 5 mM unlabelled Leu-Enk. These results show that the measurable but low first-pass extractions for enkephalins are not representative of the uptake of these peptides into the brain, but rather reflect their extreme sensitivity to enzymatic degradation with a release of the N-terminal tyrosine residue. The results also suggest that small amounts of D-Ala2-D-Leu5-Enk might cross the blood-brain barrier in an intact form.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3891014

  1. Influence of Phenylalanine on Carotenoid Aggregation

    NASA Astrophysics Data System (ADS)

    Lu, L.; Ni, X.; Luo, X.

    2015-01-01

    The carotenoids lutein and β-carotene form, in 1:1 ethanol-water mixtures H-aggregates, of different strengths. The effects of phenylalanine on these aggregates were recorded by UV-Vis absorption, steady-state fluorescence, and Raman spectra. The H-aggregate of lutein was characterized by a large 78 nm blue shift in the absorption spectra, confirming the strong coupling between hydroxyl groups of adjacent molecules. The 15 nm blue shift in the β-carotene mixture also indicates that it was assembled by weak coupling between polyenes. After adding phenylalanine, the reducing absorption strength of the aggregates of lutein and reappearance of vibrational substructure indicate that the hydroxyl and amino groups of phenylalanine may coordinate to lutein and disaggregate the H-aggregates. However, phenylalanine had no effect on aggregates of β-carotene. The Raman spectra show three bands of carotenoids whose intensities decreased with increasing phenylalanine concentration. The frequency of ν1 corresponding to the length of the conjugated region was more sensitive to the solution of lutein. This coordination of phenylalanine to lutein could increase the length of the conjugated region. In addition, phenylalanine significantly affected the excited electronic states of carotenoids, which were crucial in the energy transfer from carotenoids to chlorophyll a in vivo.

  2. Effects of running the Bostom Marathon on plasma concentrations of large neutral amino acids

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Wurtman, R. J.; Lopez G-Coviella, I.; Blusztajn, J. K.; Vacanti, C. A.; Logue, M.; During, M.; Caballero, B.; Maher, T. J.; Evoniuk, G.

    1989-01-01

    Plasma large neutral amino acid concentrations were measured in thirty-seven subjects before and after completing the Boston Marathon. Concentrations of tyrosine, phenylalanine, and methionine increased, as did their 'plasma ratios' (i.e., the ratio of each amino acid's concentration to the summed plasma concentrations of the other large neutral amino acids which compete with it for brain uptake). No changes were noted in the plasma concentrations of tryptophan, leucine, isoleucine, nor valine; however, the 'plasma ratios' of valine, leucine, and isoleucine all decreased. These changes in plasma amino acid patterns may influence neurotransmitter synthesis.

  3. Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase

    PubMed Central

    Abromaitis, Stephanie; Hefty, P. Scott; Stephens, Richard S.

    2010-01-01

    Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases. Aromatic amino acid hydroxylases (AroAA-H) hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine (L-DOPA), and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-H are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylae host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria. PMID:19141112

  4. Tyrosine Detoxification Is an Essential Trait in the Life History of Blood-Feeding Arthropods.

    PubMed

    Sterkel, Marcos; Perdomo, Hugo D; Guizzo, Melina G; Barletta, Ana Beatriz F; Nunes, Rodrigo D; Dias, Felipe A; Sorgine, Marcos H F; Oliveira, Pedro L

    2016-08-22

    Blood-feeding arthropods are vectors of infectious diseases such as dengue, Zika, Chagas disease, and malaria [1], and vector control is essential to limiting disease spread. Because these arthropods ingest very large amounts of blood, a protein-rich meal, huge amounts of amino acids are produced during digestion. Previous work on Rhodnius prolixus, a vector of Chagas disease, showed that, among all amino acids, only tyrosine degradation enzymes were overexpressed in the midgut compared to other tissues [2]. Here we demonstrate that tyrosine detoxification is an essential trait in the life history of blood-sucking arthropods. We found that silencing Rhodnius tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of insects after a blood meal. This was confirmed by using the HPPD inhibitor mesotrione, which selectively killed hematophagous arthropods but did not affect non-hematophagous insects. In addition, mosquitoes and kissing bugs died after feeding on mice that had previously received a therapeutic effective oral dose (1 mg/kg) of nitisinone, another HPPD inhibitor used in humans for the treatment of tyrosinemia type I [3]. These findings indicate that HPPD (and TAT) can be a target for the selective control of blood-sucking disease vector populations. Because HPPD inhibitors are extensively used as herbicides and in medicine, these compounds may provide an alternative less toxic to humans and more environmentally friendly than the conventional neurotoxic insecticides that are currently used, with the ability to affect only hematophagous arthropods. PMID:27476595

  5. Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity.

    PubMed

    Lovelock, Sarah L; Turner, Nicholas J

    2014-10-15

    Phenylalanine ammonia lyases (PALs) catalyse the regio- and stereoselective hydroamination of cinnamic acid analogues to yield optically enriched α-amino acids. Herein, we demonstrate that a bacterial PAL from Anabaena variabilis (AvPAL) displays significantly higher activity towards a series of non-natural substrates than previously described eukaryotic PALs. Biotransformations performed on a preparative scale led to the synthesis of the 2-chloro- and 4-trifluoromethyl-phenylalanine derivatives in excellent ee, highlighting the enormous potential of bacterial PALs as biocatalysts for the synthesis of high value, non-natural amino acids. PMID:25037641

  6. A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells*

    PubMed Central

    Ramesh, Mathangi; Krishnan, Navasona; Muthuswamy, Senthil K.; Tonks, Nicholas K.

    2015-01-01

    We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling. The study revealed a novel role for PTPD2 as a positive regulator of ERBB2 signaling. Suppression of PTPD2 attenuated the ERBB2-induced multiacinar phenotype in three-dimensional cultures specifically by inhibiting ERBB2-mediated loss of polarity and lumen filling. In contrast, overexpression of PTPD2 enhanced the ERBB2 phenotype. We also found that a lipid second messenger, phosphatidic acid, bound PTPD2 in vitro and enhanced its catalytic activity. Small molecule inhibitors of phospholipase D (PLD), an enzyme that produces phosphatidic acid in cells, also attenuated the ERBB2 phenotype. Exogenously added phosphatidic acid rescued the PLD-inhibition phenotype, but only when PTPD2 was present. These findings illustrate a novel pathway involving PTPD2 and the lipid second messenger phosphatidic acid that promotes ERBB2 function. PMID:25681440

  7. Computational study of human tyrosine hydroxylase mutants to uphold [4-(Propan-2-yl) Phenyl]Carbamic acid as a potential inhibitor.

    PubMed

    Nawaz, Muhammad S; Parveen, Zahida; Wang, Liyong; Rashid, Sajid; Fatmi, Muhammad Q; Kamal, Mohammad A

    2014-01-01

    Neurodegenerative diseases that afflict nervous system are characterized by progressive nervous system dysfunction and associated with the one-set of many diseases like Segawa's syndrome (recessive form), autosomal recessive L-dopa-responsive dystonia, L-dopa non-responsive dystonia or progressive early-onset encephalopathy and recessive L-dopa-responsive parkinsonism. It has been reported that a number of mutations in coding regions, splice sites and promoter regions of tyrosine hydroxylase (TH) are associated with many such diseases. TH is responsible for catalyzing the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. This reaction is considered as rate-limiting step in the biosynthesis of catecholamines, dopamine, norepinephrine and epinephrine, which has made TH an important target for drug development. In our previous study using comparative molecular docking approach, it was concluded that [4-(Propan-2-yl) Phenyl]Carbamic acid (PPCA) may serve as a potential inhibitor. By further extending, our focus is to determine the binding affinities of PPCA and mutated TH. 3D structures of mutated TH were predicted and subjected to molecular docking studies. PPCA was found to bind in the deep narrow groove lined with polar and aromatic amino acids in 14 out of 17 mutants under study (R202H, L205P, H215Y, G216S, T245P, F278P, T283M, R297W, R306H, C328F, A345V, L356M, T368M, Q381K, P461L, T463M and D467G). Our results corroborate efficient binding of PPCA with normal and mutated TH, indicating that PPCA might be a strong therapeutic candidate for the management of Parkinson's disease and other related disorders. It may be a valuable target for evaluation in preclinical models. PMID:25230230

  8. Phenylalanine Ammonia-Lyase from Loblolly Pine 1

    PubMed Central

    Whetten, Ross W.; Sederoff, Ronald R.

    1992-01-01

    Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from differentiating secondary xylem of loblolly pine (Pinus taeda L.). Native molecular weight of the enzyme was estimated to be 280,000, with a subunit molecular weight of 74,000; isoelectric point, 5.8; and Michaelis constant for i-phenylalanine, 27 micromolar. No evidence was obtained for the existence of isoforms of the enzyme, nor for negative cooperativity of substrate binding. Polyclonal antibodies were raised against the phenylalanine ammonia-lyase subunit and used to identify a pal clone in an expression library of xylem complementary DNA (cDNA). Polymerase chain reaction, using oligonucleotide primers made from N-terminal amino acid sequence and from the 5′ end of the clone isolated from the expression library, was also used to isolate cDNA clones. These methods yielded cDNA clones covering the protein coding region of the pal messenger RNA. Comparisons of nucleotide sequence of pal cDNAs from pine, bean, sweet potato, and rice showed 60 to 62% identity between the pine clone and the angiosperm clones. ImagesFigure 1Figure 4 PMID:16668639

  9. Raman and surface-enhanced Raman spectroscopy of amino acids and nucleotide bases for target bacterial vibrational mode identification

    NASA Astrophysics Data System (ADS)

    Guicheteau, Jason; Argue, Leanne; Hyre, Aaron; Jacobson, Michele; Christesen, Steven D.

    2006-05-01

    Raman and surface-enhanced Raman spectroscopy (SERS) studies of bacteria have reported a wide range of vibrational mode assignments associated with biological material. We present Raman and SER spectra of the amino acids phenylalanine, tyrosine, tryptophan, glutamine, cysteine, alanine, proline, methionine, asparagine, threonine, valine, glycine, serine, leucine, isoleucine, aspartic acid and glutamic acid and the nucleic acid bases adenosine, guanosine, thymidine, and uridine to better characterize biological vibrational mode assignments for bacterial target identification. We also report spectra of the bacteria Bacillus globigii, Pantoea agglomerans, and Yersinia rhodei along with band assignments determined from the reference spectra obtained.

  10. Chlamydia pneumoniae encodes a functional aromatic amino acid hydroxylase.

    PubMed

    Abromaitis, Stephanie; Hefty, P Scott; Stephens, Richard S

    2009-03-01

    Chlamydia pneumoniae is a community-acquired respiratory pathogen that has been associated with the development of atherosclerosis. Analysis of the C. pneumoniae genome identified a gene (Cpn1046) homologous to eukaryotic aromatic amino acid hydroxylases (AroAA-Hs). AroAA-Hs hydroxylate phenylalanine, tyrosine, and tryptophan into tyrosine, dihydroxyphenylalanine, and 5-hydroxytryptophan, respectively. Sequence analysis of Cpn1046 demonstrated that residues essential for AroAA-H enzymatic function are conserved and that a subset of Chlamydia species contain an AroAA-H homolog. The chlamydial AroAA-Hs are transcriptionally linked to a putative bacterial membrane transport protein. We determined that recombinant Cpn1046 is able to hydroxylate phenylalanine, tyrosine, and tryptophan with roughly equivalent activity for all three substrates. Cpn1046 is expressed within 24 h of infection, allowing C. pneumoniae to hydroxylate host stores of aromatic amino acids during the period of logarithmic bacterial growth. From these results we can conclude that C. pneumoniae, as well as a subset of other Chlamydia species, encode an AroAA-H that is able to use all three aromatic amino acids as substrates. The maintenance of this gene within a number of Chlamydia suggests that the enzyme may have an important role in shaping the metabolism or overall pathogenesis of these bacteria. PMID:19141112

  11. A new D-stereospecific amino acid amidase from Ochrobactrum anthropi.

    PubMed

    Asano, Y; Mori, T; Hanamoto, S; Kato, Y; Nakazawa, A

    1989-07-14

    A new D-stereospecific amino acid amidase has been partially purified from Ochrobactrum anthropi SCRC SV3, which had been isolated and selected from soil. The Mr of the enzyme was estimated to be about 38,000, and its isoelectric point was 5.3. The enzyme catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-amino acid and ammonia. The major substrates included D-phenylalanine amide, D-tyrosine amide, D-tryptophan amide, D-leucine amide, and D-alanine amide. PMID:2751665

  12. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU)

    PubMed Central

    Harding, Cary O.; Winn, Shelley R.; Gibson, K. Michael; Arning, Erland; Bottiglieri, Teodoro; Grompe, Markus

    2014-01-01

    Summary Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU. PMID:24487571

  13. Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU).

    PubMed

    Harding, Cary O; Winn, Shelley R; Gibson, K Michael; Arning, Erland; Bottiglieri, Teodoro; Grompe, Markus

    2014-09-01

    Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU. PMID:24487571

  14. The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

    PubMed Central

    Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg

    2015-01-01

    Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

  15. Poly(ferulic acid-co-tyrosine): Effect of the Regiochemistry on the Photophysical and Physical Properties en Route to Biomedical Applications

    PubMed Central

    2015-01-01

    The photophysical and mechanical properties of novel poly(carbonate-amide)s derived from two biorenewable resources, ferulic acid (FA) and l-tyrosine ethyl ester, were evaluated in detail. From these two bio-based precursors, a series of four monomers were generated (having amide and/or carbonate coupling units with remaining functionalities to allow for carbonate formation) and transformed to a series of four poly(carbonate-amide)s. The simplest monomer, which was biphenolic and was obtained in a single amidation synthetic step, displayed bright, visible fluorescence that was twice brighter than FA. Multidimensional fluorescence spectroscopy of the polymers in solution highlighted the strong influence that regioselectivity and the degree of polymerization have on their photophysical properties. The regiochemistry of the system had little effect on the wettability, surface free energy, and Young’s modulus (ca. 2.5 GPa) in the solid state. Confocal imaging of solvent-cast films of each polymer revealed microscopically flat surfaces with fluorescent emission deep into the visible region. Fortuitously, one of the two regiorandom polymers (obtainable from the biphenolic monomer in only an overall two synthetic steps from FA and l-tyrosine ethyl ester) displayed the most promising fluorescent properties both in the solid state and in solution, allowing for the possibility of translating this system as a self-reporting or imaging agent in future applications. To further evaluate the potential of this polymer as a biodegradable material, hydrolytic degradation studies at different pH values and temperatures were investigated. Additionally, the antioxidant properties of the degradation products of this polymer were compared with its biphenolic monomer and FA. PMID:25364040

  16. Redundant and selective roles for erythropoietin receptor tyrosines in erythropoiesis in vivo.

    PubMed

    Longmore, G D; You, Y; Molden, J; Liu, K D; Mikami, A; Lai, S Y; Pharr, P; Goldsmith, M A

    1998-02-01

    Cytokine receptors have been shown in cell culture systems to use phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or not tyrosine residues within the erythropoietin receptor (EPOR) are essential for biologic activity during hematopoiesis in vivo. A variant of the EPOR was constructed that contains both a substitution (R129C) causing constitutive receptor activation as well as replacement of all eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison between animals exposed to recombinant retroviruses expressing cEPOR and cEPORYF showed that efficient red blood cell (RBC) development in vivo is dependent on the pressence of tyrosine residues in the cytoplasmic domain of the EPOR. In addition, an inefficient EPOR tyrosine independent pathway supporting RBC development was detected. Tyrosine add-back mutants showed that multiple individual tyrosines have the capacity to restore full erythropoietic potential to the EPOR as determined in whole animals. The analysis of primary erythroid progenitors transduced with the various cEPOR tyrosine mutants and tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit-erythroid progenitor development. Thus, this receptor is characterized by striking functional redundancy of tyrosines in a biologically relevant context. However, selective tyrosine residues may be uniquely important for early signals supporting erythroid development. PMID:9446647

  17. Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells.

    PubMed

    Wehinger, Sergio; Ortiz, Rina; Díaz, María Inés; Aguirre, Adam; Valenzuela, Manuel; Llanos, Paola; Mc Master, Christopher; Leyton, Lisette; Quest, Andrew F G

    2015-05-01

    A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation. PMID:25572853

  18. Intersubunit binding domains within tyrosine hydroxylase and tryptophan hydroxylase.

    PubMed

    Yohrling, G J; Jiang, G C; Mockus, S M; Vrana, K E

    2000-08-01

    Tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin (5-HT) belongs to the aromatic amino acid hydroxylase superfamily, which includes phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). The crystal structures for both PAH and TH have been reported, but a crystallographic model of TPH remains elusive. For this reason, we have utilized the information presented in the TH crystal structure in combination with primary sequence alignments to design point mutations in potential structural domains of the TPH protein. Mutation of a TH salt bridge (K170E) was sufficient to alter enzyme macromolecular assembly. We found that the disruption of the cognate intersubunit dimerization salt bridge (K111-E223) in TPH, however, did not affect the macromolecular assembly of TPH. Enzyme peaks representing only tetramers were observed with size exclusion chromatography. By contrast, a single-point mutation within the tetramerization domain of TPH (L435A) was sufficient to disrupt the normal homotetrameric assembly of TPH. These studies indicate that, although the proposed salt bridge dimerization interface of TH is conserved in TPH, this hypothetical TPH intersubunit binding domain, K111-E223, is not required for the proper macromolecular assembly of the protein. However, leucine 435 within the tetramerization domain is necessary for the proper macromolecular assembly of TPH. PMID:10900078

  19. Tyrosine - Effects on catecholamine release

    NASA Technical Reports Server (NTRS)

    Acworth, Ian N.; During, Matthew J.; Wurtman, Richard J.

    1988-01-01

    Tyrosine administration elevates striatal levels of dopamine metabolites in animals given treatments that accelerate nigrostriatal firing, but not in untreated rats. We examined the possibility that the amino acid might actually enhance dopamine release in untreated animals, but that the technique of measuring striatal dopamine metabolism was too insensitive to demonstrate such an effect. Dopamine release was assessed directly, using brain microdialysis of striatal extracellular fluid. Tyrosine administration (50-200 mg/kg IP) did indeed cause a dose related increase in extracellular fluid dopamine levels with minor elevations in levels of DOPAC and HVA, its major metabolites, which were not dose-related. The rise in dopamine was short-lived, suggesting that receptor-mediated feedback mechanisms responded to the increased dopamine release by diminishing neuronal firing or sensitivity to tyrosine. These observations indicate that measurement of changes in striatal DOPAC and HVA, if negative, need not rule out increases in nigrostriatal dopamine release.

  20. Age-Related Psychophysiological Vulnerability to Phenylalanine in Phenylketonuria

    PubMed Central

    Leuzzi, Vincenzo; Mannarelli, Daniela; Manti, Filippo; Pauletti, Caterina; Locuratolo, Nicoletta; Carducci, Carla; Carducci, Claudia; Vanacore, Nicola; Fattapposta, Francesco

    2014-01-01

    Background: Phenylketonuria (PKU) is caused by the inherited defect of the phenylalanine hydroxylase enzyme, which converts phenylalanine (Phe) into tyrosine (Tyr). Neonatal screening programs and early treatment have radically changed the natural history of PKU. Nevertheless, an increased risk of neurocognitive and psychiatric problems in adulthood remains a challenging aspect of the disease. In order to assess the vulnerability of complex skills to Phe, we explored: (a) the effect of a rapid increase in blood Phe levels on event-related potentials (ERP) in PKU subjects during their second decade of life; (b) the association (if existing) between psychophysiological and neurocognitive features. Methods: Seventeen early-treated PKU subjects, aged 10–20, underwent ERP [mismatch negativity, auditory P300, contingent negative variation (CNV), and Intensity Dependence of Auditory Evoked Potentials] recording before and 2 h after an oral loading of Phe. Neurocognitive functioning, historical and concurrent biochemical values of blood Phe, Tyr, and Phe/Tyr ratio, were all included in the statistical analysis. Results: Event-related potential components were normally detected in all the subjects. In subjects younger than 13 CNV amplitude, W2-CNV area, P3b latency, and reaction times in motor responses were negatively influenced by Phe-loading. Independently from the psychophysiological vulnerability, some neurocognitive skills were more impaired in younger patients. No correlation was found between biochemical alterations and neurocognitive and psychophysiological findings. Conclusion: The vulnerability of the emerging neurocognitive functions to Phe suggests a strict metabolic control in adolescents affected by PKU and a neurodevelopmental approach in the study of neurocognitive outcome in PKU. PMID:25003100

  1. Effect of acute acid-base disturbances on ErbB1/2 tyrosine phosphorylation in rabbit renal proximal tubules

    PubMed Central

    Skelton, Lara A.

    2013-01-01

    The renal proximal tubule (PT) is a major site for maintaining whole body pH homeostasis and is responsible for reabsorbing ∼80% of filtered HCO3−, the major plasma buffer, into the blood. The PT adapts its rate of HCO3− reabsorption (JHCO3−) in response to acute acid-base disturbances. Our laboratory previously showed that single isolated perfused PTs adapt JHCO3− in response to isolated changes in basolateral (i.e., blood side) CO2 and HCO3− concentrations but, surprisingly, not to pH. The response to CO2 concentration can be blocked by the ErbB family tyrosine kinase inhibitor PD-168393. In the present study, we exposed enriched rabbit PT suspensions to five acute acid-base disturbances for 5 and 20 min using a panel of phosphotyrosine (pY)-specific antibodies to determine the influence of each disturbance on pan-pY, ErbB1-specific pY (four sites), and ErbB2-specific pY (two sites). We found that each acid-base treatment generated a distinct temporal pY pattern. For example, the summated responses of the individual ErbB1/2-pY sites to each disturbance showed that metabolic acidosis (normal CO2 concentration and reduced HCO3− concentration) produced a transient summated pY decrease (5 vs. 20 min), whereas metabolic alkalosis produced a transient increase. Respiratory acidosis (normal HCO3− concentration and elevated CO2 concentration) had little effect on summated pY at 5 min but produced an elevation at 20 min, whereas respiratory alkalosis produced a reduction at 20 min. Our data show that ErbB1 and ErbB2 in the PT respond to acute acid-base disturbances, consistent with the hypothesis that they are part of the signaling cascade. PMID:24133121

  2. Polyclonal antibody to soman-tyrosine

    PubMed Central

    Li, Bin; Duysen, Ellen G.; Froment, Marie-Thérèse; Masson, Patrick; Nachon, Florian; Jiang, Wei; Schopfer, Lawrence M.; Thiele, Geoffrey M.; Klassen, Lynell W.; Cashman, John; Williams, Gareth R.; Lockridge, Oksana

    2013-01-01

    Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin, but not non-phosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10−11 M. The limit of detection on Western blots was 0.01 μg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity. PMID:23469927

  3. A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-β-Tyrosine with Unique High Enantioselectivity and Retention of Configuration.

    PubMed

    Walter, Tyler; King, Zayna; Walker, Kevin D

    2016-01-12

    A recently discovered 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent tyrosine aminomutase (OsTAM) from rice [Yan, J., et al. (2015) Plant Cell 27, 1265] converts (S)-α-tyrosine to a mixture of (R)- and (S)-β-tyrosines, with high (94%) enantiomeric excess, which does not change with pH, like it does for two bacterial TAMs. The K(M) of 490 μM and the k(cat) of 0.005 s(-1) are similar for other TAM enzymes. OsTAM is unique and also catalyzes (R)-β- from (S)-α-phenylalanine. OsTAM principally retains the configuration at the reactive C(α) and C(β) centers during catalysis much like the phenylalanine aminomutase on the Taxol biosynthetic pathway in Taxus plants. PMID:26709535

  4. Therapeutic implication of L-phenylalanine aggregation mechanism and its modulation by D-phenylalanine in phenylketonuria

    PubMed Central

    Singh, Virender; Rai, Ratan Kumar; Arora, Ashish; Sinha, Neeraj; Thakur, Ashwani Kumar

    2014-01-01

    Self-assembly of phenylalanine is linked to amyloid formation toxicity in phenylketonuria disease. We are demonstrating that L-phenylalanine self-assembles to amyloid fibrils at varying experimental conditions and transforms to a gel state at saturated concentration. Biophysical methods including nuclear magnetic resonance, resistance by alpha-phenylglycine to fibril formation and preference of protected phenylalanine to self-assemble show that this behaviour of L-phenylalanine is governed mainly by hydrophobic interactions. Interestingly, D-phenylalanine arrests the fibre formation by L-phenylalanine and gives rise to flakes. These flakes do not propagate further and prevent fibre formation by L-phenylalanine. This suggests the use of D-phenylalanine as modulator of L-phenylalanine amyloid formation and may qualify as a therapeutic molecule in phenylketonuria. PMID:24464217

  5. Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development

    PubMed Central

    2013-01-01

    Background Nutritional symbioses play a central role in insects’ adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. Results We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are

  6. First-principles study on the adsorption properties of phenylalanine on carbon graphitic structures

    NASA Astrophysics Data System (ADS)

    Kang, Seoung-Hun; Kwon, Dae-Gyeon; Park, Sora; Kwon, Young-Kyun

    2015-12-01

    Using ab-initio density functional theory, we investigate the binding properties of phenylalanine, an amino acid, on graphitic carbon structures, such as graphene, nanotubes, and their modified structures. We focus especially on the effect of the adsorbate on the geometrical and the electronic structures of the absorbents. The phenylalanine molecule is found to bind weakly on pristine graphitic structures with a binding energy of 40-70 meV and not to change the electronic configuration of the graphitic structures, implying that the phenylalanine molecule may not be detected on pristine graphitic structures. On the other hand, the phenylalanine molecule exhibits a substantial increase in its binding energy up to ~2.60 eV on the magnesium-decorated boron-doped graphitic structures. We discover that the Fermi level of the system, which was shifted below the Dirac point of the graphitic structures due to p-doping by boron substitution, can be completely restored to the Dirac point because of the amino acid adsorption. This behavior implies that such modified structures can be utilized to detect phenylalanine molecules.

  7. Plasma amino-acid patterns in liver disease.

    PubMed Central

    Morgan, M Y; Marshall, A W; Milsom, J P; Sherlock, S

    1982-01-01

    Plasma amino-acid concentrations were measured in 167 patients with liver disease of varying aetiology and severity, all free of encephalopathy, and the results compared with those in 57 control subjects matched for age and sex. In the four groups of patients with chronic liver disease (26 patients with chronic active hepatitis, 23 with primary biliary cirrhosis, 11 with cryptogenic cirrhosis, and 48 with alcoholic hepatitis +/- cirrhosis) plasma concentrations of methionine were significantly increased, while concentrations of the three branched chain amino-acids were significantly reduced. In the first three groups of patients plasma concentrations of aspartate, serine, and one or both of the aromatic amino-acids tyrosine and phenylalanine were also significantly increased, while in the patients with alcoholic hepatitis +/- cirrhosis plasma concentrations of glycine, alanine, and phenylalanine were significantly reduced. In the three groups of patients with minimal, potentially reversible liver disease (31 patients with alcoholic fatty liver, 10 with viral hepatitis, and 18 with biliary disease) plasma concentrations of proline and the three branched chain amino-acids were significantly reduced. Patients with alcoholic fatty liver also showed significantly reduced plasma phenylalanine values. Most changes in plasma amino-acid concentrations in patients with chronic liver disease may be explained on the basis of impaired hepatic function, portal-systemic shunting of blood, and hyperinsulinaemia and hyperglucagonaemia. The changes in patients with minimal liver disease are less easily explained. PMID:7076013

  8. Laser Desorption Supersonic Jet Spectroscopy of Hydrated Tyrosine

    NASA Astrophysics Data System (ADS)

    Oba, Hikari; Shimozono, Yoko; Ishiuchi, Shun-Ichi; Fujii, Masaaki; Carcabal, Pierre

    2013-06-01

    The structure of tyrosine (tyr) consists of amino-acid chain and phenol, and it has roughly two possible binding sites for water, amino-acid site and phenolic OH site. Investigating how water molecule binds to tyr will give fundamental information for hydrations of peptide and protein. Resonance enhanced multi photon ionization (REMPI) spectrum of tyr-water 1:1 cluster has already been reported by de Vries and co-workers, however, no analysis on the hydrated structures has been reported. In the REMPI spectrum, two clusters of bands are observed; one appears at ˜35600 cm^{-1} energy region which is the almost same with 0-0 transitions of tyr monomer, and another is observed at ˜300 cm^{-1} lower than the former. Based on the electronic transition energy of phenylalanine and the hydrated clusters, the former is expected to be derived from a structure that water binds to amino acid site. On the other hand, it is plausibly predicted that the latter originates from a structure that water binds to phenolic OH group, because the electronic transition of mono hydrated phenol is ˜300 cm^{-1} red-shifted from the monomer. We applied IR dip spectroscopy which can measure conformer selective IR spectra to the tyr-(H_{2}O)_{1} clusters by using laser desorption supersonic jet technique to confirm the assignments. Especially in the phenolic OH bound isomer, it was found that the intra molecular hydrogen bond within amino-acid chain, which is far from the water molecule and cannot interact directly with each other, is strengthened by the hydration. A. Abio-Riziq et al., J. Phys. Chem. A, 115, 6077 (2011). Y. Shimozono, et al., Phys. Chem. Chem. Phys., (2013) DOI: 10.1039/c3cp43573c. T. Ebata et al., Phys. Chem. Chem. Phys., 8, 4783 (2006). T. Watanabe et al., J. Chem. Phys., 105, 408 (1996).

  9. [Characteristics of the effect of gamma-irradiation on the amino acid composition of collagen as modified by a gaseous atmosphere].

    PubMed

    Duzhenkova, N A; Savich, A V

    1986-01-01

    The comparative changes in the amino acid composition of calf skin collagen after gamma-irradiation (doses from 100 to 1,000 Gy) in aqueous solutions under different gas atmospheres (O2, N2O, H2, vacuum) were investigated. The radiochemical yields of collagen amino acid residues destruction were determined. Under O2 (OH X, O2-) most of amino acids are destroyed with higher yields than under N2O. Leucine, valine, isoleucine, phenylalanine, arginine were the exception because of their high reaction rate constants with OH X and hydroxylation reactions. Under H2 (e-aq, H) and in vacuum (e-aq, OH X) the mechanism of collagen radiolysis changed due to its aggregation; the destruction of those amino acids which have high reaction rate constants with water radiolysis products was mainly observed (phenylalanine, tyrosine, histidine). PMID:3774972

  10. Phenylalanine Binding Is Linked to Dimerization of the Regulatory Domain of Phenylalanine Hydroxylase

    PubMed Central

    2015-01-01

    Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer–dimer equilibrium with a dissociation constant of ∼46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme. PMID:25299136

  11. Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

    PubMed

    Strobel, H J; Russell, J B; Driessen, A J; Konings, W N

    1989-01-01

    Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors. PMID:2492498

  12. Preparation of l-phenylalanine-imprinted solid-phase extraction sorbent by Pickering emulsion polymerization and the selective enrichment of l-phenylalanine from human urine.

    PubMed

    Li, Ji; Hu, Xiaoling; Guan, Ping; Zhang, Xiaoyan; Qian, Liwei; Zhang, Nan; Du, Chunbao; Song, Renyuan

    2016-05-01

    A novel l-phenylalanine molecularly imprinted solid-phase extraction sorbent was synthesized by the combination of Pickering emulsion polymerization and ion-pair dummy template imprinting. Compared to other polymerization methods, the molecularly imprinted polymers thus prepared exhibit a high specific surface, large pore diameter, and appropriate particle size. The key parameters for solid-phase extraction were optimized, and the result indicated that the molecularly imprinted polymer thus prepared exhibits a good recovery of 98.9% for l-phenylalanine. Under the optimized conditions of the procedure, an analytical method for l-phenylalanine was well established. By comparing the performance of the molecularly imprinted polymer and a commercial reverse-phase silica gel, the obtained molecularly imprinted polymer as an solid-phase extraction sorbent is more suitable, exhibiting high precision (relative standard deviation 3.2%, n = 4) and a low limit of detection (60.0 ± 1.9 nmol·L(-1) ) for the isolation of l-phenylalanine. Based on these results, the combination of the Pickering emulsion polymerization and ion-pair dummy template imprinting is effective for preparing selective solid-phase extraction sorbents for the separation of amino acids and organic acids from complex biological samples. PMID:26991761

  13. Isotopic exchange of hydrogen in aromatic amino acids

    SciTech Connect

    Pshenichnikova, A.B.; Karnaukhova, E.N.; Mitsner, B.I.

    1993-10-20

    The kinetics of the isotopic replacement of hydrogen in the aromatic amino acids L-tryptophan, L-tyrosine, and L-phenylalanine in solutions of deuterochloric and deuterosulfuric acids in deuterium oxide were investigated by PMR spectroscopy. The reactions were shown to be of first orders with respect both to the concentration of the substrate and to the activity of the deuterium ion. The isotopic effects of hydrogen and the values of the activation energy of H-D exchange in different positions of the aromatic ring in tryptophan and tyrosine were determined. The effect of properties of the medium on the rate of the isotopic exchange of hydrogen is discussed. 17 refs., 2 figs., 2 tabs.

  14. 21 CFR 582.5590 - Phenylalanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Phenylalanine. 582.5590 Section 582.5590 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  15. 21 CFR 582.5590 - Phenylalanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Phenylalanine. 582.5590 Section 582.5590 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  16. Bioinspired bioadhesive polymers: dopa-modified poly(acrylic acid) derivatives.

    PubMed

    Laulicht, Bryan; Mancini, Alexis; Geman, Nathanael; Cho, Daniel; Estrellas, Kenneth; Furtado, Stacia; Hopson, Russell; Tripathi, Anubhav; Mathiowitz, Edith

    2012-11-01

    The one-step synthesis and characterization of novel bioinspired bioadhesive polymers that contain Dopa, implicated in the extremely adhesive byssal fibers of certain gastropods, is reported. The novel polymers consist of combinations of either of two polyanhydride backbones and one of three amino acids, phenylalanine, tyrosine, or Dopa, grafted as side chains. Dopa-grafted hydrophobic backbone polymers exhibit as much as 2.5 × the fracture strength and 2.8 × the tensile work of bioadhesion of a commercially available poly(acrylic acid) derivative as tested on live, excised, rat intestinal tissue. PMID:23008096

  17. A salicylic acid-based small molecule inhibitor for the oncogenic Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2)

    PubMed Central

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-01-01

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias and solid tumors. Thus there is considerable interest in SHP2 as a potential target for anti-cancer and anti-leukemia therapy. We report a salicylic acid-based combinatorial library approach aimed to bind both active site and unique nearby sub-pockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anti-cancer and anti-leukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors. PMID:20170098

  18. Small activating ribonucleic acid reverses tyrosine kinase inhibitor resistance in epidermal growth factor receptor‐mutant lung cancer by increasing the expression of phosphatase and tensin homolog

    PubMed Central

    Li, Meng; Peng, Zhongmin; Ren, Wangang

    2016-01-01

    Background Epidermal growth factor receptor‐tyrosine kinase inhibitors (TKI‐EGFRs) present a new prospect for the treatment of lung cancer. However, in clinical application, the majority of patients become TKI resistant within a year. More and more studies have shown that a loss of phosphatase and tensin homolog (PTEN) expression is associated with TKI resistance. An alternative method of upregulating PTEN expression may reverse TKI resistance. Methods We designed five candidate small activating ribonucleic acids (saRNAs) to target PTEN, and transfected them into H‐157 cells to screen out functional saRNA. We used reverse transcriptase‐polymerase chain reaction and Western blot to evaluate the effect of saRNA to PTEN expression. We then analyzed the growth and apoptosis of cells transfected with saRNA under the treatment of TKI to investigate whether saRNAs can reverse TKI resistance by upregulating PTEN expression. Results The functional saRNA we designed could upregulate PTEN expression. The H‐157 cells transfected with saRNA grew slower in the presence of TKI drugs than the cells that were not transfected with saRNA. The apoptosis rate was also obviously higher. Conclusions Our study proves that loss of PTEN expression is an important mechanism of TKI resistance. It is possible to control TKI resistance by upregulating PTEN expression using RNA activation technology. PMID:27385992

  19. New platform for controlled and sustained delivery of the EGF receptor tyrosine kinase inhibitor AG1478 using poly(lactic-co-glycolic acid) microspheres

    PubMed Central

    Robinson, Rebecca; Bertram, James P.; Reiter, Jill L.; Lavik, Erin B.

    2015-01-01

    Inhibition of the epidermal growth factor receptor (EGFR) has been shown to reduce tumor growth and metastases and promote axon regeneration in the central nervous system. Current strategies for inhibiting EGFR include the administration of reversible or irreversible small-molecule tyrosine kinase inhibitors (TKIs). However, to be effective in vivo constant and sustained delivery is required. This study explored the feasibility of encapsulating the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) in poly(lactic-co-glycolic acid) (PLGA) microspheres to achieve sustained delivery of the TKI. We characterized microspheres prepared using three different emulsion methods: solid-in-oil-in-water, oil-in-water, and oil-in-water with co-solvent. Addition of a co-solvent increased the loading and release of AG1478, and significantly (P<0.001) decreased the size of the microspheres which facilitates administration of the spheres. On average, sustained delivery of AG1478 from microspheres was achieved for six months. However, the addition of a co-solvent prolonged release for over nine months (266 days). In addition, AG1478 retained its bioactivity upon delivery, and inhibited EGFR in both immortalized rat fibroblasts and in EGFR-amplified human carcinoma cells. These results demonstrate that AG1478 can be encapsulated in PLGA and retain bioactivity; thereby providing a new platform for controlled administration of EGFR TKIs. PMID:20055747

  20. Construction and evaluation of a novel bifunctional phenylalanine-formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration.

    PubMed

    Jiang, Wei; Fang, Bai-Shan

    2016-05-01

    Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of L-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of L-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH-FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5-9.0). The bifunctional enzyme can produce 153.9 mM L-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to L-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH-FDH with coenzyme regeneration for phenylpyruvic acid analysis and L-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field. PMID:26819086

  1. Effect of L-aspartyl-L-phenylalanine methyl ester on leukotriene biosynthesis in macrophage cells.

    PubMed

    Hardcastle, J E; Bruch, R J

    1997-09-01

    Macrophage cells treated with L-aspartyl-L-phenylalanine methyl ester (aspartame) produced leukotrienes and other arachidonic acid metabolites. Leukotriene C4, leukotriene B4, and 15-hydroxyeicosatetraenoic acid were the major metabolites detected. The aspartame-treated macrophage cell cultures produced three times as much arachidonic acid metabolites as did the untreated control cell cultures. Leukotriene C4 was produced in the largest amount by the aspartame-treated cells. PMID:9384524

  2. A new physiological role for Pdr12p in Saccharomyces cerevisiae: export of aromatic and branched-chain organic acids produced in amino acid catabolism.

    PubMed

    Hazelwood, Lucie A; Tai, Siew Leng; Boer, Viktor M; de Winde, Johannes H; Pronk, Jack T; Daran, Jean Marc

    2006-09-01

    Saccharomyces cerevisiae can use a broad range of compounds as sole nitrogen source. Many amino acids, such as leucine, tyrosine, phenylalanine and methionine, are utilized through the Ehrlich pathway. The fusel acids and alcohols produced from this pathway, along with their derived esters, are important contributors to beer and wine flavor. It is unknown how these compounds are exported from the cell. Analysis of nitrogen-source-dependent transcript profiles via microarray analysis of glucose-limited, aerobic chemostat cultures revealed a common upregulation of PDR12 in cultures grown with leucine, methionine or phenylalanine as sole nitrogen source. PDR12 encodes an ABC transporter involved in weak-organic-acid resistance, which has hitherto been studied in the context of resistance to exogenous organic acids. The hypothesis that PDR12 is involved in export of natural products of amino acid catabolism was evaluated by analyzing the phenotype of null mutants in PDR12 or in WAR1, its positive transcriptional regulator. The hypersensitivity of the pdr12Delta and war1Delta strains for some of these compounds indicates that Pdr12p is involved in export of the fusel acids, but not the fusel alcohols derived from leucine, isoleucine, valine, phenylalanine and tryptophan. PMID:16911515

  3. A study of placental transfer mechanisms in nonhuman primates using (/sup 14/C)phenylalanine

    SciTech Connect

    Pueschel, S.M.; Boylan, J.M.; Jackson, B.T.; Piasecki, G.J.

    1982-02-01

    Placental transfer mechanisms were investigated in pregnant Macaca Fascicularis and Macaca mulatta during the gestational age of 120 to 130 days. These primates underwent an operative procedure that allowed continuous fetal blood sampling. The administration of (/sup 14/C)phenylalanine into the maternal circulation revealed a significant increase of radioactive material in the fetal circulation, indicating an active placental transport mechanism unidirectional to the fetus. When (/sup 14/C)phenylalanine was injected into the fetus, radioactive aromatic amino acids in the maternal circulation increased only slightly over time, resembling a simple diffusion process.

  4. The influence of the cell free solution of lactic acid bacteria on tyramine production by food borne-pathogens in tyrosine decarboxylase broth.

    PubMed

    Toy, Nurten; Özogul, Fatih; Özogul, Yesim

    2015-04-15

    The function of cell-free solutions (CFSs) of lactic acid bacteria (LAB) on tyramine and other biogenic amine production by different food borne-pathogens (FBPs) was investigated in tyrosine decarboxylase broth (TDB) using HPLC. Cell free solutions were prepared from four LAB strains. Two different concentrations which were 50% (5 ml CFS+5 ml medium/1:1) and 25% (2.5 ml CFS+7.5 ml medium/1:3) CFS and the control without CFS were prepared. Both concentration of CFS of Streptococcus thermophilus and 50% CFS of Pediococcus acidophilus inhibited tyramine production up to 98% by Salmonella paratyphi A. Tyramine production by Escherichia coli was also inhibited by 50% CFS of Lactococcus lactis subsp. lactis and 25% CFS of Leuconostoc lactis. subsp. cremoris. The inhibitor effect of 50% CFS of P. acidophilus was the highest on tyramine production (55%) by Listeria monocytogenes, following Lc. lactis subsp. lactis and Leuconostoc mesenteroides subsp. cremoris (20%) whilst 25% CFS of Leu. mes. subsp. cremoris and Lc. lactis subsp. lactis showed stimulator effects (160%). The stimulation effects of 50% CFS of S. thermophilus and Lc. lactis subsp. lactis were more than 70% by Staphylococcus aureus comparing to the control. CFS of LAB strains showed statistically inhibitor effect since lactic acid inhibited microbial growth, decreased pH quickly and reduced the formation of AMN and BAs. Consequently, in order to avoid the formation of high concentrations of biogenic amines in fermented food by bacteria, it is advisable to use CFS for food and food products. PMID:25465993

  5. Biphenylsulfonacetic Acid Inhibitors of the Human Papillomavirus Type 6 E1 Helicase Inhibit ATP Hydrolysis by an Allosteric Mechanism Involving Tyrosine 486

    PubMed Central

    White, Peter W.; Faucher, Anne-Marie; Massariol, Marie-Josée; Welchner, Ewald; Rancourt, Jean; Cartier, Mireille; Archambault, Jacques

    2005-01-01

    Human papillomaviruses (HPVs) are the causative agents of benign and malignant lesions of the epithelium. Despite their high prevalence, there is currently no antiviral drug for the treatment of HPV-induced lesions. The ATPase and helicase activities of the highly conserved E1 protein of HPV are essential for viral DNA replication and pathogenesis and hence are considered valid antiviral targets. We recently described novel biphenylsulfonacetic acid inhibitors of the ATPase activity of E1 from HPV type 6 (HPV6). Based on kinetics and mutagenesis studies, we now report that these compounds act by an allosteric mechanism. They are hyperbolic competitive inhibitors of the ATPase activity of HPV6 E1 and also inhibit its helicase activity. Compounds in this series can also inhibit the ATPase activity of the closely related enzyme from HPV11; however, the most potent inhibitors of HPV6 E1 are significantly less active against the type 11 protein. We identified a single critical residue in HPV6 E1, Tyr-486, substituted by a cysteine in HPV11, which is primarily responsible for this difference in inhibitor potency. Interestingly, HPV18 E1, which also has a tyrosine at this position, could be inhibited by biphenylsulfonacetic acid derivatives, thereby raising the possibility that this class of inhibitors could be optimized as antiviral agents against multiple HPV types. These studies implicate Tyr-486 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts. PMID:16304143

  6. Effect of total hepatectomy and administration of branched-chain amino acids on regional norepinephrine, dopamine, and amino acids in rat brain.

    PubMed Central

    Herlin, P M; James, J H; Nachbauer, C A; Fischer, J E

    1983-01-01

    In rats after total hepatectomy, the effect of infusing glucose alone or combined with branched-chain amino acids on amino acid concentrations in plasma and cerebral cortex and on catecholamine levels in eight different regions of the brain was studied. Infusion of branched-chain amino acids reduced the accumulation of tryptophan, phenylalanine, and tyrosine in plasma, while in cerebral cortex, the concentrations of phenylalanine and tyrosine were normalized and that of tryptophan was reduced greatly. In rats with hepatectomy and glucose infusion alone, norepinephrine levels were decreased in seven of eight brain regions with the exception of striatum, while dopamine was reduced significantly in striatum only. Infusion of branched-chain amino acids resulted in higher norepinephrine in cortex, mesencephalon, and hypothalamus and higher striatal dopamine 18 hours after hepatectomy. Thus, infusing branched-chain amino acids and reducing the accumulation of various neutral amino acids in brain may partially prevent the loss of norepinephrine from brain after total hepatectomy. PMID:6870374

  7. Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

    PubMed Central

    Yun, Yeo Hong; Koo, Ja Sun

    2015-01-01

    Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi. PMID:26539050

  8. Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes.

    PubMed

    Yun, Yeo Hong; Koo, Ja Sun; Kim, Seong Hwan; Kong, Won Sik

    2015-09-01

    Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi. PMID:26539050

  9. Production and applications of rosmarinic acid and structurally related compounds.

    PubMed

    Kim, Gun-Dong; Park, Yong Seek; Jin, Young-Ho; Park, Cheung-Seog

    2015-03-01

    Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a naturally occurring hydroxylated compound commonly found in species of the subfamily Nepetoideae of the Lamiaceae and Boraginaceae, such as Rosmarinus officinalis, Salvia officinalis, and Perilla frutescens. RA is biosynthesized from the amino acids L-phenylalanine and L-tyrosine by eight enzymes that include phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. RA can also be chemically produced by the esterification of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA and its numerous derivatives containing one or two RA with other aromatic moieties are well known and include lithospermic acid, yunnaneic acid, salvianolic acid, and melitric acid. Recently, RA and its derivatives have attracted interest for their biological activities, which include anti-inflammatory, anti-oxidant, anti-angiogenic, anti-tumor, and anti-microbial functions. Clinically, RA attenuates T cell receptor-mediated signaling, attenuates allergic diseases like allergic rhinitis and asthma, and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like symptoms, protects from neurotoxicity, and slows the development of Alzheimer's disease. These attributes have increased the demand for the biotechnological production and application of RA and its derivatives. The present review discusses the function and application of RA and its derivatives including the molecular mechanisms underlying clinical efficacy. PMID:25620368

  10. Biosynthesis and Metabolic Fate of Phenylalanine in Conifers.

    PubMed

    Pascual, María B; El-Azaz, Jorge; de la Torre, Fernando N; Cañas, Rafael A; Avila, Concepción; Cánovas, Francisco M

    2016-01-01

    The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined. PMID:27468292

  11. Biosynthesis and Metabolic Fate of Phenylalanine in Conifers

    PubMed Central

    Pascual, María B.; El-Azaz, Jorge; de la Torre, Fernando N.; Cañas, Rafael A.; Avila, Concepción; Cánovas, Francisco M.

    2016-01-01

    The amino acid phenylalanine (Phe) is a critical metabolic node that plays an essential role in the interconnection between primary and secondary metabolism in plants. Phe is used as a protein building block but it is also as a precursor for numerous plant compounds that are crucial for plant reproduction, growth, development, and defense against different types of stresses. The metabolism of Phe plays a central role in the channeling of carbon from photosynthesis to the biosynthesis of phenylpropanoids. The study of this metabolic pathway is particularly relevant in trees, which divert large amounts of carbon into the biosynthesis of Phe-derived compounds, particularly lignin, an important constituent of wood. The trunks of trees are metabolic sinks that consume a considerable percentage of carbon and energy from photosynthesis, and carbon is finally immobilized in wood. This paper reviews recent advances in the biosynthesis and metabolic utilization of Phe in conifer trees. Two alternative routes have been identified: the ancient phenylpyruvate pathway that is present in microorganisms, and the arogenate pathway that possibly evolved later during plant evolution. Additionally, an efficient nitrogen recycling mechanism is required to maintain sustained growth during xylem formation. The relevance of phenylalanine metabolic pathways in wood formation, the biotic interactions, and ultraviolet protection is discussed. The genetic manipulation and transcriptional regulation of the pathways are also outlined. PMID:27468292

  12. Synthesis and characterization of nonconventional surfactants of aromatic amino acid-glycerol ethers: effect of the amino acid moiety on the orientation and surface properties of these soap-type amphiphiles.

    PubMed

    Varka, Evdoxia-Maria A; Heli, Maria G; Coutouli-Argyropoulou, Evdoxia; Pegiadou, Sofia A

    2006-11-01

    The synthesis, characterization, and surface properties of soap-type amphiphiles comprising alkyl chains of 10-16 carbon atoms linked through an ether group to a glycerol-amino acid hydrophilic head group is described. The surface properties of members of this series derived from histidine and tyrosine were compared with those of phenylalanine and tryptophan derivatives described previously and with those of conventional soaps. In all cases, the amino acid derivatives showed superior surface properties, and an interesting differentiation was discovered regarding the orientation of tryptophan derivatives. PMID:16847987

  13. Plasma Amino Acid Concentrations Predict Mortality in Patients with End-Stage Liver Disease

    PubMed Central

    Kinny-Köster, Benedict; Bartels, Michael; Becker, Susen; Scholz, Markus; Thiery, Joachim

    2016-01-01

    Background The liver plays a key role in amino acid metabolism. In former studies, a ratio between branched-chain and aromatic amino acids (Fischer’s ratio) revealed associations with hepatic encephalopathy. Furthermore, low concentrations of branched-chain amino acids were linked to sarcopenia in literature. Encephalopathy and sarcopenia are known to dramatically worsen the prognosis. Aim of this study was to investigate a complex panel of plasma amino acids in the context of mortality in patients with end-stage liver disease. Methods 166 patients evaluated for orthotopic liver transplantation were included. 19 amino acids were measured from citrated plasma samples using mass spectrometry. We performed survival analysis for plasma amino acid constellations and examined the relationship to established mortality predictors. Results 33/166 (19.9%) patients died during follow-up. Lower values of valine (p<0.001), Fischer’s ratio (p<0.001) and valine to phenylalanine ratio (p<0.001) and higher values of phenylalanine (p<0.05) and tyrosine (p<0.05) were significantly associated with mortality. When divided in three groups, the tertiles discriminated cumulative survival for valine (p = 0.016), phenylalanine (p = 0.024) and in particular for valine to phenylalanine ratio (p = 0.003) and Fischer’s ratio (p = 0.005). Parameters were also significantly correlated with MELD and MELD-Na score. Conclusions Amino acids in plasma are valuable biomarkers to determine increased risk of mortality in patients with end-stage liver disease. In particular, valine concentrations and constellations composed of branched-chain and aromatic amino acids were strongly associated with prognosis. Due to their pathophysiological importance, the identified amino acids could be used to examine individual dietary recommendations to serve as potential therapeutic targets. PMID:27410482

  14. Carbohydrate/protein selection in a single meal correlated with plasma tryptophan and tyrosine ratios to neutral amino acids in fasting individuals.

    PubMed

    Møller, S E

    1986-01-01

    Plasma ratios of tryptophan (Trp) and tyrosine (Tyr) to their respective competing large neutral amino acids (LNAA) for brain uptake, serum insulin and plasma glucose concentrations were determined in 31 fasting healthy female subjects, and in two smaller groups of smokers and oral contraceptive users, who were subsequently allowed to compose individual breakfast meals from a selection of 25 dietary products. Additional blood samples were collected at 2 hr after the meal. Smokers consumed less carbohydrate (-22%) and total calories (-23%) and showed decreased basal serum insulin level, when compared to controls on the same age. Females on oral contraceptives consumed significantly more carbohydrate (+54%) and total calories (+32%) than comparable controls. In the 31 females there was no significant correlation between any of the biological variables and the intake of fat or total calories. The ratio of carbohydrate/protein eaten was significantly and directly correlated with age and with the sum of plasma ratios Trp/LNAA and Tyr/LNAA, and these independent variables associated with 37% of the variance in the ratio carbohydrate/protein consumed, as evaluated by multiple regression analysis. After the meal, the plasma ratio Tyr/LNAA was increased, whereas the ratio Trp/LNAA was decreased in subjects whose ratio carbohydrate/protein consumed was below the mean of the full sample, whereas subjects who consumed meals with a high ratio carbohydrate/protein showed an increase in plasma ratio Trp/LNAA. It is concluded that biological variables in man are significantly associated with the choice between nutrients with different carbohydrate and protein contents for breakfast. The changes in the plasma ratios Trp/LNAA and Tyr/LNAA after consumption were generally moderate. PMID:3797484

  15. Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements.

    PubMed

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Ferreira-Leitão, Viridiana Santana; da Silva Bon, Elba Pinto

    2012-12-01

    This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used. PMID:23103512

  16. Radiolysis of N-acetyl amino acids as model compounds for radiation degradation of polypeptides

    NASA Astrophysics Data System (ADS)

    Wayne Garrett, R.; Hill, David J. T.; Ho, Sook-Ying; O'Donnell, James H.; O'Sullivan, Paul W.; Pomery, Peter J.

    Radiation chemical yields of (i) the volatile radiolysis products and (ii) the trapped free radicals from the y-radiolysis of the N-acetyl derivatives of glycine, L-valine, L-phenylalanine and L-tyrosine in the polycrystalline state have been determined at room temperature (303 K). Carbon dioxide was found to be the major molecular product for all these compounds with G(CO 2) varying from 0.36 for N-acetyl-L-tyrosine to 8 for N-acetyl-L-valine. There was evidence for some scission of the N-C α bond, indicated by the production of acetamide and the corresponding aliphatic acid, but the determination reaction was found to be of much lesser importance than the decarboxylation reaction. A protective effect of the aromatic ring in N-acetyl-L-phenylalanine and in N-acetyl-L-tyrosine was indicated by the lower yields of volatile products for these compounds. The yields of trapped free radicals were found to vary with the nature of the amino acid side chain, increasing with chain length and chain branching. The radical yields were decreased by incorporation of an aromatic moiety in the side chain, this effect being greater for the tyrosyl side chain than for the phenyl side chain. The G(R·) values showed a good correlation with G(CO 2) indicating that a common reaction may be involved in radical production and carbon dioxide formation.

  17. Differential Effects of Low-Phenylalanine Protein Sources on Brain Neurotransmitters and Behavior in C57Bl/6-Pahenu2 Mice

    PubMed Central

    Sawin, Emily A.; Murali, Sangita G.; Ney, Denise M.

    2014-01-01

    Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase, which metabolizes phenylalanine (phe) to tyrosine. A low-phe diet plus amino acid (AA) formula is necessary to prevent cognitive impairment; glycomacropeptide (GMP) contains minimal phe and provides a palatable alternative to the AA formula. Our objective was to assess neurotransmitter concentrations in brain and the behavioral phenotype of PKU mice (Pahenu2 on the C57Bl/6 background) and how this is affected by low-phe protein sources. Wild type (WT) and PKU mice, both male and female, were fed high-phe casein, low-phe AA, or low-phe GMP diets between 3–18 weeks of age. Behavioral phenotype was assessed using the open field and marble burying tests, and brain neurotransmitter concentration measured using HPLC with electrochemical detection system. Data were analyzed by 3-way ANOVA with genotype, sex, and diet as the main treatment effects. Brain mass and the concentrations of catecholamines and serotonin were reduced in PKU mice compared to WT mice; the low-phe AA and GMP diets improved these parameters in PKU mice. Relative brain mass was increased in female PKU mice fed the GMP diet compared to the AA diet. PKU mice exhibited hyperactivity and impaired vertical exploration compared to their WT littermates during the open field test. Regardless of genotype or diet, female mice demonstrated increased vertical activity time and increased total ambulatory and horizontal activity counts compared with male mice. PKU mice fed the high-phe casein diet buried significantly fewer marbles than WT control mice fed casein; this was normalized in PKU mice fed the low-phe AA and GMP diets. In summary, C57Bl/6-Pahenu2 mice showed an impaired behavioral phenotype and reduced brain neurotransmitter concentrations that were improved by the low-phe AA or GMP diets. These data support lifelong adherence to a low-phe diet for PKU. PMID:24560888

  18. Nuclear magnetic resonance studies of phenylalanine analog interactions with normal and sicklen hemoglobin

    SciTech Connect

    Lee, Y.H.

    1985-01-01

    Several phenylalanine derivatives have been found to inhibit the gelation of deoxygenated sickle hemoglobin (deoxy HbS). Proton and /sup 19/F-NMR techniques were used to monitor the interaction of selected phenylalanine derivatives with the Hb molecule by using fluorine containing phenylalanine derivatives, Hb labeled at the ..beta..93 position with N-(2,2,2-trifluoroethyl) iodoacetamide (IA-F/sub 3/), and by monitoring the relaxation rates of the C2 and C4 histidine protons. The results show that the /sup 19/F spin-spin relaxation times of L-phenylalanin-4-fluorobenzylamide (PheNBz1-F), which has a deoxy HbS antigelling activity comparable to that of the amino acid, tryptophan, are affected much more strongly by interaction with Hb than are those of glycin-4-fluorobenzylamide (GlyNBz1-F). In contrast, it is shown that N-(2,2,5,5-tetramethylpyrrolidin-1-oxy-3-carboxyl)-L-phenylalanine t-butyl ester (SL-Phe) exhibits specific binding to Hb, and an antigelling activity more than two orders of magnitude greater than that of phenylalanine. These results indicate that the fluorine nuclei strongly influenced by the presence of spin label nitroxide are located in a conformation within a few angstroms of the SL-Phe binding site. Proton NMR relaxation measurements of the C2 and C4 proton resonances from the ..beta..2, 4b143 and ..beta..146 histidine residues show significant and selective effects from the binding of SL-Phe to Hb, indicating that the SL-Phe binding site must be close to the side chains of these three residues. The strong antigelation activity of SL-Phe suggests that this binding site may be one of the intermolecular contact sites of importance to the deoxy HbS aggregation process.

  19. Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase.

    PubMed

    Katayama, T; Suzuki, H; Koyanagi, T; Kumagai, H

    2000-11-01

    Tyrosine phenol-lyase (Tpl), which can synthesize 3, 4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that the tpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrR gene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tpl were screened for by use of the lac reporter system in E. coli. The most increased transcription of tpl was observed for the strain with the mutant tyrR allele involving amino acid substitutions of alanine, cysteine, and glycine for valine-67, tyrosine-72, and glutamate-201, respectively. A tyrR-deficient derivative of E. herbicola was constructed and transformed with a plasmid carrying the mutant tyrR allele (V67A Y72C E201G substitutions). The resultant strain expressed Tpl without the addition of tyrosine to the medium and produced as much of it as was produced by the wild-type strain grown under tyrosine-induced conditions. The regulatory properties of the mutant TyrR(V67A), TyrR(Y72C), TyrR(E201G), and TyrR(V67A Y72C E201G) proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrR(V67A) protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector. PMID:11055921

  20. The isolation and properties of phenylalanine hydroxylase from human liver

    PubMed Central

    Woo, Savio L. C.; Gillam, Shirley Su; Woolf, Louis I.

    1974-01-01

    Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The Km values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria. PMID:4854919

  1. Amino acid profile during exercise and training in Standardbreds.

    PubMed

    Westermann, C M; Dorland, L; Wijnberg, I D; de Sain-van der Velden, M G M; van Breda, E; Barneveld, A; de Graaf-Roelfsema, E; Keizer, H A; van der Kolk, J H

    2011-08-01

    The objective of this study is to assess the influence of acute exercise, training and intensified training on the plasma amino acid profile. In a 32-week longitudinal study using 10 Standardbred horses, training was divided into four phases, including a phase of intensified training for five horses. At the end of each phase, a standardized exercise test, SET, was performed. Plasma amino acid concentrations before and after each SET were measured. Training significantly reduced mean plasma aspartic acid concentration, whereas exercise significantly increased the plasma concentrations of alanine, taurine, methionine, leucine, tyrosine and phenylalanine and reduced the plasma concentrations of glycine, ornithine, glutamine, citrulline and serine. Normally and intensified trained horses differed not significantly. It is concluded that amino acids should not be regarded as limiting training performance in Standardbreds except for aspartic acid which is the most likely candidate for supplementation. PMID:20863542

  2. EFFECT OF TETRACYCLINES ON THE INTRACELLULAR AMINO ACIDS OF MOLDS.

    PubMed

    FREEMAN, B A; CIRCO, R

    1963-07-01

    Freeman, Bob A. (University of Chicago, Chicago, Ill.) and Richard Circo. Effect of tetracyclines on the intracellular amino acids of molds. J. Bacteriol. 86:38-44. 1963.-The tetracycline antibiotics were shown to alter the amino acid metabolism of molds whose growth is not markedly affected. Eight molds were grown in the presence of these antiobiotics; four exhibited a general reduction in the concentration of the intracellular amino acids, except for glutamic acid and alanine. In most of these four cultures, the tetracyclines also caused the complete disappearance of arginine, lysine, proline, phenylalanine, and tyrosine from the intracellular amino acid pool. The significance of these observations and the usefulness of the method in the study of the mechanisms of antibiotic action are discussed. PMID:14051820

  3. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  4. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids.

    PubMed

    Hesse, Almut; Weller, Michael G

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  5. Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro

    PubMed Central

    Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product. PMID:27558633

  6. Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro.

    PubMed

    Ding, Dongqin; Liu, Yongfei; Xu, Yiran; Zheng, Ping; Li, Haixing; Zhang, Dawei; Sun, Jibin

    2016-01-01

    L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product. PMID:27558633

  7. Selective separation of sodium ions from a mixture with phenylalanine by Donnan dialysis with a profiled sulfogroup cation exchange membrane

    NASA Astrophysics Data System (ADS)

    Vasil'eva, V. I.; Goleva, E. A.

    2013-11-01

    The possibility of separating ions of metal from a mixture with ampholyte (an amino acid) by Donnan dialysis with an MK-40 sulfogroup cation exchange membrane is demonstrated. Conditions ensuring the selectivity and intensity of the mass transfer of sodium ions from a mixture with bipolar phenylalanine ions into a diffusate containing hydrochloric acid through a cation exchange membrane are found.

  8. Charge Migration in Phenylalanine Initiated by Attosecond Pulses

    NASA Astrophysics Data System (ADS)

    Greenwood, Jason; Trabattoni, Andrea; Ayuso, David; Belshaw, Louise; de Camillis, Simone; Anumula, Sunil; Frassetto, Fabio; Poletto, Luca; Palacios, Alicia; Declava, Piero; Martin, Fernando; Calegari, Francesca; Nisoli, Mauro

    2015-05-01

    In the past few years attosecond techniques have been implemented for the investigation of ultrafast dynamics in molecules. The generation of isolated attosecond pulses characterized by a relatively high photon flux has opened up new possibilities in the study of molecular dynamics. We report on experimental and theoretical results of ultrafast charge dynamics in a biochemically relevant molecule, namely, the amino acid phenylalanine. The data represent the first experimental demonstration of the generation and observation of a charge migration process in a complex molecule, where electron dynamics precede nuclear motion. The application of attosecond technology to the investigation of electron dynamics in biologically relevant molecules represents a multidisciplinary work, which can open new research frontiers: those in which few femtosecond and even sub-femtosecond electron processes determine the fate of biomolecules.

  9. Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

    PubMed Central

    Dankort, D L; Wang, Z; Blackmore, V; Moran, M F; Muller, W J

    1997-01-01

    A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites. PMID:9271418

  10. Biosynthesis of the Aromatic Amino Acids.

    PubMed

    Pittard, James; Yang, Ji

    2008-09-01

    This chapter describes in detail the genes and proteins of Escherichia coli involved in the biosynthesis and transport of the three aromatic amino acids tyrosine, phenylalanine, and tryptophan. It provides a historical perspective on the elaboration of the various reactions of the common pathway converting erythrose-4-phosphate and phosphoenolpyruvate to chorismate and those of the three terminal pathways converting chorismate to phenylalanine, tyrosine, and tryptophan. The regulation of key reactions by feedback inhibition, attenuation, repression, and activation are also discussed. Two regulatory proteins, TrpR (108 amino acids) and TyrR (513 amino acids), play a major role in transcriptional regulation. The TrpR protein functions only as a dimer which, in the presence of tryptophan, represses the expression of trp operon plus four other genes (the TrpR regulon). The TyrR protein, which can function both as a dimer and as a hexamer, regulates the expression of nine genes constituting the TyrR regulon. TyrR can bind each of the three aromatic amino acids and ATP and under their influence can act as a repressor or activator of gene expression. The various domains of this protein involved in binding the aromatic amino acids and ATP, recognizing DNA binding sites, interacting with the alpha subunit of RNA polymerase, and changing from a monomer to a dimer or a hexamer are all described. There is also an analysis of the various strategies which allow TyrR in conjunction with particular amino acids to differentially affect the expression of individual genes of the TyrR regulon. PMID:26443741

  11. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  12. Aromatic amino acids as precursors of antimicrobial metabolites in Geotrichum candidum.

    PubMed

    Naz, Saima; Gueguen-Minerbe, Marielle; Cretenet, Marina; Vernoux, Jean-Paul

    2013-07-01

    Geotrichum candidum ATCC 204307 was previously found to generate phenyllactic acid (PLA) and indoleacetic acid (ILA) in complex culture media. In this study, a relationship between concentrations of PLA, ILA, and hydroxy PLA (OH-PLA) and initial concentrations of phenylalanine, tryptophan, and tyrosine, added respectively as unique sources of nitrogen in synthetic medium, was established. Phenylpyruvic acid (PPA), an intermediate compound of PLA metabolism, was able to induce not only PLA but also phenylethyl alcohol (PEA) production when used separately as initial substrate. Under pH, temperature, and salt concentrations used for cheese-making, phenylalanine was found to be the most efficient substrate for antimicrobial metabolite production. In excess of substrate, different yeast strains of Geotrichum candidum, Yarrowia lipolytica, Candida natalensis, and Candida catenulata were shown here to produce 1.6 ± 0.5-5.0 ± 0.2 mM of PLA from phenylalanine, 5.0 ± 0.1-10.9 ± 0.3 mM of ILA from tryptophan, and 1.3 ± 0.3-7.0 ± 0.02 of PLA and 0.1 ± 0.0-2.22 ± 0.09 mM of PEA from PPA. Geotrichum candidum ATCC 204307 was the highest producer. This is the first time these antimicrobial metabolites PLA, OH-PLA, ILA, and PEA are being reported as the reaction products of aromatic amino acids catabolism in G. candidum. PMID:23590565

  13. Tyrosine hydroxylase- and/or aromatic L-amino acid decarboxylase-expressing neurons in the rat arcuate nucleus: ontogenesis and functional significance.

    PubMed

    Ugrumov, M; Melnikova, V; Ershov, P; Balan, I; Calas, A

    2002-07-01

    This study has evaluated in vivo, ex vivo and in vitro the ontogenesis and functional significance of the neurons of the arcuate nucleus (AN) expressing either individual enzymes of dopamine (DA) synthesis, tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) as well as both of them in rats from the 17th embryonic day (E) till adulthood. Immunocytochemistry, image analysis, confocal microscopy, high performance liquid chromatography with electrochemical detection and radioimmunoassay were used to solve this problem. Monoenzymatic TH-containing neurons were initially observed on E18 located in the ventrolateral AN whereas the neurons expressing only AADC or both AADC and TH first appeared on E20 in the dorsomedial AN. On E21, the monoenzymatic TH- or AADC-expressing neurons comprised more than 99% of the whole neuron population expressing the DA-synthesizing enzymes. In spite of an extremely small number (<1%) of the neurons expressing both enzymes (DArgic neurons), the dissected AN (ex vivo) and its primary cell culture (in vitro) contained a surprisingly high amount of DA and L-dihydroxyphenylalanine (L-DOPA) which were released in response to membrane depolarization. Furthermore, DA production in the AN of fetuses occurred to be sufficient to provide an inhibitory control of prolactin secretion, as in adults. The above data suggest that DA could be synthesized, at least in the AN of fetuses, by monoenzymatic neurons containing either TH or AADC, in co-operation. This hypothesis may be extended to adult animals as their AN contained the same populations of the neurons expressing DA-synthesizing enzymes as in fetuses though the proportion of true DArgic neurons increased up to 38%. During ontogenesis, the monoenzymatic TH- and AADC-containing neurons established axosomatic and axo-axonal junctions that might facilitate the L-DOPA transport from the former to the latter. Moreover, the monoenzymatic AADC-expressing neurons project their axons to

  14. Organization and evolution of the rat tyrosine hydroxylase gene

    SciTech Connect

    Brown, E.R.; Coker, G.T. III; O'Malley, K.L.

    1987-08-11

    This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylases genes are members of a gene family which has a common evolutionary origin. The authors predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family.

  15. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed Central

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-01-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7741705

  16. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-05-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. PMID:7741705

  17. On the reported optical activity of amino acids in the Murchison meteorite

    NASA Technical Reports Server (NTRS)

    Bada, J. L.; Ho, M.-S.; Steinberg, S.; Cronin, J. R.; Kvenvolden, K. A.; Lawless, J. G.; Miller, S. L.; Oro, J.

    1983-01-01

    It is shown that the explanation of terrestrial contamination of the Murchison meteorite is consistent with the analysis of extracts from the meteorite reported by Engel and Nagy (EN) (1982) and is much more probable than their suggestion that the excess of L-enantiomers for several protein amino acids is due to asymmetric synthesis or decomposition. The low abundance of serine and threonine reported by EN may be due to their decomposition during the derivatization procedure, and the absence of methionine, tyrosine, and phenylalanine can be attributed to various causes. The amount of contamination in EN's extracts are estimated from a mass balance of the amino acid enantiomers, and it is found that the amino acids in the HCl could be due entirely to contamination while in the water extract the amount of contamination ranges from about 40 to 97 percent, depending on the amino acid. The argument that contaminants were preferentially extracted by EN's procedure cannot account for the failure to detect methionine, tyrosine, and phenylalanine.

  18. Alterations in amino acid concentrations in the plasma and muscle in human subjects during 24 h of simulated adventure racing.

    PubMed

    Borgenvik, Marcus; Nordin, Marie; Mikael Mattsson, C; Enqvist, Jonas K; Blomstrand, Eva; Ekblom, Björn

    2012-10-01

    This investigation was designed to evaluate changes in plasma and muscle levels of free amino acids during an ultra-endurance exercise and following recovery. Nine male ultra-endurance trained athletes participated in a 24-h standardized endurance trial with controlled energy intake. The participants performed 12 sessions of running, kayaking and cycling (4 × each discipline). Blood samples were collected before, during and after exercise, as well as after 28 h of recovery. Muscle biopsies were taken before the test and after exercise, as well as after 28 h of recovery. During the 24-h exercise, plasma levels of branched-chain (BCAA), essential amino acids (EAA) and glutamine fell 13, 14 and 19% (P < 0.05), respectively, whereas their concentrations in muscle were unaltered. Simultaneously, tyrosine and phenylalanine levels rose 38 and 50% (P < 0.05) in the plasma and 66 and 46% (P < 0.05) in muscle, respectively. After the 24-h exercise, plasma levels of BCAA were positively correlated with muscle levels of glycogen (r (2) = 0.73, P < 0.05), as was the combined concentrations of muscle tyrosine and phenylalanine with plasma creatine kinase (R (2) = 0.55, P < 0.05). Following 28-h of recovery, plasma and muscle levels of amino acids had either returned to their initial levels or were elevated. In conclusion, ultra-endurance exercise caused significant changes elevations in plasma and muscle levels of tyrosine and phenylalanine, which suggest an increase in net muscle protein breakdown during exercise. There was a reduction in plasma concentrations of EAA and glutamine during exercise, whereas no changes were detected in their muscle concentration after exercise. PMID:22350359

  19. Mis-regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase does not account for growth inhibition by phenylalanine in Agmenellum quadruplicatum.

    PubMed

    Jensen, R A; Stenmark-Cox, S; Ingram, L O

    1974-12-01

    The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l-phenylalanine

  20. Human milk nonprotein nitrogen components: changing patterns of free amino acids and urea in the course of early lactation.

    PubMed

    Harzer, G; Franzke, V; Bindels, J G

    1984-08-01

    Free amino acids and urea were analyzed in 78 human milk samples obtained during the first 5 wk of lactation from 10 mothers delivering at term. Significant differences (p less than 0.05) in the concentrations between colostral and mature milk were found for glutamic acid, glutamine, alanine, glycine, cystine, and phosphoethanolamine which increased, and with serine, phosphoserine, aspartic acid + asparagine, arginine, lysine, isoleucine, phenylalanine, proline, methionine, tryptophan, and beta-alanine which decreased. Some of these changes occurred within the first 5 days of lactation, so that differences between transitional and mature milk became negligible (glutamic acid, alanine, and serine, aspartic acid + asparagine, lysine, isoleucine, methionine, tryptophan, respectively). No significant differences between any of the three stages of lactation were found regarding the concentrations of total free amino acids, urea, taurine, threonine, valine, leucine, histidine, and tyrosine. Possible relevances for free amino acids, including nonprotein ones, in human milk are discussed. PMID:6147084

  1. 3,4-Dihydroxy-l-phenylalanine as a biomarker of oxidative damage in proteins: improved detection using cloud-point extraction and HPLC.

    PubMed

    McPherson, Peter A C; Türemen, Bryn T

    2014-09-26

    Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker for oxidized proteins is protein-bound 3,4-dihydroxyl-l-phenylalanine (l-DOPA), which is formed on 3'-hydroxylation of tyrosine residues by hydroxyl radicals. Existing methodologies to measure protein-bound l-DOPA employ lengthy acid hydrolysis steps (ca. 16h) which may cause artifactual protein oxidation, followed by HPLC with detection based on the intrinsic fluorescence of l-DOPA. We report a novel method for the measurement of protein-bound l-DOPA which involves rapid hydrolysis followed by pre-column concentration of 6-aminoquinolyl-derivatives using cloud-point extraction. The derivatized material is resolved by reversed-phase HPLC in less than 30min and has derivatization chemistry compatible with both UV and fluorescent detection, providing detection down to the femtomole level. The method provides identical results to those found with highly specific ELISA-based techniques and requires only basic instrumentation. The stability of the 6-aminoquinolyl-derivatives together with the fast and sensitive nature of the assay will be appealing to those who require large sample throughput. PMID:25157806

  2. The Effect of Copper And Zinc Nanoparticles on the Growth Parameters, Contents of Ascorbic Acid, and Qualitative Composition of Amino Acids and Acylcarnitines in Pistia stratiotes L. (Araceae)

    NASA Astrophysics Data System (ADS)

    Olkhovych, Olga; Volkogon, Mykola; Taran, Nataliya; Batsmanova, Lyudmyla; Kravchenko, Inna

    2016-04-01

    The paper covers the research of copper and zinc nanoparticle effect on the content of ascorbic acid, and quantitative and qualitative composition of amino acids and acylcarnitines in Pistia stratiotes L. plants. Plant exposition to copper nanoparticles led to the decrease in (1) the amount of ascorbic acid, (2) the total content of amino acids (by 25 %), and (3) the amount of all studied amino acids except for the glycine amino acid. At this, the amount of 5-oxoproline, arginine, leucine, ornithine, phenylalanine, proline, serine, and tyrosine was two times lower than in control plants. The reduction of the contents of 8 out of 12 investigated acylcarnitines (namely C0, C2, C3, C5, C6, C8, C16, C18:1) was observed in plants under the influence of copper nanoparticles. The result of plants incubation with zinc nanoparticles was the decrease in (1) the amount of ascorbic acid, (2) the total content of amino acids (by 15 %), (3) the content of leucine, methionine, phenylalanine, proline, and tyrosine (more than twice), and (4) the content of 10 acylcarnitines (C0, C2, C3, C4, C5, C10, C16, C18, C18:1, C18:2). The observed reduction in amino acid contents may negatively affect plants adaptive reactions associated with de novo synthesis of stress proteins. At the same time, the decrease in the content of acylcarnitines, responsible for fatty acid transportation, may lead to the changes in the activity and direction of lipid metabolism in plants and reduce plant's ability to use free fatty acids as the oxidation substrate for cell reparation.

  3. The Effect of Copper And Zinc Nanoparticles on the Growth Parameters, Contents of Ascorbic Acid, and Qualitative Composition of Amino Acids and Acylcarnitines in Pistia stratiotes L. (Araceae).

    PubMed

    Olkhovych, Olga; Volkogon, Mykola; Taran, Nataliya; Batsmanova, Lyudmyla; Kravchenko, Inna

    2016-12-01

    The paper covers the research of copper and zinc nanoparticle effect on the content of ascorbic acid, and quantitative and qualitative composition of amino acids and acylcarnitines in Pistia stratiotes L. plants. Plant exposition to copper nanoparticles led to the decrease in (1) the amount of ascorbic acid, (2) the total content of amino acids (by 25 %), and (3) the amount of all studied amino acids except for the glycine amino acid. At this, the amount of 5-oxoproline, arginine, leucine, ornithine, phenylalanine, proline, serine, and tyrosine was two times lower than in control plants. The reduction of the contents of 8 out of 12 investigated acylcarnitines (namely C0, C2, C3, C5, C6, C8, C16, C18:1) was observed in plants under the influence of copper nanoparticles. The result of plants incubation with zinc nanoparticles was the decrease in (1) the amount of ascorbic acid, (2) the total content of amino acids (by 15 %), (3) the content of leucine, methionine, phenylalanine, proline, and tyrosine (more than twice), and (4) the content of 10 acylcarnitines (C0, C2, C3, C4, C5, C10, C16, C18, C18:1, C18:2). The observed reduction in amino acid contents may negatively affect plants adaptive reactions associated with de novo synthesis of stress proteins. At the same time, the decrease in the content of acylcarnitines, responsible for fatty acid transportation, may lead to the changes in the activity and direction of lipid metabolism in plants and reduce plant's ability to use free fatty acids as the oxidation substrate for cell reparation. PMID:27107771

  4. 21 CFR 862.1555 - Phenylalanine test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Phenylalanine test system. 862.1555 Section 862.1555 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1555 Phenylalanine test system....

  5. 21 CFR 862.1555 - Phenylalanine test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Phenylalanine test system. 862.1555 Section 862.1555 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1555 Phenylalanine test system....

  6. 21 CFR 862.1555 - Phenylalanine test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Phenylalanine test system. 862.1555 Section 862.1555 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1555 Phenylalanine test system....

  7. 21 CFR 862.1555 - Phenylalanine test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Phenylalanine test system. 862.1555 Section 862.1555 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1555 Phenylalanine test system....

  8. Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.

    PubMed

    Utreras, Elías; Jiménez-Mateos, Eva Maria; Contreras-Vallejos, Erick; Tortosa, Elena; Pérez, Mar; Rojas, Sebastián; Saragoni, Lorena; Maccioni, Ricardo B; Avila, Jesús; González-Billault, Christian

    2008-01-01

    Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration. PMID:18075266

  9. Multiple tyrosine metabolites are GPR35 agonists

    PubMed Central

    Deng, Huayun; Hu, Haibei; Fang, Ye

    2012-01-01

    Both kynurenic acid and 2-acyl lysophosphatidic acid have been postulated to be the endogenous agonists of GPR35. However, controversy remains whether alternative endogenous agonists exist. The molecular targets accounted for many nongenomic actions of thyroid hormones are mostly unknown. Here we report the agonist activity of multiple tyrosine metabolites at the GPR35. Tyrosine metabolism intermediates that contain carboxylic acid and/or catechol functional groups were first selected. Whole cell dynamic mass redistribution (DMR) assays enabled by label-free optical biosensor were then used to characterize their agonist activity in native HT-29. Molecular assays including β-arrestin translocation, ERK phosphorylation and receptor internalization confirmed that GPR35 functions as a receptor for 5,6-dihydroxyindole-2-carboxylic acid, 3,3′,5′-triiodothyronine, 3,3′,5-triiodothyronine, gentisate, rosmarinate, and 3-nitrotyrosine. These results suggest that multiple tyrosine metabolites are alternative endogenous ligands of GPR35, and GPR35 may represent a druggable target for treating certain diseases associated with abnormality of tyrosine metabolism. PMID:22523636

  10. Genetic Engineering Activates Biosynthesis of Aromatic Fumaric Acid Amides in the Human Pathogen Aspergillus fumigatus

    PubMed Central

    Kalb, Daniel; Heinekamp, Thorsten; Lackner, Gerald; Scharf, Daniel H.; Dahse, Hans-Martin; Brakhage, Axel A.

    2014-01-01

    The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both l-tyrosine and l-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-l-tyrosine and fumaryl-l-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds. PMID:25527545

  11. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

    PubMed Central

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

    2016-01-01

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

  12. OMEGA-3 LONG-CHAIN POLYUNSATURATED FATTY ACIDS IN OLDER CHILDREN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenylketonuria (PKU), the most prevalent inborn error of metabolism, is usually secondary to low hepatic activity of phenylalanine hydroxylase, the enzyme that catalyzes conversion of phenylalanine to tyrosine. Growth and development of infants and children with PKU who are managed by mandatory n...

  13. Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

    NASA Astrophysics Data System (ADS)

    Sarma, Bani Kanta

    2013-09-01

    The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by nO → σ*S-OH orbital interactions, which force the -OH group to adopt a position trans to the S⋯O interaction, leading to an almost linear arrangement of the O⋯S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S⋯N or S⋯O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

  14. Effect of muscle glycogen on glucose, lactate and amino acid metabolism during exercise and recovery in human subjects

    PubMed Central

    Blomstrand, Eva; Saltin, Bengt

    1999-01-01

    Eight subjects performed two-legged exercise, one leg with low and the other with normal muscle glycogen content. The purpose was to study the effect of low initial muscle glycogen content on the metabolic response during 1 h of exercise and 2 h of recovery. This model allows direct comparison of net fluxes of substrates and metabolites over the exercising legs receiving the same arterial inflow.Muscle glycogen breakdown during exercise was 60% lower in the leg with a reduced pre-exercise glycogen concentration and the rate of glucose uptake during exercise was 30% higher.The amount of pyruvate that was oxidized during exercise was calculated to be approximately 450 mmol in the low-glycogen leg and 750 mmol in the normal-glycogen leg, which suggests more fat and amino acid oxidation in the low-glycogen leg.During exercise, there was a significant release of amino acids not metabolized in the muscle, e.g. tyrosine and phenylalanine, only from the low-glycogen leg, suggesting an increased rate of net protein degradation in this leg.The release of tyrosine and phenylalanine from the low-glycogen leg during the exercise period and the change in their muscle concentrations yield a net tyrosine and phenylalanine production rate of 1.4 and 1.5 mmol h−1, respectively. The net rate of protein degradation was then calculated to be 7–12 g h−1.The results suggest that the observed differences in metabolism between the low-glycogen and the normal-glycogen leg are induced by the glycogen level per se, since the legs received the same arterial supply of hormones and substrates. PMID:9831734

  15. Adsorption dynamics of L-glutamic acid copolymers at a heptane/water interface.

    PubMed

    Beverung, C J; Radke, C J; Blanch, H W

    1998-02-16

    Random copolymers of glutamic acid (glu-ala, glu-leu, glu-phe, glu-tyr) were employed to investigate the relationship between side chain structure and peptide charge on adsorption behavior at an oil/water boundary. Adsorption of a series of glutamate copolymers at a heptane/water interface was examined by the dynamic pendant-drop method to determine interfacial tension. Incorporation of leucine or phenylalanine into a glutamate copolymer results in greater tension reduction than incorporation of alanine or tyrosine. These effects are amplified at pH values near the isoelectric point of glutamate, where macroscopic adsorbed films of glu-leu and glu-phe exhibit gel-like properties in response to interfacial area compression. Differences in interfacial tension behavior of glu-tyr and glu-phe indicate the importance of the tyrosine p-hydroxyl group on adsorption and aggregation at the oil/water interface. PMID:9540205

  16. On Spontaneously Pulsating Proline-Phenylalanine Peptide Microfibers.

    PubMed

    Godziek, Agnieszka; Maciejowska, Anna; Talik, Ewa; Wrzalik, Roman; Sajewicz, Mieczysław; Kowalska, Teresa

    2016-01-01

    Earlier, we have collected an experimental evidence showing that low molecular weight chiral carboxylic acids (amino acids included) can spontaneously undergo an oscillatory chiral conversion and an oscillatory condensation in abiotic aqueous and non-aqueous liquid systems, stored for certain amount of time under mild external conditions. These earlier findings are summarized in the introductory part of this study. In the second part, a preliminary report is given on spontaneous pulsation of peptide microfibers in the aged proline-phenylalanine (Pro-Phe) solution in 70% aqueous acetonitrile. The experimental evidence originates from a number of advanced analytical techniques. In view of our earlier and present findings, a presumption is made that the mechanism of spontaneous pulsation (formation and decay) of Pro-Phe microfibers is directly related to the oscillatory chiral conversion and oscillatory peptidization. The entity of the discussed results pointing out to spontaneous and uncontrolled instability of peptide structures might be a bad prognostic for employing such structures in nanobiotechnology. PMID:26521956

  17. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat

    PubMed Central

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn’t result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  18. Genomic Characterization of Phenylalanine Ammonia Lyase Gene in Buckwheat.

    PubMed

    Thiyagarajan, Karthikeyan; Vitali, Fabio; Tolaini, Valentina; Galeffi, Patrizia; Cantale, Cristina; Vikram, Prashant; Singh, Sukhwinder; De Rossi, Patrizia; Nobili, Chiara; Procacci, Silvia; Del Fiore, Antonella; Antonini, Alessandro; Presenti, Ombretta; Brunori, Andrea

    2016-01-01

    Phenylalanine Ammonia Lyase (PAL) gene which plays a key role in bio-synthesis of medicinally important compounds, Rutin/quercetin was sequence characterized for its efficient genomics application. These compounds possessing anti-diabetic and anti-cancer properties and are predominantly produced by Fagopyrum spp. In the present study, PAL gene was sequenced from three Fagopyrum spp. (F. tataricum, F. esculentum and F. dibotrys) and showed the presence of three SNPs and four insertion/deletions at intra and inter specific level. Among them, the potential SNP (position 949th bp G>C) with Parsimony Informative Site was selected and successfully utilised to individuate the zygosity/allelic variation of 16 F. tataricum varieties. Insertion mutations were identified in coding region, which resulted the change of a stretch of 39 amino acids on the putative protein. Our Study revealed that autogamous species (F. tataricum) has lower frequency of observed SNPs as compared to allogamous species (F. dibotrys and F. esculentum). The identified SNPs in F. tataricum didn't result to amino acid change, while in other two species it caused both conservative and non-conservative variations. Consistent pattern of SNPs across the species revealed their phylogenetic importance. We found two groups of F. tataricum and one of them was closely related with F. dibotrys. Sequence characterization information of PAL gene reported in present investigation can be utilized in genetic improvement of buckwheat in reference to its medicinal value. PMID:26990297

  19. Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

    PubMed

    Bazewicz, Christopher G; Liskov, Melanie T; Hines, Kevin J; Brewer, Scott H

    2013-08-01

    We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-L-phenylalanine (pN₃CH₂Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN₃CH₂Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN₃Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN₃CH₂Phe to different protein environments to be measured. The photostability of pN₃CH₂Phe was also measured relative to the photoreactive UAA, pN₃Phe. PMID:23865850

  20. Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

    SciTech Connect

    Saidha, T.; Hanfstingl, U.; Schiff, J.A. )

    1989-04-01

    Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

  1. Expanding the utility of 4-cyano-L-phenylalanine as a vibrational reporter of protein environments.

    PubMed

    Bazewicz, Christopher G; Lipkin, Jacob S; Smith, Emily E; Liskov, Melanie T; Brewer, Scott H

    2012-09-01

    The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-L-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers ((15)N, (13)C, (13)C(15)N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-L-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-L-phenylalanine. PMID:22909326

  2. The M1 family of vertebrate aminopeptidases: role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B.

    PubMed

    Cadel, Sandrine; Darmon, Cécile; Pernier, Julien; Hervé, Guy; Foulon, Thierry

    2015-02-01

    Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases. PMID:25530263

  3. Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

    PubMed Central

    McMahon, K A; Wilson, N J; Marks, D C; Beecroft, T L; Whitty, G A; Hamilton, J A; Csar, X F

    2001-01-01

    M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells. PMID:11513742

  4. Novel post-translational incorporation of tyrosine in PMA-activated polymorphonuclear leukocytes (PMN)

    SciTech Connect

    Nath, J.; Oliver, C.; Ohno, Y.; Gallin, J.I.

    1986-03-05

    During studies undertaken to determine whether stimulation of tubulin tyrosinolation occurs in PMA-activated PMN, a distinctly different and novel post-translational incorporation of tyrosine into multiple PMN proteins was observed. The reaction also occurred in organelle-depleted neutrophil cytoplasts and was highly exaggerated in organelle-enriched karyogranuloplasts. The incorporation was specific for tyrosine, did not require extracellular Ca/sup 2 +/ and was inhibited in the presence of a variety of reducing agents, intracellular scavengers of oxygen radicals and inhibitors of peroxidase-mediated reactions. The PMA-induced incorporation of tyrosine was completely absent in PMN from patients with chronic granulomatous disease, but occurred normally in PMN of a patient with myeloperoxidase deficiency. Moreover, the incorporation of tyrosine was blocked by N-acetyl-L-tyrosine but not by phenylalanine suggesting a requirement for the phenolic group. A two-fold increase in stable protein carbonyl derivatives was demonstrated suggesting an increased oxidative modification of the proteins. SDS urea PAGE and reversed phase HPLC did not reveal any detectable changes in the extent of protein cross-linking. The PMN tyrosine pool was approximately 900 ..mu..M and yet only 1 ..mu..M tyrosine was added in these experiments. The functional significance of this reaction is not yet clear.

  5. Experiments on the origins of optical activity. [in amino acids

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Flores, J. J.

    1975-01-01

    An investigation was conducted concerning the asymmetric adsorption of phenylalanine enantiomers by kaolin. No preferential adsorption of either phenylalanine enantiomer could be detected and there was no resolution of the racemic phenylalanine by kaolin. The attempted asymmetric polymerization of aspartic acid by kaolin is also discussed along with a strontium-90 bremsstrahlung radiolysis of leucine.

  6. Virulent Hessian fly larvae manipulate the free amino acid content of host wheat plants.

    PubMed

    Saltzmann, Kurt D; Giovanini, Marcelo P; Zheng, Cheng; Williams, Christie E

    2008-11-01

    Gall-forming insects induce host plants to form specialized structures (galls) that provide immature life stages of the insect access to host plant nutrients and protection from natural enemies. Feeding by larvae of the Hessian fly (Mayetiola destructor Say) causes susceptible host wheat plants to produce a gall-like nutritive tissue that supports larval growth and development. To determine if changes in host plant free amino acid levels are associated with virulent Biotype L Hessian fly larval feeding, we quantified free amino acid levels in crown tissues of susceptible Newton wheat plants 1, 4, and 7 days after Hessian fly egg hatch. Hessian fly-infested susceptible plants were more responsive than resistant plants or uninfested controls, showing higher concentrations of alanine, glutamic acid, glycine, phenylalanine, proline, and serine 4 days after egg hatch. This 4-day post-hatch time point corresponds to the maturation of nutritive tissue cells in susceptible plants and the onset of rapid larval growth. By 7 days after egg hatch, when virulent second instars are actively feeding on the contents of nutritive tissue cells, the aromatic amino acids phenylalanine and tyrosine were more abundant compared to uninfested controls, but the levels of other free amino acids were no longer elevated. Changes in free amino acid abundance described in this report were associated with increased levels of mRNA encoded by wheat genes involved in amino acid synthesis and transport. PMID:18841417

  7. Poly-dopamine-beta-cyclodextrin: A novel nanobiopolymer towards sensing of some amino acids at physiological pH.

    PubMed

    Hasanzadeh, Mohammad; Sadeghi, Sattar; Bageri, Leyla; Mokhtarzadeh, Ahad; Karimzadeh, Ayub; Shadjou, Nasrin; Mahboob, Soltanali

    2016-12-01

    A novel nanobiopolymer film was electrodeposited on the surface of glassy carbon through cyclic voltammetry from dopamine, β-cyclodextrin, and phosphate buffer solution in physiological pH (7.40). The electrochemical behavior of polydopamine-Beta-cyclodextrin modified glassy carbon electrode was investigated for electro-oxidation and determination of some amino acids (l-Cysteine, l-Tyrosine, l-Glycine, and l-Phenylalanine). The modified electrode was applied for selected amino acid detection at physiological pH using cyclic voltammetry, differential pulse voltammetry and chronoamperometry, chronocoulometery. The linear concentration range of the proposed sensor for the l-Glycine, l-Cysteine, l-Tyrosine, and l-Phenylalanine were 0.2-70, 0.06-0.2, 0.01-0.1, and 0.2-10μM, while low limit of quantifications were 0.2, 0.06, 0.01, and 0.2μM, respectively. The modified electrode shows many advantages as an amino acid sensor such as simple preparation method without using any specific electron transfer mediator or specific reagent, good sensitivity, short response time, and long term stability. PMID:27612722

  8. Effect of thenardite on the direct detection of aromatic amino acids: implications for the search for life in the solar system

    NASA Astrophysics Data System (ADS)

    Doc Richardson, C.; Hinman, Nancy W.; Scott, Jill R.

    2009-10-01

    With the discovery of Na-sulphate minerals on Mars and Europa, recent studies using these minerals have focused on their ability to assist in the detection of bio/organic signatures. This study further investigates the ability of thenardite (Na2SO4) to effectively facilitate the ionization and identification of aromatic amino acids (phenylalanine, tyrosine and tryptophan) using a technique called geomatrix-assisted laser desorption/ionization in conjunction with a Fourier transform ion cyclotron resonance mass spectrometry. This technique is based on the ability of a mineral host to facilitate desorption and ionization of bio/organic molecules for detection. Spectra obtained from each aromatic amino acid alone and in combination with thenardite show differences in ionization mechanism and fragmentation patterns. These differences are due to chemical and structural differences between the aromatic side chains of their respective amino acid. Tyrosine and tryptophan when combined with thenardite were observed to undergo cation-attachment ([M+Na]+), due to the high alkali ion affinity of their aromatic side chains. In addition, substitution of the carboxyl group hydrogen by sodium led to formation of [M-H+Na]Na+ peaks. In contrast, phenylalanine mixed with thenardite showed no evidence of Na+ attachment. Understanding how co-deposition of amino acids with thenardite can affect the observed mass spectra is important for future exploration missions that are likely to use laser desorption mass spectrometry to search for bio/organic compounds in extraterrestrial environments.

  9. Effect of Thenardite on the Direct Detection of Aromatic Amino Acids: Implications for the Search for Life in the Solar System

    SciTech Connect

    C. Doc Richardson; Nancy W. Hinman; Jill R. Scott

    2009-10-01

    With the discovery of Na-sulfate minerals on Mars and Europa, recent studies using these minerals have focused on their ability to assist in the detection of bio/organic signatures. This study further investigates the ability of thenardite (Na2SO4) to effectively facilitate the ionization and identification of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) using a technique called geomatrix-assisted laser desorption/ionization (GALDI) in conjunction with a Fourier transform mass spectrometry (FTICR-MS). This technique is based on the ability of a mineral host to facilitate the ionization and detection of bio/organic molecules. Spectra obtained from each aromatic amino acid alone and in combination with thenardite show differences in ionization mechanism and fragmentation patterns. These differences are due to chemical and structural differences between the aromatic side chains of their respective amino acid. Tyrosine and tryptophan when combined with thenardite were observed to undergo cation-attachment ([M+Na]+), due to the high alkali affinity of their aromatic side chains. Subsequent cation substitution of the carboxyl group led to formation double cation-attached peaks ([M-H+Na]Na+). In contrast, phenylalanine mixed with thenardite showed no evidence of Na+ interaction. Understanding how codeposition of amino acids with thenardite can affect the observed mass spectra is important for future exploration missions that are likely to use laser desorption mass spectrometry to search for bio/organic compounds in extraterrestrial environments.

  10. Phenylalanine Is Required to Promote Specific Developmental Responses and Prevents Cellular Damage in Response to Ultraviolet Light in Soybean (Glycine max) during the Seed-to-Seedling Transition

    PubMed Central

    Sullivan, Joe H.; Muhammad, DurreShahwar; Warpeha, Katherine M.

    2014-01-01

    UV-radiation elicits a suite of developmental (photomorphogenic) and protective responses in plants, but responses early post-germination have received little attention, particularly in intensively bred plants of economic importance. We examined germination, hypocotyl elongation, leaf pubescence and subcellular responses of germinating and/or etiolated soybean (Glycine max (L.) Merr.) seedlings in response to treatment with discrete wavelengths of UV-A or UV-B radiation. We demonstrate differential responses of germinating/young soybean seedlings to a range of UV wavelengths that indicate unique signal transduction mechanisms regulate UV-initiated responses. We have investigated how phenylalanine, a key substrate in the phenylpropanoid pathway, may be involved in these responses. Pubescence may be a key location for phenylalanine-derived protective compounds, as UV-B irradiation increased pubescence and accumulation of UV-absorbing compounds within primary leaf pubescence, visualized by microscopy and absorbance spectra. Mass spectrometry analysis of pubescence indicated that sinapic esters accumulate in the UV-irradiated hairs compared to unirradiated primary leaf tissue. Deleterious effects of some UV-B wavelengths on germination and seedling responses were reduced or entirely prevented by inclusion of phenylalanine in the growth media. Key effects of phenylalanine were not duplicated by tyrosine or tryptophan or sucrose, nor is the specificity of response due to the absorbance of phenylalanine itself. These results suggest that in the seed-to-seedling transition, phenylalanine may be a limiting factor in the development of initial mechanisms of UV protection in the developing leaf. PMID:25549094

  11. Phenylalanine prevents acute poisoning by ochratoxina in mice.

    PubMed

    Creppy, E E; Schlegel, M; Röschenthaler, R; Dirheimer, G

    1980-07-01

    Ochratoxin-A (OT-A) a mycotoxin isolated from Aspergillus ochraceus is toxic for animals and man causing a fatal chronic kidney disease and liver damages. OT-A is a competitive inhibitor of phenylalanine in the phenylalanyl-tRNA synthetase-catalyzed reaction thus inhibiting protein synthesis. This inhibition can be reversed by phenylalanine. When injected intraperitoneally (i.p.) to mice a dose of 0.8 mg of OT-A is lethal within 24h. However, when this lethal dose is injected together with 1 mg of phenylalanine 100% of the animals survive. When phenylalanine was injected 30 min after OT-A the doses required for the survival of 92% of the animals had to be about 25 times higher. PMID:7414623

  12. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Macdonald, R. E.; Lanyi, J. K.

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

  13. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

    PubMed Central

    Ni, Zhongqiu; Zhou, Lanxia; Li, Xu; Zhang, Jing; Dong, Shouliang

    2015-01-01

    A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis. PMID:26536589

  14. Phenylalanine ab initio models for the simulation of skin natural moisturizing factor

    NASA Astrophysics Data System (ADS)

    Carvalho, B. G.; Raniero, L. J.; Martin, A. A.; Favero, P. P.

    2013-04-01

    In this study, we evaluated models that can be used to simulate amino acids in biological environments via density functional theory (DFT). The goal was to obtain realistic representations that combine computational economy and result quality when compared to experimental data. We increased the complexity of the models by using a model of an amino acid in a vacuum, followed by a water-solvated amino acid model. To consider pH variation, we simulated zwitterionic and nonionic amino acid configurations. The amino acid chosen for testing was phenylalanine, an aromatic amino acid present in high concentrations in the natural moisturizing factor of skin that plays a fundamental role in ultraviolet protection and vitiligo disease. To validate the models, vibrational modes and electronic properties were calculated and compared to experimental results.

  15. Phenylalanine functionalized zwitterionic monolith for hydrophobic interaction electrochromatography.

    PubMed

    Wang, Jiabin; Jia, Wenchao; Lin, Xucong; Wu, Xiaoping; Xie, Zenghong

    2013-12-01

    A novel phenylalanine (Phe) functionalized zwitterionic monolith for hydrophobic electrochromatography was prepared by a two-step procedure involving the synthesis of glycidyl methacrylate based polymer monolith and subsequent on-column chemical modification with Phe via ring-opening reaction of epoxides. Benefitting from the hydrophobicity of both methacrylate-based matrix and aromatic group of Phe, this monolith could exhibit good hydrophobic interaction for the separation. Typical RP chromatographic behavior was observed toward various solutes. The well-controlled cathodic or anodic EOF of the prepared column could be facilely switched by altering the pH values of running buffers. The separation mechanism of this Phe functionalized zwitterionic monolith is discussed in detail. Two mixed-mode mechanisms of RP/cation exchange and RP/anion exchange could be further realized on the same monolith in different pH condition of the mobile phase. Versatile separation capabilities of neutral, basic, and acidic analytes have been successfully achieved in this zwitterionic monolith by CEC method. PMID:24242631

  16. Self-assembly of phenylalanine-based molecules.

    PubMed

    German, Helen W; Uyaver, Sahin; Hansmann, Ulrich H E

    2015-03-01

    Using molecular dynamics, we study the self-assembly of phenylalanine with charged end-groups at various temperatures and concentrations. As in the case of diphenylalanine, we observe the formation of nanotubes; however, phenylalanine aggregates in layers of four, not six, molecules. The observed aggregates are consistent with recent experimental measurements of fibrils obtained from mice with phenylketonuria. We investigate the stability and the mechanism by which these tubular structures form and discuss potential toxicity mechanisms. PMID:25347763

  17. On the asymmetric adsorption of phenylalanine enantiomers by kaolin.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Flores, J.

    1973-01-01

    The attempt is described to verify a recent report that kaolin adsorbs D- and L-phenylalanine enantiomers to different extents from aqueous solutions at both pH 5.8 and pH 2. No evidence whatsoever could be found for the differential adsorption of D- versus L-phenylalanine by kaolin from either pH 6 or pH 2 solutions.

  18. Determination of phenylalanine isotope ratio enrichment by liquid chromatography/time- of-flight mass spectrometry.

    PubMed

    Wu, Zhanpin; Zhang, Xiao-Jun; Cody, Robert B; Wolfe, Robert R

    2004-01-01

    The application of time-of-flight mass spectrometry to isotope ratio measurements has been limited by the relatively low dynamic range of the time-to-digital converter detectors available on commercial LC/ToF-MS systems. Here we report the measurement of phenylalanine isotope ratio enrichment by using a new LC/ToF-MS system with wide dynamic range. Underivatized phenylalanine was injected onto a C18 column directly with 0.1% formic acid/acetonitrile as the mobile phase. The optimal instrument parameters for the time-of-flight mass spectrometer were determined by tuning the instrument with a phenylalanine standard. The accuracy of the isotope enrichment measurement was determined by the injection of standard solutions with known isotope ratios ranging from 0.02% to 9.2%. A plot of the results against the theoretical values gave a linear curve with R2 of 0.9999. The coefficient of variation for the isotope ratio measurement was below 2%. The method is simple, rapid, and accurate and presents an attractive alternative to traditional GC/MS applications. PMID:15531795

  19. Changes on grape phenolic composition induced by grapevine foliar applications of phenylalanine and urea.

    PubMed

    Portu, J; López-Alfaro, I; Gómez-Alonso, S; López, R; Garde-Cerdán, T

    2015-08-01

    Grapevines may require the input of nitrogen to grow and to guarantee an appropriate grape composition. Recently there has been a growing interest in foliar fertilization, which entails a fast and efficient assimilation of the products. The aim of this work was to study the influence of foliar applications of phenylalanine and urea, at two different doses, on grape anthocyanins, flavonols, flavan-3-ols, phenolic acids, and stilbenes. All treatments were applied at veraison and one week later at doses of 0.9 and 1.5 kg N/ha. The results showed that the synthesis of phenolic compounds was favoured by foliar applications of phenylalanine and urea. The application of the lowest dose of urea was the most effective treatment, increasing the content of several anthocyanins and flavonols. Moreover, none of the foliar treatments worsened the grape phenolic composition. In conclusion, foliar application of phenylalanine and especially urea, could be an interesting management tool for improving grape quality and their health-promoting properties. PMID:25766815

  20. Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

    PubMed Central

    Kovács, Klaudia; Bánóczi, Gergely; Varga, Andrea; Szabó, Izabella; Holczinger, András; Hornyánszky, Gábor; Zagyva, Imre

    2014-01-01

    The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia. PMID:24475062

  1. A new precursor for the preparation of 6-[18F]-fluoro-L-m-tyrosine (FMT): Efficient synthesis and comparison of radiolabeling

    SciTech Connect

    VanBrocklin, Henry F.; Blagoev, Milan; Hoepping, Alexander; O'Neil, James P.; Klose, Manuela; Schubiger, Pius A.; Ametamey, Simon

    2004-01-09

    For the electrophilic preparation of 6-[18F]-Fluoro-L-m-tyrosine (FMT), a PET tracer for measuring changes in dopaminergic function in movement disorders, a novel precursor, N-(tert-butoxycarbonyl)-3-(tert-butoxycarbonyloxy)-6-trimethylstannnyl-L-phenylalanine ethyl ester, was synthesized in four steps and 26 percent yield starting from L-m-tyrosine. FMT produced by two methods at two institutions was comparable in decay corrected yield, 25-26 percent, and quality (chemical, enantiomeric, and radiochemical purity and specific activity) as that obtained with the original N-trifluoroacetyl-3-acetyl-6-trimethylstannyl-L-m-tyrosine ethyl ester FMT precursor.

  2. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    PubMed

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids. PMID:25199802

  3. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts. PMID:24910972

  4. Differential effects of low-phenylalanine protein sources on brain neurotransmitters and behavior in C57Bl/6-Pah(enu2) mice.

    PubMed

    Sawin, Emily A; Murali, Sangita G; Ney, Denise M

    2014-04-01

    Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase, which metabolizes phenylalanine (phe) to tyrosine. A low-phe diet plus amino acid (AA) formula is necessary to prevent cognitive impairment; glycomacropeptide (GMP) contains minimal phe and provides a palatable alternative to the AA formula. Our objective was to assess neurotransmitter concentrations in the brain and the behavioral phenotype of PKU mice (Pah(enu2) on the C57Bl/6 background) and how this is affected by low-phe protein sources. Wild type (WT) and PKU mice, both male and female, were fed high-phe casein, low-phe AA, or low-phe GMP diets between 3 and 18 weeks of age. Behavioral phenotype was assessed using the open field and marble burying tests, and brain neurotransmitter concentrations were measured using HPLC with electrochemical detection system. Data were analyzed by 3-way ANOVA with genotype, sex, and diet as the main treatment effects. Brain mass and the concentrations of catecholamines and serotonin were reduced in PKU mice compared to WT mice; the low-phe AA and GMP diets improved these parameters in PKU mice. Relative brain mass was increased in female PKU mice fed the GMP diet compared to the AA diet. PKU mice exhibited hyperactivity and impaired vertical exploration compared to their WT littermates during the open field test. Regardless of genotype or diet, female mice demonstrated increased vertical activity time and increased total ambulatory and horizontal activity counts compared with male mice. PKU mice fed the high-phe casein diet buried significantly fewer marbles than WT control mice fed casein; this was normalized in PKU mice fed the low-phe AA and GMP diets. In summary, C57Bl/6-Pah(enu2) mice showed an impaired behavioral phenotype and reduced brain neurotransmitter concentrations that were improved by the low-phe AA or GMP diets. These data support lifelong adherence to a low-phe diet for PKU. PMID:24560888

  5. A capillary electrophoresis method for the determination of selected biogenic amines and amino acids in mammalian decomposition fluid.

    PubMed

    Swann, L M; Forbes, S L; Lewis, S W

    2010-06-15

    A simple capillary zone electrophoresis method for the determination of selected biogenic amines (tyramine and tryptamine) and amino acids (tryptophan, phenylalanine and tyrosine) in mammalian decomposition fluids is presented. Separations were carried out in a fused silica capillary (75microm i.d., total length 65cm, effective length 56cm) with detection by ultraviolet absorbance spectrophotometry at 200nm. In order to improve resolution and total analysis time, the method was subjected to optimisation utilising a chemometric approach. A screening design was carried out followed by a central composite design (CCD), using peak resolution and total analysis time as response factors. The influences of four experimental variables (pH, background electrolyte concentration, percentage of organic modifier (methanol) and applied voltage) were investigated. Optimum separation conditions were determined to be; a background electrolyte of boric acid (70mM) adjusted to pH 9.5 with 0.1M sodium hydroxide with 32% methanol (v/v). Applied voltage was 30kV, with the resulting current being less than 26microA. Under these conditions the analytes were separated within 12min. Tryptamine, tyramine, tryptophan, tyrosine and phenylalanine were identified by migration time and spiking in porcine decomposition fluids. PMID:20441960

  6. Tyrosine Administration Decreases Vulnerability to Ventricular Fibrillation in the Normal Canine Heart

    NASA Astrophysics Data System (ADS)

    Scott, Neal A.; Desilva, Regis A.; Lown, Bernard; Wurtman, Richard J.

    1981-02-01

    Intravenous infusion of tyrosine (1, 2, or 4 milligrams per kilogram) for 20 to 30 minutes caused dose-dependent increases in the ventricular fibrillation threshold in normal dogs. Administration of valine, a neutral amino acid that competes with tyrosine for uptake at the blood-brain barrier, in a dose equimolar to the most effective dose of tyrosine, slightly decreased the ventricular fibrillation threshold when given alone and significantly blocked elevation of the ventricular fibrillation threshold after tyrosine infusion. Hence, tyrosine, presumably acting in the central nervous system, can protect against certain ventricular arrhythmias.

  7. Dopamine release in rat striatum - Physiological coupling to tyrosine supply

    NASA Technical Reports Server (NTRS)

    During, Matthew J.; Acworth, Ian N.; Wurtman, Richard J.

    1989-01-01

    Intracerebral microdialysis was used to monitor dopamine release in rat striatal extracellular fluid following the intraperitoneal administration of dopamine's precursor amino acid, L-tyrosine. Dopamine concentrations in dialysates increased transiently after tyrosine (50-100 mg/kg) administration. Pretreatment with haloperidol or the partial lesioning of nigrostriatal neurons enhanced the effect of tyrosine on dopamine release, and haloperidol also prolonged this effect. These data suggest that nigrostriatal dopaminergic neurons are responsive to changes in precursor availability under basal conditions, but that receptor-mediated feedback mechanisms limit the magnitude and duration of this effect.

  8. The effects of plant growth regulators and L-phenylalanine on phenolic compounds of sweet basil.

    PubMed

    Koca, Nülüfer; Karaman, Şengül

    2015-01-01

    The effects of methyl jasmonate (MeJA), spermine (Spm), epibrassinolide (EBL) and l-phenylalanine on sweet basil (Ocimum basilicum L.) were studied to determine the amount of phenolic compounds and enzymatic activity of phenylalanine ammonia-lyase (PAL). Total phenolic and total flavonoid contents of sweet basils were determined by a spectrophotometer, and individual phenolic compounds and activity of PAL were analysed by HPLC/UV. The highest total phenolic (6.72 mg GAE/g) and total flavonoid contents (0.92 mg QE/g) obtained from 1.0 mM Spm+MeJA application. Rosmarinic acid (RA) and caffeic acid contents significantly enhanced after the applications but no such differences observed in chicoric acid content or PAL activity. RA was the main phenolic acid in all samples and its concentration varied from 1.04 to 2.70 mg/gFW. As a result the combinations of Spm+MeJA and EBL+MeJA can induce secondary metabolites effectively and those interactions play important role in the production of phytochemicals in plants. PMID:25053088

  9. Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

    NASA Technical Reports Server (NTRS)

    Conlay, L. A.; Maher, T. J.; Roberts, C. H.; Wurtman, R. J.

    1988-01-01

    Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord were examined. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial (H-3)tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of (H-3)tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.

  10. DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

    PubMed Central

    Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

    2011-01-01

    The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

  11. The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment.

    PubMed

    Shang, Xuefang; Du, Jinge; Yang, Wancai; Liu, Yun; Fu, Zhiyuan; Wei, Xiaofang; Yan, Ruifang; Yao, Ningcong; Guo, Yaping; Zhang, Jinlian; Xu, Xiufang

    2014-05-01

    Two nano-material-containing azo groups have been designed and developed, and the binding ability of nano-materials with various amino acids has been characterized by UV-vis and fluorescence titrations. Results indicated that two nano-materials showed the strongest binding ability for homocysteine among twenty normal kinds of amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, tyrosine and homocysteine). The reason for the high sensitivity for homocysteine was that two nano-materials containing an aldehyde group reacted with SH in homocysteine and afforded very stable thiazolidine derivatives. Theoretical investigation further illustrated the possible binding mode in host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. Thus, the two nano-materials can be used as optical sensors for the detection of homocysteine. PMID:24656358

  12. Quantification of Five Clinically Important Amino Acids by HPLC-Triple TOF™ 5600 Based on Pre-column Double Derivatization Method.

    PubMed

    Deng, Shuang; Scott, David; Garg, Uttam

    2016-01-01

    Phenylalanine, tyrosine, glycine, cystine, and phosphoethanolamine are commonly measured amino acids in various physiological fluids to diagnose or follow-up various inborn errors of metabolism. The gold standard method for the amino acids quantitation has been ion exchange chromatography with ninhydrin post-column derivatization. However, this method is very laborious and time consuming. In recent years, liquid-chromatography mass spectrometry is being increasingly used for the assay of amino acids. Pre-column butyl derivatization with reverse phase chromatography has been widely used for mass spectrometry analysis of amino acids. Phosphoethanolamine is not butylated and cannot be measured by this method. Nevertheless, phosphoethanolamine can be dansyl-derivatized using dansyl chloride. We developed a double derivatization method by using butanol and dansyl chloride to derivatize carboxylic and amino groups separately, and then combining the derivatives to simultaneously measure these five amino acids using TOF-MS scan. Stable isotope-labeled internal standards were used. PMID:26602116

  13. Tyrosine isomers and hormonal signaling: A possible role for the hydroxyl free radical in insulin resistance

    PubMed Central

    Molnár, Gergő A; Mikolás, Esztella Zsóka; Szijártó, István András; Kun, Szilárd; Sélley, Eszter; Wittmann, István

    2015-01-01

    Oxidative stress processes play a major role in the development of the complications associated with diabetes and other diseases via non-enzymatic glycation, the hexosamine pathway, the polyol pathway and diacylglycerol-protein kinase C. Oxidative stress may lead to the production of hydroxyl free radicals, which can attack macromolecules, such as lipids, nucleic acids or amino acids. Phenylalanine (Phe) can be enzymatically converted to the physiological para-tyrosine (p-Tyr); however, a hydroxyl free radical attack on Phe may yield meta- and ortho-tyrosine (m- and o-Tyr, respectively) in addition to p-Tyr. Hence, m- and o-Tyr may be regarded as markers of hydroxyl free radical-induced damage. Their accumulation has been described; e.g., this accumulation has been found in the urine of patients with diabetes mellitus (DM) and/or chronic kidney disease, in cataract lenses, in vessel walls, in irradiated food and in amniotic fluid, and it may serve as an indicator of oxidative stress. The use of resveratrol to treat patients with type 2 DM led to a decrease in the urinary excretion of o-Tyr and concomitantly led to an improvement in insulin signaling and insulin sensitivity. Literature data also suggest that m- and o-Tyr may interfere with intracellular signaling. Our group has shown that erythropoietin (EPO) has insulin-like metabolic effects on fat cells in addition to its ability to promote the proliferation of erythroid precursor cells. We have shown that the supplementation of cell culture medium with m- and o-Tyr inhibits erythroblast cell proliferation, which could be ameliorated by p-Tyr. Additionally, in vivo, the o-Tyr/p-Tyr ratio is higher in patients with renal replacement therapy and a greater need for EPO. However, the o-Tyr/p-Tyr ratio was an independent determinant of EPO-resistance indices in our human study. The o-Tyr content of blood vessel walls inversely correlates with insulin- and acetylcholine-induced vasodilation, which could be further

  14. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency

    PubMed Central

    Singh, Rani H.; Rohr, Fran; Frazier, Dianne; Cunningham, Amy; Mofidi, Shideh; Ogata, Beth; Splett, Patricia L.; Moseley, Kathryn; Huntington, Kathleen; Acosta, Phyllis B.; Vockley, Jerry; Van Calcar, Sandra C.

    2014-01-01

    The effectiveness of a phenylalanine-restricted diet to improve the outcome of individuals with phenylalanine hydroxylase deficiency (OMIM no. 261600) has been recognized since the first patients were treated 60 years ago. However, the treatment regime is complex, costly, and often difficult to maintain for the long term. Improvements and refinements in the diet for phenylalanine hydroxylase deficiency have been made over the years, and adjunctive therapies have proven to be successful for certain patients. Yet evidence-based guidelines for managing phenylalanine hydroxylase deficiency, optimizing outcomes, and addressing all available therapies are lacking. Thus, recommendations for nutrition management were developed using evidence from peer-reviewed publications, gray literature, and consensus surveys. The areas investigated included choice of appropriate medical foods, integration of adjunctive therapies, treatment during pregnancy, monitoring of nutritional and clinical markers, prevention of nutrient deficiencies, providing of access to care, and compliance strategies. This process has not only provided assessment and refinement of current nutrition management and monitoring recommendations but also charted a direction for future studies. This document serves as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylalanine hydroxylase deficiency. Genet Med 16 2, 121–131. PMID:24385075

  15. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency.

    PubMed

    Singh, Rani H; Rohr, Fran; Frazier, Dianne; Cunningham, Amy; Mofidi, Shideh; Ogata, Beth; Splett, Patricia L; Moseley, Kathryn; Huntington, Kathleen; Acosta, Phyllis B; Vockley, Jerry; Van Calcar, Sandra C

    2014-02-01

    The effectiveness of a phenylalanine-restricted diet to improve the outcome of individuals with phenylalanine hydroxylase deficiency (OMIM no. 261600) has been recognized since the first patients were treated 60 years ago. However, the treatment regime is complex, costly, and often difficult to maintain for the long term. Improvements and refinements in the diet for phenylalanine hydroxylase deficiency have been made over the years, and adjunctive therapies have proven to be successful for certain patients. Yet evidence-based guidelines for managing phenylalanine hydroxylase deficiency, optimizing outcomes, and addressing all available therapies are lacking. Thus, recommendations for nutrition management were developed using evidence from peer-reviewed publications, gray literature, and consensus surveys. The areas investigated included choice of appropriate medical foods, integration of adjunctive therapies, treatment during pregnancy, monitoring of nutritional and clinical markers, prevention of nutrient deficiencies, providing of access to care, and compliance strategies. This process has not only provided assessment and refinement of current nutrition management and monitoring recommendations but also charted a direction for future studies. This document serves as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylalanine hydroxylase deficiency. PMID:24385075

  16. Kinetic Characterization of O-Phospho-L-Tyrosine Phosphohydrolase Activity of Two Fungal Phytases.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal phytases belonging to 'Histidine Acid Phosphatase' or HAP class of phosphomonoesterase that catalyzes the hydrolysis of phytic acid could also hydrolyze O-phospho-tyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phospho-tyrosine hydrola...

  17. [Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].

    PubMed

    Mosin, O V; Shvets, V I; Skladnev, D A; Ignatov, I

    2014-01-01

    The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions. PMID:25249528

  18. Isolation and characterization of the human tyrosine hydroxylase gene: identification of 5' alternative splice sites responsible for multiple mRNAs

    SciTech Connect

    O'Malley, K.L.; Anhalt, M.J.; Martin, B.M.; Kelsoe, J.R.; Winfield, S.L.; Ginns, E.I.

    1987-11-03

    A full-length genomic clone for human tyrosine hydroxylase (L-tyrosine, tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.2) has been isolated. A human brain genomic library constructed in EMBL3 was screened by using a rat cDNA for tyrosine hydroxylase as a probe. Out of one million recombinant phage, one clone was identified that hybridized to both 5' and 3' rat cDNA probes. Restriction endonuclease mapping, Southern blotting, and sequence analysis revealed that, like its rodent counterpart, the human gene is single copy, contains 13 primary exons, and spans approximately 8 kilobases (kb). In contrast to the rat gene, human tyrosine hydroxylase undergoes alternative RNA processing within intron 1, generating at least three distinct mRNAs. A comparison of the human tyrosine hydroxylase and phenylalanine hydroxylase genes indicates that although both probably evolved from a common ancestral gene, major changes in the size of introns have occurred since their divergence.

  19. Novel 2,7-Substituted (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acids: Peroxisome Proliferator-Activated Receptor γ Partial Agonists with Protein-Tyrosine Phosphatase 1B Inhibition.

    PubMed

    Otake, Kazuya; Azukizawa, Satoru; Takeda, Shigemitsu; Fukui, Masaki; Kawahara, Arisa; Kitao, Tatsuya; Shirahase, Hiroaki

    2015-01-01

    A novel series of 2,7-substituted 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and biologically evaluated. (S)-2-(2-Furylacryloyl)-7-[2-(2-methylindane-2-yl)-5-methyloxazol-4-yl]methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid tert-butylamine salt (13jE) was identified as a potent human peroxisome proliferator-activated receptor γ (PPARγ)-selective agonist (EC50=85 nM) and human protein-tyrosine phosphatase 1B (PTP-1B) inhibitor (IC50=1.0 µM). Compound 13jE partially activated PPARγ, but not PPARα or PPARδ, and antagonized farglitazar, a full PPARγ agonist. Cmax after the oral administration of 13jE at 10 mg/kg was 28.6 µg/mL (53 µM) in male Sprague-Dawley (SD) rats. Repeated administration of 13jE and rosiglitazone for 14 d at 10 mg/kg/d decreased plasma glucose and triglyceride levels significantly in male KK-A(y) mice. Rosiglitazone, but not 13jE, significantly increased the plasma volume and liver weight. In conclusion, 13jE showed stronger hypoglycemic and hypolipidemic effects and weaker hemodilution and hepatotoxic effects than rosiglitazone, suggesting that its safer efficacy may be due to its partial PPARγ agonism and PTP-1B inhibition. PMID:26633022

  20. Experimental and quantum chemical modeling studies of the interactions of L-phenylalanine with divalent transition metal cations.

    PubMed

    Mandal, Shilpi; Das, Gunajyoti; Askari, Hassan

    2014-09-22

    Encoded by the UUU and UUC codons of the genetic code, L-phenylalanine (LPA) serves as an important precursor for tyrosine and various other compounds that are necessary to support life on earth. Here, we report the synthesis (both in solid and solvent phases) and characterization of the Ni(2+), Cu(2+), and Zn(2+) complexes of LPA by several analytical, spectral, thermal, and electrochemical techniques. The results reveal that the products formed by following the two synthetic approaches are the same, and the metal ions bind to the LPA molecules in a 1:2 molar ratio (M(+2)/LPA). Complementary geometries of the metal complexes are modeled involving the most predominant LPA conformers predicted at the MP2/6-311++G(d,p) level. The gaseous and aqueous phase interaction enthalpies and free energies; theoretical IR and UV-vis spectra; HOMO-LUMO energy gaps; dipole moments; Wiberg bond indices as well as the partial atomic charges in LPA and its metallic complexes are calculated and evaluated using B3LYP/6-311++G(d,p) as the main computational method. This study also incorporates analyses on the efficacy of the DFT-D2 level in describing dispersion contributions, performance of the BHandHLYP functional for the open-shell Cu(2+)-LPA system, and relative metal binding affinities of the singlet versus triplet states of the Ni(2+)-LPA complex. Metal-π interactions established via the aromatic side chain of LPA add to the thermodynamic stability of the complexes, whereas metal coordination induces considerable intrinsic structural rearrangements in the molecular geometry of LPA. The LPA binding affinity order of the three Lewis acids investigated emerges as Cu(2+) > Ni(2+) > Zn(2+), paralleling the Irving-Williams series. The illustrative evidence offered by the present work suggests that the B3LYP/6-311++G(d,p) level in combination with an empirical dispersion-correction term performs well in describing the vibrational frequencies and cation-π interactions, which are

  1. Preparation of a Selective L-Phenylalanine Imprinted Polymer Implicated in Patients with Phenylketonuria

    PubMed Central

    Najafizadeh, Parvaneh; Ebrahimi, Soltan Ahmad; Panjehshahin, Mohammad Reza; Rezayat Sorkhabadi, Seyed Mahdi

    2014-01-01

    Background: Molecular imprinting is a method for synthesizing polymers with structure-selective adsorption properties with applications such as, selectivity binding, drug delivery systems and anti-bodies. The present study aims at optimizing the preparation of molecularly imprinted polymer (MIP) against l-phenylalanine, in order to increase phenylalanine-binding in Enzymatic Intestinal Simulated Fluid (ESIF). Methods: The MIP for l-phenylalanine, as a water-soluble template, was successfully synthesized without derivatization. Synthesization was done by a UV polymerization method in which methacrylic acid (MAA), as a functional monomer, and ethylene glycol dimethacrylate (EGDMA), as a cross-linker, were used in the presence of five different porogenic solvents including; acetonitrile, tetrahydrofuran (THF), chloroform, toluene and dimethyl sulfoxide (DMSO). The selectivity of the MIP was examined using 19 different amino acids in human serum and was evaluated by HPLC. In addition, morphological studies were conducted using SEM. Results: The results showed that the obtained MIP with acetonitrile had the highest capacity and selectivity compared with other solvents. The data indicated that Phe-binding to MIP was significantly more than the former binding to NIP in EISF (P≤0.05). Moreover, in comparison with NIP and control group, MIP showed a better selectivity and binding for Phe. This could be used for the reduction of Phe in human serum samples of Phenylketonuria. Conclusion: Our findings suggest that the MIP against Phe prepared with acetonitrile, showed a good selectivity and binding, which caused a reduction of blood Phe concentration in enzymatic simulated intestinal fluid and human serum sample of Phenylketonuria. PMID:25429178

  2. Changes in free amino acids and polyamine levels in Satsuma leaves in response to Asian citrus psyllid infestation and water stress.

    PubMed

    Malik, Nasir S A; Perez, Jose L; Kunta, Madhurababu; Patt, Joseph M; Mangan, Robert L

    2014-12-01

    The effects of biotic and abiotic stresses on changes in amino acids and polyamine levels in Satsuma orange (Citrus unshiu; cultivar Owari) leaves were investigated. Asian citrus psyllids Diaphorina citri (Kuwayama) (ACP) infestation was used to induce biotic stress while a water deficit was imposed to induce abiotic stress. Potted trees were infested by placing 50 psyllids on 3 citrus leaves enclosed in nylon mesh bags for 5 d. A parallel set of plants were kept water stressed by maintaining the soil at 20% water holding capacity for 5 d. Levels of total free amino acids were higher in water stressed and ACP infested leaves. Polyamine putrescine increased in infested leaves but not in water stressed leaves. Proline was the most abundant amino acid and its levels significantly increased by both biotic and abiotic stresses. Proline levels in infested leaves were significantly higher than the water stressed leaves. Histidine, methionine, asparagine, arginine, serine, and leucine levels also increased significantly in infested leaves, but in water stressed leaves only leucine, methionine, and threonine increased. Levels of amino acids, such as tyrosine, isoleucine, phenylalanine, glutamic acid, and alanine, declined in infested leaves. Under water stress asparagine, phenylalanine, serine, and histidine also declined compared to controls. This indicates that while proteolysis occurred under both stresses, metabolic conversion of amino acids was different under the two stresses. In ACP infested leaves some amino acids may be used as feeding material and/or converted into secondary metabolites for defense. PMID:24178691

  3. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  4. A Survey of Aspartate Phenylalanine and Glutamate Phenylalanine Interactions in the Protein Data Bank: Searching for Anion Pairs

    SciTech Connect

    Philip, Vivek M; Harris, Jason B; Adams, Rachel M; Nguyen, Don; Spires, Jeremy; Howell, Elizabeth E.; Hinde, Robert J

    2011-01-01

    Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242 8249]. To study the role of anion interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification of Phe Asp or Glu pairs separated by less than 7 in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura Morokuma energy calculations were performed on roughly 19000 benzene formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (2 to 7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion pairs are found throughout protein structures, in helices as well as strands. Numerous pairs also had nearby cation interactions as well as potential stacking. While more than 1000 structures did not contain an anion pair, the 3134 remaining structures contained approximately 2.6 anion pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.

  5. The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors

    PubMed Central

    Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias

    2015-01-01

    BACKGROUND: Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. OBJECTIVE: The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [18F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. METHODS: Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and 18F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. RESULTS: Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and 18F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. CONCLUSION: Age, tumor volume, and 18F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased

  6. Conservative tryptophan mutants of the protein tyrosine phosphatase YopH exhibit impaired WPD-loop function and crystallize with divanadate esters in their active sites.

    PubMed

    Moise, Gwendolyn; Gallup, Nathan M; Alexandrova, Anastassia N; Hengge, Alvan C; Johnson, Sean J

    2015-10-27

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  7. Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

    PubMed Central

    Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

    2016-01-01

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  8. Insulin Therapy of Nondiabetic Septic Patients Is Predicted by para-Tyrosine/Phenylalanine Ratio and by Hydroxyl Radical-Derived Products of Phenylalanine

    PubMed Central

    Kun, Szilárd; Molnár, Gergő A.; Sélley, Eszter; Szélig, Lívia; Bogár, Lajos; Csontos, Csaba; Miseta, Attila; Wittmann, István

    2015-01-01

    Hydroxyl radical converts Phe to para-, meta-, and ortho-Tyr (p-Tyr, m-Tyr, o-Tyr), while Phe is converted enzymatically to p-Tyr in the kidney and could serve as substrate for gluconeogenesis. Pathological isoforms m- and o-Tyr are supposed to be involved in development of hormone resistances. Role of Phe and the three Tyr isoforms in influencing insulin need was examined in 25 nondiabetic septic patients. Daily insulin dose (DID) and insulin-glucose product (IGP) were calculated. Serum and urinary levels of Phe and Tyr isoforms were determined using a rpHPLC-method. Urinary m-Tyr/p-Tyr ratio was higher in patients with DID and IGP over median compared to those below median (P = 0.005 and P = 0.01, resp.). Urinary m-Tyr and m-Tyr/p-Tyr ratio showed positive correlation with DID (P = 0.009 and P = 0.023, resp.) and with IGP (P = 0.004 and P = 0.008, resp.). Serum Phe was a negative predictor, while serum p-Tyr/Phe ratio was positive predictor of both DID and IGP. Urinary m-Tyr and urinary m-Tyr/p-Tyr, o-Tyr/p-Tyr, and (m-Tyr+o-Tyr)/p-Tyr ratios were positive predictors of both DID and IGP. Phe and Tyr isoforms have a predictive role in carbohydrate metabolism of nondiabetic septic patients. Phe may serve as substrate for renal gluconeogenesis via enzymatically produced p-Tyr, while hydroxyl radical derived Phe products may interfere with insulin action. PMID:26576228

  9. The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

    PubMed Central

    Wunder, Tobias; Martin, Roman; Löffelhardt, Wolfgang; Schleiff, Enrico; Steiner, Jürgen M

    2007-01-01

    Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has

  10. Effective atomic numbers and electron densities of bacteriorhodopsin and its comprising amino acids in the energy range 1 keV-100 GeV

    NASA Astrophysics Data System (ADS)

    Ahmadi, Morteza; Lunscher, Nolan; Yeow, John T. W.

    2013-04-01

    Recently, there has been an interest in fabrication of X-ray sensors based on bacteriorhodopsin, a proton pump protein in cell membrane of Halobacterium salinarium. Therefore, a better understanding of interaction of X-ray photons with bacteriorhodopsin is required. We use WinXCom program to calculate the mass attenuation coefficient of bacteriorhodopsin and its comprising amino acids for photon energies from 1 keV to 100 GeV. These amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, Asx1, Asx2, Glx1 and Glx2. We then use that data to calculate effective atomic number and electron densities for the same range of energy. We also emphasize on two ranges of energies (10-200 keV and 1-20 MeV) in which X-ray imaging and radiotherapy machines work.

  11. Para-Tyrosine Supplementation Improves Insulin- and Liraglutide- Induced Vasorelaxation in Cholesterol-Fed Rats.

    PubMed

    Sélley, Eszter; Kun, Szilárd; Kürthy, Mária; Kovács, Tibor; Wittmann, István; Molnár, Gergo A

    2015-01-01

    Former data of our workgroup indicated that the accumulation of oxidized amino acids (meta- and ortho-tyrosine) due to oxidative stress may play an important role in the impaired insulininduced vasoactive properties of different arterial segments. There are evidences, that incorporation of these amino acids into cellular proteins leads to certain hormonal resistances, which might be restored by supplementation with the physiologic isoform, para-tyrosine. Rats in the control group were kept on a regular diet, rats in the cholesterol-fed group received high-fat diet, while the third group of rats received high-fat diet with para-tyrosine supplementation for 16 weeks. Plasma cholesterol level was significantly higher in the cholesterol-fed group, while the level of cholesterol in the cholesterol+para-tyrosine group did not differ significantly from that of the controls. Plasma level of insulin after glucose stimulation was decreased in the cholesterol-fed group, while that in the para-tyrosine supplemented group did not differ significantly from the controls. Vascular para-, meta- and ortho-tyrosine content was measured with HPLC. Elevated vascular meta-tyrosine/para-tyrosine ratio of cholesterol fed rats could be avoided by para-tyrosine supplementation. Vascular response of the thoracic aorta to insulin and liraglutide was assessed by a DMT multi-myograph. Cholesterol feeding resulted in vascular insulin-and liraglutide resistance, which was restored by para-tyrosine supplementation. Incorporation of the oxidative stress induced pathological tyrosine isoforms leads to vascular-hormone-resistances. We show that the physiological amino acid para-tyrosine is capable of restoring hypercholesterolemia-induced increased meta-tyrosine content of the vascular wall, thus attenuating functional vascular damage. PMID:26202368

  12. Arabidopsis thaliana NIP7;1: An Anther-Specific Boric Acid Transporter of the Aquaporin Superfamily Regulated by an Unusual Tyrosine in Helix 2 of the Transport Pore

    SciTech Connect

    Li, Tian; Choi, Won-Gyu; Baudry, Jerome Y; Roberts, Daniel M

    2011-01-01

    Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9 11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.

  13. Umbilical uptake of amino acids in the unstressed fetal lamb.

    PubMed Central

    Lemons, J A; Adcock, E W; Jones, M D; Naughton, M A; Meschia, G; Battaglia, F C

    1976-01-01

    The whole blood concentrations of 22 amino acids were measured in a chronic, unstressed fetal lamb preparations. Samples were taken daily from the umbilical artery, umbilical vein, and maternal artery over the latter quarter of gestation. 73 sets of samples (from the umbilical artery and vein and the maternal artery) from 13 animals were analyzed for amino acid levels. Oxygen contents were determined simultaneously in 48 sets (umbilical artery and vein) to relate fetal oxygen consumption to amino acid uptake via the umbilical circulation. The results indicate that there is no umbilical uptake of the acidic amino acids, glutamate and aspartate; there is, in fact, a net flux of glutamate out of the fetus into the placenta. As both of these amino acids are major constituents of body proteins, the data indicate that they are formed within the fetus. The umbilical uptake of some neutral and basic amino acids (e.g., valine, leucine, isoleucine, arginine, phenylalanine, and tyrosine) is in considerable excess of estimated growth requirements, suggesting that some amino acids undergo extensive transamination and oxidative degradation in the fetus. Finally, the net uptake of nitrogen, carbon, and calories by the growing ovine fetus in the form of amino acids, glucose, and lactate is compared to estimated requirements as determined in previous studies. PMID:1033209

  14. Electronic Relaxation of the Phenylalanine Residue in Gas Phase Peptides: Role of the Neighbouring Amide Groups in the Photophysics

    NASA Astrophysics Data System (ADS)

    Loquais, Y.; Biswal, H. S.; Tardivel, B.; Brenner, V.; Mons, M.; Gloaguen, E.; Jouvet, C.; Broquier, M.; Malis, M.; Ljubic, I.; Doslic, N.

    2012-06-01

    Protein absorption in the near UV is mainly due to the presence of aromatic systems on the side chain of three residues: phenylalanine, tryptophan and tyrosine. It is generally expected that the photophysics of these UV chromophores depends on their immediate environment within the molecule and thus on the conformation of these flexible molecules. This property may in particular be used as an optical diagnostic of the conformational state of the peptide chain. The structure of peptide chains isolated in the gas phase can be characterized by UV and IR laser spectroscopy. These measurements allow us to distinguish the spectral contributions of the different conformers and thus provide us with an elegant way to address the issue of the conformational dependence on the photophysics. For this purpose, the dynamics of relaxation of the ππ* excited state of several peptides containing a phenylalanine residue have been studied using two-colour resonant two-photon ionization (2C-R2PI) in the ns time scale at CEA and ps at CLUPS and laser-induced fluorescence as well. The lifetime of the ππ* excited state is found to strongly depend on the conformation adopted by the molecule and on the excess energy in the excited state, with measured lifetimes ranging from 1 ns to 80 ns. W. Chin; F. Piuzzi; I. Dimicoli and M. Mons, Phys. Chem. Chem. Phys., 8, pp 1033-1048 (2006)

  15. The isolation and properties of phenylalanine hydroxylase from rat liver

    PubMed Central

    Gillam, Shirley Su; Woo, Savio L. C.; Woolf, Louis I.

    1974-01-01

    Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The Km and Vmax. values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide. PMID:4854920

  16. Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson's disease.

    PubMed

    Azzouz, Mimoun; Martin-Rendon, Enca; Barber, Robert D; Mitrophanous, Kyriacos A; Carter, Emma E; Rohll, Jonathan B; Kingsman, Susan M; Kingsman, Alan J; Mazarakis, Nicholas D

    2002-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra. This loss leads to complete dopamine depletion in the striatum and severe motor impairment. It has been demonstrated previously that a lentiviral vector system based on equine infectious anemia virus (EIAV) gives rise to highly efficient and sustained transduction of neurons in the rat brain. Therefore, a dopamine replacement strategy using EIAV has been investigated as a treatment in the 6-hydroxydopamine (6-OHDA) animal model of PD. A self-inactivating EIAV minimal lentiviral vector that expresses tyrosine hydroxylase (TH), aromatic amino acid dopa decarboxylase (AADC), and GTP cyclohydrolase 1 (CH1) in a single transcription unit has been generated. In cultured striatal neurons transduced with this vector, TH, AADC, and CH1 proteins can all be detected. After stereotactic delivery into the dopamine-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and effective production of catecholamines were detected, resulting in significant reduction of apomorphine-induced motor asymmetry compared with control animals (p < 0.003). Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that the delivery of three catecholaminergic synthetic enzymes by a single lentiviral vector can achieve functional improvement and thus open the potential for the use of this vector for gene therapy of late-stage PD patients. PMID:12451130

  17. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics*

    PubMed Central

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-01-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  18. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics.

    PubMed

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-09-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  19. Neutron inelastic scattering by amino acids

    SciTech Connect

    Thaper, C.L.; Sinha, S.K.; Dasannacharya, B.A.

    1982-01-01

    Inelastic neutron scattering experiments on normal, N-deuterated glycine, normal and N-deuterated alanine, L-valine, L-tyrosine and, L-phenylalanine at 100 K, are reported. Coupling of the external modes to different hydrogens is discussed.

  20. Feto-maternal plasma phenylalanine concentration gradient from 19 weeks gestation to term.

    PubMed

    Schoonheyt, W E; Clarke, J T; Hanley, W B; Johnson, J M; Lehotay, D C

    1994-03-01

    Plasma phenylalanine concentrations in fetal blood, obtained by cordocentesis, were compared with simultaneous peripheral venous plasma phenylalanine levels in mothers. The feto-maternal phenylalanine concentration ratio showed a gradual decrease from 19 weeks of gestation to term with an overall ratio of 1.35 +/- 0.42 (mean +/- S.D., n = 14). PMID:8088005

  1. Replacement of the N-terminal tyrosine residue in opioid peptides with 3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid (Dcp) results in novel opioid antagonists.

    PubMed

    Lu, Yixin; Lum, Tze Keong; Leow Augustine, Yoon Wui; Weltrowska, Grazyna; Nguyen, Thi M-D; Lemieux, Carole; Chung, Nga N; Schiller, Peter W

    2006-08-24

    3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist. PMID:16913729

  2. Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2S)-Naringenin Production from l-Tyrosine in Escherichia coli

    PubMed Central

    Wu, Junjun; Yu, Oliver; Du, Guocheng

    2014-01-01

    Malonyl coenzyme A (malonyl-CoA) is an important precursor for the synthesis of natural products, such as polyketides and flavonoids. The majority of this cofactor often is consumed for producing fatty acids and phospholipids, leaving only a small amount of cellular malonyl-CoA available for producing the target compound. The tuning of malonyl-CoA into heterologous pathways yields significant phenotypic effects, such as growth retardation and even cell death. In this study, fine-tuning of the fatty acid pathway in Escherichia coli with antisense RNA (asRNA) to balance the demands on malonyl-CoA for target-product synthesis and cell health was proposed. To establish an efficient asRNA system, the relationship between sequence and function for asRNA was explored. It was demonstrated that the gene-silencing effect of asRNA could be tuned by directing asRNA to different positions in the 5′-UTR (untranslated region) of the target gene. Based on this principle, the activity of asRNA was quantitatively tailored to balance the need for malonyl-CoA in cell growth and the production of the main flavonoid precursor, (2S)-naringenin. Appropriate inhibitory efficiency of the anti-fabB/fabF asRNA improved the production titer by 431% (391 mg/liter). Therefore, the strategy presented in this study provided a useful tool for the fine-tuning of endogenous gene expression in bacteria. PMID:25239896

  3. [Role of NO signal in ABA-induced phenolic acids accumulation in Salvia miltiorrhiza hairy roots].

    PubMed

    Shen, Lihong; Ren, Jiahui; Jin, Wenfang; Wang, Ruijie; Ni, Chunhong; Tong, Mengjiao; Liang, Zongsuo; Yang, Dongfeng

    2016-02-01

    To investigate roles of nitric oxide (NO) signal in accumulations of phenolic acids in abscisic.acid (ABA)-induced Salvia miltiorrhiza hairy roots, S. miltiorrhiza hairy roots were treated with different concentrations of sodium nitroprusside (SNP)-an exogenous NO donor, for 6 days, and contents of phenolic acids in the hairy roots are determined. Then with treatment of ABA and NO scavenger (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide, c-PTIO) or NO synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME), contents of phenolic acids and expression levels of three key genes involved in phenolic acids biosynthesis were detected. Phenolic acids production in S. miltiorrhiza hairy roots was most significantly improved by 100 µmoL/L SNP. Contents of RA and salvianolic acid B increased by 3 and 4 folds. ABA significantly improved transcript levels of PAL (phenylalanine ammonia lyase), TAT (tyrosine aminotransferase) and RAS (rosmarinic acid synthase), and increased phenolic acids accumulations. However, with treatments of ABA+c-PTIO or ABA+L-NAME, accumulations of phenolic acids and expression levels of the three key genes were significantly inhibited. Both NO and ABA can increase accumulations of phenolic acids in S. miltiorrhiza hairy roots. NO signal probably mediates the ABA-induced phenolic acids production. PMID:27382772

  4. Optical sensing of phenylalanine in urine via extraction with magnetic molecularly imprinted poly(ethylene-co-vinyl alcohol) nanoparticles

    NASA Astrophysics Data System (ADS)

    Hsu, Chung-Yi; Lee, Mei-Hwa; Thomas, James L.; Shih, Ching-Ping; Hung, Tzu-Lin; Whang, Thou-Jen; Lin, Hung-Yin

    2015-07-01

    Incorporation of superparamagnetic nanoparticles into molecularly imprinted polymers (MIPs) is useful for both bioseparations and for concentration and sensing of biomedically relevant target molecules in physiological fluids, through the application of a magnetic field. In this study, we combined the separation and concentration of a target (phenylalanine) in urine, using magnetic molecularly imprinted polymeric composite nanoparticles, with optical sensing, to improve assay sensitivity. This target is important as a catecholamine precursor, and as an important amino acid constituent of proteins. Poly(ethylene-co-vinyl alcohol)s were imprinted with target molecules, and showed a high imprinting effectiveness (target binding compared with binding to non-imprinted polymer particles.) Fluorescence spectrophotometry was used to measure binding of the target, and also binding of possible interfering compounds. These measurements suggest that functional groups on phenylalanine dominate the selectivity of the synthesized MIPs. Finally, the composite nanoparticles were used to separate and sense the target molecule in urine by Raman scattering microscopy.

  5. The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase

    PubMed Central

    Zhang, Shengnan; Huang, Tao; Ilangovan, Udayar; Hinck, Andrew P.; Fitzpatrick, Paul F.

    2014-01-01

    Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1-71) and a well-folded C-terminal portion (residues 72-159). The structure of a truncated version of the regulatory domain containing residues 65-159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65-159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH. PMID:24361276

  6. Tyrosine motifs are required for prestin basolateral membrane targeting

    PubMed Central

    Zhang, Yifan; Moeini-Naghani, Iman; Bai, JunPing; Santos-Sacchi, Joseph; Navaratnam, Dhasakumar S.

    2015-01-01

    ABSTRACT Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure. PMID:25596279

  7. Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes

    PubMed Central

    Vargas-Tah, Alejandra; Gosset, Guillermo

    2015-01-01

    The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement. PMID:26347861

  8. Molecular recognition of α-cyclodextrin (CD) to choral amino acids based on methyl orange as a molecular probe

    NASA Astrophysics Data System (ADS)

    Yuexian, Fan; Yu, Yang; Shaomin, Shuang; Chuan, Dong

    2005-03-01

    The molecular recognition interaction of α-CD to chiral amino acids was investigated by using spectrophotometry based on methyl orange as a molecular probe. The molecular recognition ability depended on the inclusion formation constants. The molecular recognition of α-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine ≈ DL-β-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L- iso-leucine > L-leucine ≈ L-methionine ≈ DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, Δ G, Δ H, Δ S, were determined. The experimental results indicated that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative or minor positive entropic contribution. The inclusion interaction between α-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K.

  9. Molecular recognition of alpha-cyclodextrin (CD) to choral amino acids based on methyl orange as a molecular probe.

    PubMed

    Yuexian, Fan; Yu, Yang; Shaomin, Shuang; Chuan, Dong

    2005-03-01

    The molecular recognition interaction of alpha-CD to chiral amino acids was investigated by using spectrophotometry based on methyl orange as a molecular probe. The molecular recognition ability depended on the inclusion formation constants. The molecular recognition of alpha-CD to aromatic amino acids was the order: DL-tryptophan > L-tryptophan > L-phenylalanine > L-tyrosine approximately DL-beta-3,4-dihydroxy-phenylalanine; whereas for aliphatic amino acids, the order was: L-iso-leucine > L-leucine approximately L-methionine approximately DL-mehtionine > D-leucine. The effect of temperature on the inclusion interaction was examined and the thermodynamic parameters of inclusion process, delta G, delta H, delta S, were determined. The experimental results indicated that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative or minor positive entropic contribution. The inclusion interaction between alpha-CD and amino acids satisfied the law of enthalpy-entropy compensation. The compensation temperature was 291 K. PMID:15683802

  10. Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis.

    PubMed

    Zhu, Longbao; Cui, Wenjing; Fang, Yueqin; Liu, Yi; Gao, Xinxing; Zhou, Zhemin

    2013-05-01

    The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg(-1) and the k cat/K m was 1.9 × 10(4) mM(-1) s(-1), exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry. PMID:23338700

  11. Enrichment and detection of tyrosine-nitrated proteins.

    PubMed

    Dekker, Frank; Abello, Nicolas; Wisastra, Rosalina; Bischoff, Rainer

    2012-08-01

    Nitrotyrosine is a post-translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine-nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al(3+). In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol. PMID:22851496

  12. Characterization of the PEST family protein tyrosine phosphatase BDP1.

    PubMed

    Kim, Y W; Wang, H; Sures, I; Lammers, R; Martell, K J; Ullrich, A

    1996-11-21

    Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues. PMID:8950995

  13. Novel Anthraquinone-based Derivatives as Potent Inhibitors for Receptor Tyrosine Kinases

    PubMed Central

    Stasevych, M.; Zvarych, V.; Lunin, V.; Halenova, T.; Savchuk, O.; Dudchak, O.; Vovk, M.; Novikov, V.

    2015-01-01

    The influence of new derivatives of 9,10-anthraquinone with benzoylthiourea, thiazole, triazole and amino acid fragments on the activity of membrane-associated tyrosine kinases was investigated. Inhibitors of protein tyrosine kinase activity of the membrane fraction, as promising agents to search for new potential anticancer agents among the studied compounds, were discovered. PMID:26798182

  14. [13C]-Specific labeling of 8-2' linked (-)-cis-blechnic, (-)-trans-blechnic and (-)-brainic acids in the fern Blechnum spicant

    NASA Technical Reports Server (NTRS)

    Davin, Laurence B.; Wang, Chang-Zeng; Helms, Gregory L.; Lewis, Norman G.

    2003-01-01

    In vivo administration experiments using stable (13C) and radio (14C) labeled precursors established that the optically active 8-2' linked lignans, (-)-cis-blechnic, (-)-trans-blechnic and (-)-trans-brainic acids, were directly derived from L-phenylalanine, cinnamate, and p-coumarate but not either from tyrosine or acetate. The radiochemical time course data suggest that the initial coupling product is (-)-cis-blechnic acid, which is then apparently converted into both (-)-trans-blechnic and (-)-trans-brainic acids in vivo. These findings provide additional evidence for vascular plant proteins engendering distinct but specific phenolic radical-radical coupling modes, i.e., for full control over phenylpropanoid coupling in vivo, whether stereoselective or regiospecific.

  15. The cellular protective effects of rosmarinic acid: from bench to bedside.

    PubMed

    Nabavi, Seyed Fazel; Tenore, Gian Carlo; Daglia, Maria; Tundis, Rosa; Loizzo, Monica Rosa; Nabavi, Seyed Mohammad

    2015-01-01

    Rosemary (Rosmarinus officinalis L.) is one of the most common household herbs, used as spices in a variety of foods, and employed in traditional medicine for its healing properties. Rosemary is a rich source of active antioxidant constituents such as phenolic diterpenes, flavonoids and phenolic acids. Caffeic acid and rosmarinic acid are the most important bioactive constituents. Rosmarinic acid is the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid and is widely identified in different plant species. Chemical structure of rosmarinic acid contains two phenolic rings: one of them is derived from phenylalanine via caffeic acid and the other from tyrosine via dihydroxyphenyl-lactic acid. Its large-scale production is obtained from plant cell cultures of Coleus blumei Benth. It is easily absorbed through gastrointestinal tract as well as the skin. Rosmarinic acid is one of the most important and well known natural antioxidant compounds, which possesses neuroprotective effects in different models of neuroinflammation, neurodegeneration, as well as chemicalinduced neurotoxicity and oxidative stress. Therefore, in present review, we aim to discuss about chemistry, sources, biotechnological production and neuroprotective actions of rosmarinic acid with emphasis on its possible molecular mechanisms of neuroprotection. PMID:25578431

  16. Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.

    PubMed Central

    Barghouthi, S; Young, R; Olson, M O; Arceneaux, J E; Clem, L W; Byers, B R

    1989-01-01

    Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species. PMID:2522922

  17. Characterization and differential expression analysis of artichoke phenylalanine ammonia-lyase-coding sequences.

    PubMed

    De Paolis, Angelo; Pignone, Domenico; Morgese, Anita; Sonnante, Gabriella

    2008-01-01

    Sequences encoding phenylalanine ammonia-lyase were isolated from artichoke, by using a sequence homology strategy, by screening a genomic library and by 3'-rapid amplification of cDNA end (RACE) technology. These analyses and Southern blots suggested that, in artichoke, phenylalanine ammonia-lyase (PAL) is encoded by a small gene family. The sequences isolated from genomic DNA possess two exons and one intron at the conserved position as in most plant pal characterized to date. The 3'-RACE analysis also indicated that each member of the artichoke pal gene family was present as a pool of transcripts, different in the length of 3'-untranslated region. The deduced amino acid sequences were highly similar to those of PAL from lettuce and sunflower. One of the artichoke pal genes was completely sequenced, and its 5' upstream region contained TATA, CAAT box and cis regulatory elements identified in other phenylpropanoid pathway genes as playing a role in UV and elicitor induction. The expression of three of the identified artichoke pal sequences was evaluated in different plant parts, in developmental stages and after wounding, using gene-specific primers/probe combinations in real-time polymerase chain reaction assays. The three putative genes were differentially expressed in the plant parts analysed and were developmentally regulated. Moreover, after leaf mechanical injury, all of them were differentially regulated. The possible involvement of the single pal genes in different physiological processes is discussed. PMID:18251868

  18. Fluorescence of Zn-Al-Eu ternary layered hydroxide response to phenylalanine

    NASA Astrophysics Data System (ADS)

    Chen, Yufeng; Li, Fei; Yu, Gensheng; Yang, Xiaojing

    2012-02-01

    We reported the fluorescence of a Zn-Al-Eu ternary layered double hydroxide (LDH) response to an amino acid (phenylalanine) for the first time. As shown in fluorescence, the red emissions attributed to 5D 0- 7F J transitions ( J = 1, 2, 3, 4) of Eu 3+ ions were quenched by the phenylalanine (Phe), and a strong blue emission at around 445 nm appeared. The fluorescent changes may be due to ligand-to-metal charges transfer, which was caused by the interaction between the Zn-Al-Eu LDH and Phe. This interaction was manifested by markedly different chemical shift positions of the Zn 3p 3/2, Al 2p, Eu 4d 3/2, O 1s, and C 1s peaks in the XPS spectra from those of the Zn-Al-Eu LDH and Zn-Al-Eu/Phe composite. Furthermore, the interaction between the LDH and Phe was supported by the results of X-ray diffraction (XRD) measurements, Fourier transform infrared (FT-IR) spectra, and thermogravimetric and differential thermogravimetric (TG-DTG) analysis. The fluorescence of Zn-Al-Eu LDH response to Phe may be potential application in biological techniques.

  19. Three-dimensional tertiary structure of yeast phenylalanine transfer RNA

    NASA Technical Reports Server (NTRS)

    Kim, S. H.; Sussman, J. L.; Suddath, F. L.; Quigley, G. J.; Mcpherson, A.; Wang, A. H. J.; Seeman, N. C.; Rich, A.

    1974-01-01

    Results of an analysis and interpretation of a 3-A electron density map of yeast phenylalanine transfer RNA. Some earlier detailed assignments of nucleotide residues to electron density peaks are found to be in error, even though the overall tracing of the backbone conformation of yeast phenylalanine transfer RNA was generally correct. A new, more comprehensive interpretation is made which makes it possible to define the tertiary interactions in the molecule. The new interpretation makes it possible to visualize a number of tertiary interactions which not only explain the structural role of most of the bases which are constant in transfer RNAs, but also makes it possible to understand in a direct and simple fashion the chemical modification data on transfer RNA. In addition, this pattern of tertiary interactions provides a basis for understanding the general three-dimensional folding of all transfer RNA molecules.

  20. Raman spectra of amino acids and their aqueous solutions

    NASA Astrophysics Data System (ADS)

    Zhu, Guangyong; Zhu, Xian; Fan, Qi; Wan, Xueliang

    2011-03-01

    Amino acids are the basic "building blocks" that combine to form proteins and play an important physiological role in all life-forms. Amino acids can be used as models for the examination of the importance of intermolecular bonding in life processes. Raman spectra serve to obtain information regarding molecular conformation, giving valuable insights into the topology of more complex molecules (peptides and proteins). In this paper, amino acids and their aqueous solution have been studied by Raman spectroscopy. Comparisons of certain values for these frequencies in amino acids and their aqueous solutions are given. Spectra of solids when compared to those of the solute in solution are invariably much more complex and almost always sharper. We present a collection of Raman spectra of 18 kinds of amino acids ( L-alanine, L-arginine, L-aspartic acid, cystine, L-glutamic acid, L-glycine, L-histidine, L-isoluecine, L-leucine, L-lysine, L-phenylalanine, L-methionone, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine) and their aqueous solutions that can serve as references for the interpretation of Raman spectra of proteins and biological materials.

  1. Amino Acid and Vitamin Requirements of Several Bacteroides Strains

    PubMed Central

    Quinto, Grace

    1966-01-01

    Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins. PMID:16349673

  2. Self-assembling peptide of D-amino acids boosts selectivity and antitumor efficacy of 10-hydroxycamptothecin.

    PubMed

    Liu, Jianfeng; Liu, Jinjian; Chu, Liping; Zhang, Yumin; Xu, Hongyan; Kong, Deling; Yang, Zhimou; Yang, Cuihong; Ding, Dan

    2014-04-23

    D-peptides, which consist of D-amino acids and can resist the hydrolysis catalyzed by endogenous peptidases, are one of the promising candidates for construction of peptide materials with enhanced biostability in vivo. In this paper, we report on a self-assembling supramolecular nanostructure of D-amino acid-based peptide Nap-G(D)F(D)F(D)YGRGD (D-fiber, (D)F meant D-phenylalanine, (D)Y meant D-tyrosine), which were used as carriers for 10-hydroxycamptothecin (HCPT). Transmission electron microscopy observations demonstrated the filamentous morphology of the HCPT-loaded peptides (d-fiber-HCPT). The better selectivity and antitumor activity of D-fiber-HCPT than L-fiber-HCPT were found in the in vitro and in vivo antitumor studies. These results highlight that this model D-fiber system holds great promise as vehicles of hydrophobic drugs for cancer therapy. PMID:24660962

  3. Biomolecular Interactions and Biological Responses of Emerging Two-Dimensional Materials and Aromatic Amino Acid Complexes.

    PubMed

    Mallineni, Sai Sunil Kumar; Shannahan, Jonathan; Raghavendra, Achyut J; Rao, Apparao M; Brown, Jared M; Podila, Ramakrishna

    2016-07-01

    The present work experimentally investigates the interaction of aromatic amino acids viz., tyrosine, tryptophan, and phenylalnine with novel two-dimensional (2D) materials including graphene, graphene oxide (GO), and boron nitride (BN). Photoluminescence, micro-Raman spectroscopy, and cyclic voltammetry were employed to investigate the nature of interactions and possible charge transfer between 2D materials and amino acids. Graphene and GO were found to interact strongly with aromatic amino acids through π-π stacking, charge transfer, and H-bonding. Particularly, it was observed that both physi and chemisorption are prominent in the interactions of GO/graphene with phenylalanine and tryptophan while tyrosine exhibited strong chemisorption on graphene and GO. In contrast, BN exhibited little or no interactions, which could be attributed to localized π-electron clouds around N atoms in BN lattice. Lastly, the adsorption of amino acids on 2D materials was observed to considerably change their biological response in terms of reactive oxygen species generation. More importantly, these changes in the biological response followed the same trends observed in the physi and chemisorption measurements. PMID:27281436

  4. Optically active aromatic amino acids. Part VI. Synthesis and properties of (Leu5)-enkephalin analogues containing O-methyl-L-tyrosine1 with ring substitution at position 3'.

    PubMed

    Arnold, Z S; Schiller, P W

    2000-06-01

    Twelve new [Tyr(Me)1, Leu5]-enkephalin analogues with substituents at position 3' of the Tyr ring have been synthesized using traditional solution methods. The substituents were -CO2H, -CONH2, -CO2Me, -(E)-CH=NOH, -(E)-CH=NOMe and CH2OH. The analogues were C-terminated with methyl esters, amides or as free acids. In the in vitro biological assays a remarkable agonist activity to the opiate receptor mu in guinea pig ileum (GPI) relative to Leu-ENK was shown by the following: Leu-ENK, 100; [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), 8.1; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), 26.2; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), 2.9; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), 4.7; and [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), 5.6. The agonist effect was naltrexone- or naloxone-reversible. The masking of the hydroxyl group in (E)-hydroxyiminomethyl group of analogue (VI) by O-methylation has totally abolished its GPI agonist activity. It seems that the (E)-CH=NOH group shows affinity and plays an analogous role to the phenol group Tyr1 in leucine-enkephalin and in the tyramine group of the opiate alkaloids. The analogues: [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), [Tyr(Me)(3'-CO2H)1, Leu-OMe5]-ENK (II), [Tyr(Me)(3'-CO2Me)1, Leu-NH2(5)]-ENK (III), [Tyr(Me)(3'-CO2H)1, Leu-NH2(5)]-ENK (IV), [Tyr(Me)(3'-CONH2)1, Leu-NH2(5)]-ENK (V), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), [Tyr(Me)(3'-(E)-CH=NOMe)1, Leu-OMe5]-ENK (IX), [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), [Tyr(Me)(3'-CH2OH)1, Leu-OH5]-ENK (XI) and [Tyr(Me)(3'-CH2OH)1, Leu-NH2(5)]-ENK (XII) under testing had no significant agonist activity to the enkephalinergic receptor in mouse vas deferens (MVD). All methyl esters of synthesized analogues of [Leu5]-ENK showed higher activity to mu receptors than structurally identical C-terminal amides. It is a surprising result since usually C-terminate amides are stronger

  5. Temperature-responsive molecular recognition chromatography using phenylalanine and tryptophan derived polymer modified silica beads.

    PubMed

    Hiruta, Yuki; Kanazashi, Ryosuke; Ayano, Eri; Okano, Teruo; Kanazawa, Hideko

    2016-02-01

    Temperature-responsive polymers incorporating molecular-recognition sites were developed as stationary phases for high-performance liquid chromatography (HPLC). The grafted stationary phases consisted of functional copolymers composed of N-isopropylacrylamide (NIPAAm) and N-acryloyl aromatic amino acid methyl esters, i.e., phenylalanine and tryptophan methyl esters (Phe-OMe and Trp-OMe). Three novel temperature-responsive polymers, P(NIPAAm-co-Phe-OMe5), P(NIPAAm-co-Phe-OMe10), and P(NIPAAm-co-Trp-OMe5), were synthesized. These copolymers exhibited a reversible hydrophilic/hydrophobic phase transition at their lower critical solution temperatures (LCSTs). The polymers were grafted onto aminopropyl silica using an activated ester-amine coupling method, and were packed into a stainless steel column, which was connected to an HPLC system. Temperature-responsive chromatography was conducted using water as the sole mobile phase. More hydrophobic analytes were retained longer, and the retention times of aromatic steroids and aromatic amino acids were dramatically increased. This indicated that π-π interactions occurred between the phenyl or indole moieties of phenylalanine or tryptophan, respectively, and the aromatic compounds. Furthermore, the retention times of compounds with hydrogen bond acceptors were higher with P(NIPAAm-co-Trp-OMe5), which contained indole as a hydrogen bond donor, than with P(NIPAAm-co-Phe-OMe5). This indicated that hydrogen bonding occurred between the stationary phase and the analytes. These results indicate that hydrophobic, π-π, and hydrogen bonding interactions all affected the separation mode of the temperature-responsive chromatography, and led to selective separation with molecular recognition. Both temperature-response and molecular recognition characteristics are present in the proposed separation system that utilizes a temperature-responsive polymer bearing aromatic amino acid derivatives. PMID:26646169

  6. Efficient Preparation of Enantiopure D-Phenylalanine through Asymmetric Resolution Using Immobilized Phenylalanine Ammonia-Lyase from Rhodotorula glutinis JN-1 in a Recirculating Packed-Bed Reactor

    PubMed Central

    Huang, Nan; Cui, Wenjing; Liu, Zhongmei; Xiao, Ke; Zhou, Zhemin

    2014-01-01

    An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h−1 and 0.32 g L−1 h−1, respectively. The optical purity (eeD) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99%) in the scaled-up reactor reached 7.2 g L−1 h−1. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine. PMID:25268937

  7. Efficient preparation of enantiopure D-phenylalanine through asymmetric resolution using immobilized phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1 in a recirculating packed-bed reactor.

    PubMed

    Zhu, Longbao; Zhou, Li; Huang, Nan; Cui, Wenjing; Liu, Zhongmei; Xiao, Ke; Zhou, Zhemin

    2014-01-01

    An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (eeD) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99%) in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine. PMID:25268937

  8. Elevational Variation in Soil Amino Acid and Inorganic Nitrogen Concentrations in Taibai Mountain, China.

    PubMed

    Cao, Xiaochuang; Ma, Qingxu; Zhong, Chu; Yang, Xin; Zhu, Lianfeng; Zhang, Junhua; Jin, Qianyu; Wu, Lianghuan

    2016-01-01

    Amino acids are important sources of soil organic nitrogen (N), which is essential for plant nutrition, but detailed information about which amino acids predominant and whether amino acid composition varies with elevation is lacking. In this study, we hypothesized that the concentrations of amino acids in soil would increase and their composition would vary along the elevational gradient of Taibai Mountain, as plant-derived organic matter accumulated and N mineralization and microbial immobilization of amino acids slowed with reduced soil temperature. Results showed that the concentrations of soil extractable total N, extractable organic N and amino acids significantly increased with elevation due to the accumulation of soil organic matter and the greater N content. Soil extractable organic N concentration was significantly greater than that of the extractable inorganic N (NO3--N + NH4+-N). On average, soil adsorbed amino acid concentration was approximately 5-fold greater than that of the free amino acids, which indicates that adsorbed amino acids extracted with the strong salt solution likely represent a potential source for the replenishment of free amino acids. We found no appreciable evidence to suggest that amino acids with simple molecular structure were dominant at low elevations, whereas amino acids with high molecular weight and complex aromatic structure dominated the high elevations. Across the elevational gradient, the amino acid pool was dominated by alanine, aspartic acid, glycine, glutamic acid, histidine, serine and threonine. These seven amino acids accounted for approximately 68.9% of the total hydrolyzable amino acid pool. The proportions of isoleucine, tyrosine and methionine varied with elevation, while soil major amino acid composition (including alanine, arginine, aspartic acid, glycine, histidine, leucine, phenylalanine, serine, threonine and valine) did not vary appreciably with elevation (p>0.10). The compositional similarity of many

  9. Elevational Variation in Soil Amino Acid and Inorganic Nitrogen Concentrations in Taibai Mountain, China

    PubMed Central

    Yang, Xin; Zhu, Lianfeng; Zhang, Junhua; Jin, Qianyu; Wu, Lianghuan

    2016-01-01

    Amino acids are important sources of soil organic nitrogen (N), which is essential for plant nutrition, but detailed information about which amino acids predominant and whether amino acid composition varies with elevation is lacking. In this study, we hypothesized that the concentrations of amino acids in soil would increase and their composition would vary along the elevational gradient of Taibai Mountain, as plant-derived organic matter accumulated and N mineralization and microbial immobilization of amino acids slowed with reduced soil temperature. Results showed that the concentrations of soil extractable total N, extractable organic N and amino acids significantly increased with elevation due to the accumulation of soil organic matter and the greater N content. Soil extractable organic N concentration was significantly greater than that of the extractable inorganic N (NO3−-N + NH4+-N). On average, soil adsorbed amino acid concentration was approximately 5-fold greater than that of the free amino acids, which indicates that adsorbed amino acids extracted with the strong salt solution likely represent a potential source for the replenishment of free amino acids. We found no appreciable evidence to suggest that amino acids with simple molecular structure were dominant at low elevations, whereas amino acids with high molecular weight and complex aromatic structure dominated the high elevations. Across the elevational gradient, the amino acid pool was dominated by alanine, aspartic acid, glycine, glutamic acid, histidine, serine and threonine. These seven amino acids accounted for approximately 68.9% of the total hydrolyzable amino acid pool. The proportions of isoleucine, tyrosine and methionine varied with elevation, while soil major amino acid composition (including alanine, arginine, aspartic acid, glycine, histidine, leucine, phenylalanine, serine, threonine and valine) did not vary appreciably with elevation (p>0.10). The compositional similarity of many

  10. Cross-linked enzyme aggregates of phenylalanine ammonia lyase: novel biocatalysts for synthesis of L-phenylalanine.

    PubMed

    Cui, Jian-Dong; Zhang, Si; Sun, Li-Mei

    2012-06-01

    Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL-CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40 % saturation), followed by cross-linking for 1 h with 0.2 % (v/v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of L-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of L: -phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates. PMID:22622644

  11. Induction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress.

    PubMed

    Kamalipourazad, Maryam; Sharifi, Mohsen; Maivan, Hassan Zare; Behmanesh, Mehrdad; Chashmi, Najmeh Ahmadian

    2016-10-01

    Manipulation of cell culture media by elicitors is one of most important strategies to inducing secondary metabolism for the production of valuable metabolites. In this investigation, inducing effect of chitosan on physiological, biochemical, and molecular parameters were investigated in cell suspension cultures of Scrophularia striata Boiss. The results showed that chitosan concentration and time of elicitation are determinants of the effectiveness of the elicitor. Accumulation of aromatic amino acids (phenylalanine [Phe] and tyrosine [Tyr]), phenylpropanoid compounds (phenolic acids [PAs] and echinacoside [ECH]), hydrogen peroxide (H2O2) production, phenylalanine ammonia-lyase (PAL) activity and gene expression, and antioxidant enzymes (superoxide dismutase [SOD], peroxidase [POX], catalase [CAT]) activities were altered by changing the exposure time of elicitation. Results showed that, upon elicitation with chitosan, oxidative events were induced, antioxidant responses of S. striata cells were boosted through enhanced activity of an effective series of scavenging enzymes (SOD, CAT, and POX), and biosynthesis of non-enzymatic antioxidants (ECH and PAs [cinnamic, p-coumaric and, caffeic acids]). The increase in amino acid content and PAL activity at early days of exposure to chitosan was related with rises in phenolic compounds. These results provide evidence that chitosan by up-regulation of PAL gene differentially improves the production of phenylpropanoid compounds, which are of medical commercial value with good biotechnological prospects. PMID:27392152

  12. Partitioning of amino acids in the aqueous biphasic system containing the water-miscible ionic liquid 1-butyl-3-methylimidazolium bromide and the water-structuring salt potassium citrate.

    PubMed

    Zafarani-Moattar, Mohammed Taghi; Hamzehzadeh, Sholeh

    2011-07-01

    In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio-molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)-a new class of non-volatile alternative solvents. ABS composed of the hydrophilic IL {1-butyl-3-methylimidazolium bromide ([C4 mim]Br)} and potassium citrate-which is easily degraded-represents a clean media to green separation of bio-molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL-based ABS, the distribution of five model amino acids (L-tryptophan, L-phenylalanine, L-tyrosine, L-leucine, and L-valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting-out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL-based ABS, a model was established to describe the partition coefficient of three model amino acids, L-tryptophan, L-phenylalanine, and L-valine, and employed to predict the partition coefficient of two other model amino acids, L-tyrosine and L-leucine. PMID:21509956

  13. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  14. ELECTRON SPIN RESONANCE OF IRRADIATED SINGLE CRYSTALS OF L-PHENYLALANINE-HCL*

    PubMed Central

    Fasanella, Edwin L.; Gordy, Walter

    1969-01-01

    Single crystals of L-phenylalanine-HCl have been observed with electron spin resonance after irradiation with a cobalt 60 γ-ray source. The predominant signals observed are from long-lived benzyl radicals. The results indicate that one form of radiation damage to proteins containing this amino acid is breakage of the carbon-carbon bond to release the benzyl radical from the polypeptide chain. Hyperfine structure due to proton couplings of the two methylene hydrogens and of the hydrogens on the ring of the benzyl radical is observed and analyzed to give the electron spin density on the various carbons. The spin density on the methylene carbon is found to be 0.71; that on carbon C(2), C(4), or C(6) is 0.17. The sum of the spin densities on C(1), C(3), and C(5) is —0.22. PMID:4312748

  15. Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

    PubMed Central

    Kazlauskas, A; Durden, D L; Cooper, J A

    1991-01-01

    Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis. Images PMID:1653029

  16. Application of Pre-Column Labeling Liquid Chromatography for Canine Plasma-Free Amino Acid Analysis.

    PubMed

    Azuma, Kazuo; Hirao, Yoshiko; Hayakawa, Yoshihiro; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Ito, Norihiko

    2016-01-01

    Plasma-free amino acid (PFAA) levels are a useful metric for diagnosing cancer and providing a prognosis. However, the use of analysis of PFAA levels has been limited in the veterinary medicine field. We addressed the application of liquid chromatography (LC) using a pre-column labeling technique for analysis of canine PFAA levels. This method significantly shortened the analysis time relative to conventional methods. No diurnal fluctuations were detected at 9:00 AM in most PFAA levels, and food intake increased the levels of some PFAAs, including valine, leucine, tyrosine, phenylalanine, and proline. These results indicate that LC with pre-column labeling is useful for measuring canine PFAA levels, for which time of day and interval after food intake must be taken into consideration. PMID:26771650

  17. Application of Pre-Column Labeling Liquid Chromatography for Canine Plasma-Free Amino Acid Analysis

    PubMed Central

    Azuma, Kazuo; Hirao, Yoshiko; Hayakawa, Yoshihiro; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Ito, Norihiko

    2016-01-01

    Plasma-free amino acid (PFAA) levels are a useful metric for diagnosing cancer and providing a prognosis. However, the use of analysis of PFAA levels has been limited in the veterinary medicine field. We addressed the application of liquid chromatography (LC) using a pre-column labeling technique for analysis of canine PFAA levels. This method significantly shortened the analysis time relative to conventional methods. No diurnal fluctuations were detected at 9:00 AM in most PFAA levels, and food intake increased the levels of some PFAAs, including valine, leucine, tyrosine, phenylalanine, and proline. These results indicate that LC with pre-column labeling is useful for measuring canine PFAA levels, for which time of day and interval after food intake must be taken into consideration. PMID:26771650

  18. Determination of amino acids in Chinese rice wine by fourier transform near-infrared spectroscopy.

    PubMed

    Shen, Fei; Niu, Xiaoying; Yang, Danting; Ying, Yibin; Li, Bobin; Zhu, Geqing; Wu, Jian

    2010-09-01

    Chinese rice wine is abundant in amino acids. The possibility of quantitative detection of 16 free amino acids (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and arginine) in Chinese rice wine by Fourier transform near-infrared (NIR) spectroscopy was investigated for the first time in this study. A total of 98 samples from vintage 2007 rice wines with different aging times were analyzed by NIR spectroscopy in transmission mode. Calibration models were developed using partial least-squares regression (PLSR) with high-performance liquid chromatography (HPLC) by postcolumn derivatization and diode array detection as a reference method. To validate the calibration models, full cross (leave-one-out) validation was employed. The results showed that the calibration statistics were good (rcal>0.94) for all amino acids except proline, histidine, and arginine. The correlation coefficient in cross validation (rcv) was >0.81 for 12 amino acids. The residual predictive deviation (RPD) value obtained was >1.5 in all amino acids except proline and arginine, and it was >2.0 in 6 amino acids. The results obtained in this study indicated that NIR spectroscopy could be used as an easy, rapid, and novel tool to quantitatively predict free amino acids in Chinese rice wine without sophisticated methods. PMID:20707307

  19. Reassessment of Phenylalanine Tolerance in Adults with Phenylketonuria is Needed as Body Mass Changes

    PubMed Central

    MacLeod, Erin L.; Gleason, Sally T.; van Calcar, Sandra C.; Ney, Denise M.

    2009-01-01

    Lifelong treatment of phenylketonuria (PKU) includes a phenylalanine (phe) restricted diet that provides sufficient phe for growth and maintenance plus phe-free amino acid formula to meet requirements for protein, energy and micronutrients. Phe tolerance (mg phe/kg body weight/day) is the amount of phe those with PKU can consume and maintain acceptable blood phe levels; it requires individual assessment because of varying phenylalanine hydroxylase activity. The objective was to reassess phe tolerance in 8 adults with PKU considering phe requirements, blood phe levels, genotype and phe tolerance at 5 years of age. Subjects had not received a personalized assessment of phe tolerance in several years, and 5 subjects were overweight, body mass index (BMI) 25–28. With the guidance of a metabolic dietitian, 7 subjects increased phe tolerance (by 15–173%) without significantly increasing blood phe concentration. Increased phe tolerance was associated with both improved dietary compliance and inadequate phe intake at the onset of the protocol compared with current requirements. Improved dietary compliance reflected increased consumption of protein equivalents from amino acid formula and increased frequency of formula intake, from 2.2 to 3 times per day. Predictors of higher final phe tolerance following reassessment included being male and having a lower BMI (R2=0.588). This suggests that the rising trend of overweight and obesity may affect assessment of phe tolerance in adults. Therefore, interaction with the metabolic dietitian to reassess phe tolerance in relation to body mass is essential throughout adulthood to insure adequate intake of phe to support protein synthesis and prevent catabolism. PMID:19747868

  20. Reassessment of phenylalanine tolerance in adults with phenylketonuria is needed as body mass changes.

    PubMed

    MacLeod, Erin L; Gleason, Sally T; van Calcar, Sandra C; Ney, Denise M

    2009-12-01

    Lifelong treatment of phenylketonuria (PKU) includes a phenylalanine (phe) restricted diet that provides sufficient phe for growth and maintenance plus phe-free amino acid formula to meet requirements for protein, energy and micronutrients. Phe tolerance (mg phe/kg body weight/day) is the amount of phe those with PKU can consume and maintain acceptable blood phe levels; it requires individual assessment because of varying phenylalanine hydroxylase activity. The objective was to reassess phe tolerance in eight adults with PKU considering phe requirements, blood phe levels, genotype and phe tolerance at 5 years of age. Subjects had not received a personalized assessment of phe tolerance in several years, and five subjects were overweight, body mass index (BMI) 25-28. With the guidance of a metabolic dietitian, seven subjects increased phe tolerance (by 15-173%) without significantly increasing blood phe concentration. Increased phe tolerance was associated with both improved dietary compliance and inadequate phe intake at the onset of the protocol compared with current requirements. Improved dietary compliance reflected increased consumption of protein equivalents from amino acid formula and increased frequency of formula intake, from 2.2 to 3 times per day. Predictors of higher final phe tolerance following reassessment included being male and having a lower BMI (R(2)=0.588). This suggests that the rising trend of overweight and obesity may affect assessment of phe tolerance in adults. Therefore, interaction with the metabolic dietitian to reassess phe tolerance in relation to body mass is essential throughout adulthood to insure adequate intake of phe to support protein synthesis and prevent catabolism. PMID:19747868

  1. Effect of the “Ribonucleic Acid Control” Locus in Escherichia coli on T4 Bacteriophage-Specific Ribonucleic Acid Synthesis

    PubMed Central

    Sköld, Ola

    1970-01-01

    Amino acid control of ribonucleic acid (RNA) synthesis in bacteria is known to be governed genetically by the rel locus. We investigated whether the rel gene of the host would also exert its effect on the regulation of phage-specific RNA synthesis in T4 phage-infected Escherichia coli cells. Since T-even phage infection completely shuts off host macromolecular synthesis, phage RNA synthesis could be followed specifically by the cumulative incorporation of radioactivity from labeled precursors into RNA of infected cells. Labeled uracil was shown to accumulate in phage-specific RNA for 30 to 35 min after infection, a phenomenon which probably reflects an expansion of the labile phage-RNA pool. Amino acid starvation was effected by the use of auxotrophic bacterial strains or thienylalanine. The latter substance is an amino acid analogue which induces a chemical auxotrophy by inhibiting the biosynthesis of phenylalanine, tyrosine, and tryptophan. Phage RNA synthesis was strictly dependent on the presence of amino acids, whereas phage deoxyribonucleic acid synthesis was not. By the use of several pairs of bacterial strains which were isogenic except for the rel gene, it was demonstrated that amino acid dependence was related to the allelic state of this gene. If the rel gene was mutated, amino acid starvation did not restrict phage RNA synthesis. PMID:4914097

  2. Intracellular Growth Is Dependent on Tyrosine Catabolism in the Dimorphic Fungal Pathogen Penicillium marneffei

    PubMed Central

    Boyce, Kylie J.; McLauchlan, Alisha; Schreider, Lena; Andrianopoulos, Alex

    2015-01-01

    During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells. PMID:25812137

  3. Neuroradiological findings of an adolescent with early treated phenylketonuria: is phenylalanine restriction enough?

    PubMed Central

    Beckhauser, Mayara Thays; Peruchi, Mirella Maccarini; Rozone de Luca, Gizele; Lin, Katia; Esteves, Sofia; Vilarinho, Laura; Lin, Jaime

    2011-01-01

    Phenylketonuria is caused by mutations in the enzyme phenylalanine hydroxylase gene, that can result in abnormal concentrations of phenylalanine on blood, resulting in metabolites that can cause brain damage. The treatment is based on dietary restriction of phenylalanine, and noncompliance with treatment may result in damage of the brain function. Brain abnormalities can be seen on magnetic resonance imaging of these individuals. Studies indicate that the appearance of abnormalities in white matter reflects high levels of phenylalanine on the blood. This case will show the clinical and neuroradiological aspects of a teenager with constant control of phenylalanine levels. Despite the continuous monitoring and early treatment, the magnetic resonance imaging identified impressive abnormalities in the white matter. This leads us to one question: is the restriction of phenylalanine sufficient to prevent changes in the white matter in patients with phenylketonuria? PMID:24765287

  4. Degradation of Tyrosine in Anaerobically Stored Piggery Wastes and in Pig Feces

    PubMed Central

    Spoelstra, Sierk F.

    1978-01-01

    Radioactively labeled compounds that might be intermediates in the anaerobic degradation of tyrosine were added to pig feces and to stored piggery wastes. Changes in the compounds were followed by using thin-layer and gas chromatography. In feces, p-cresol and 3-phenylpropionic acid were the end products of tyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, and minor quantities of phenylpropionic acid were formed. Schemes were proposed for the degradation of tyrosine in pig feces and in mixed wastes. PMID:16345325

  5. Investigating the inclusion properties of aromatic amino acids complexing beta-cyclodextrins in model peptides.

    PubMed

    Caso, Jolanda Valentina; Russo, Luigi; Palmieri, Maddalena; Malgieri, Gaetano; Galdiero, Stefania; Falanga, Annarita; Isernia, Carla; Iacovino, Rosa

    2015-10-01

    Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. β-cyclodextrins (β-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the β-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the β-CD. PMID:25985927

  6. Synthesis and analysis of 2-[211At]-L-phenylalanine and 4-[211At]-L-phenylalanine and their uptake in human glioma cell cultures in-vitro.

    PubMed

    Meyer, Geerd J; Walte, Almut; Sriyapureddy, Siva R; Grote, Michaela; Krull, Doris; Korkmaz, Zekiye; Knapp, Wolfram H

    2010-06-01

    2-[211At]-L-phenylalanine and 4-[211At]-L-phenylalanine were prepared from the corresponding iodo and bromo derivatives using the Cu(+)-assisted nucleophilic exchange. 4-[211At]-L-phenylalanine was additionally prepared by destannylation of the BOC-derivatized 4-tributylstannyl-L-phenylalanine. Radiochemical yields of 2-[211At]-L-phenylalanine and 4-[211At]-L-phenylalanine by nucleophilic exchange were 52-74% and 65-85%. Radiochemical yield of 4-[211At]-L-phenylalanine by electrophilic destannylation was 35-50%. HPLC sequence analysis showed that 2-[211At]-L-phenylalanine followed the halogen sequence (Fphenylalanine eluted between 4-Br-L-phenylalanine and 4-I-L-phenylalanine (Fphenylalanine and 4-[131I]-L-phenylalanine in DBTRG-05MG glioma cells was inhibited by l-phenylalanine 7-fold and 6-fold, respectively. PMID:20137958

  7. Solid state radiolysis of amino acids in an astrochemical perspective

    NASA Astrophysics Data System (ADS)

    Cataldo, Franco; Angelini, Giancarlo; Iglesias-Groth, Susana; Manchado, Arturo

    2011-01-01

    The aliphatic amino acids L-alanine and L-leucine and the aromatic amino acids L-phenylalanine, L-tyrosine and L-tryptophan were irradiated in the solid state to a dose of 3.2 MGy. The degree of decomposition was measured by differential scanning calorimetry (DSC). Furthermore the degree of radioracemization was measured by optical rotatory dispersion (ORD) spectroscopy. From the DSC measurement a radiolysis rate constant k and the half life T1/2 for each amino acid have been determined and extrapolated to a dose of 14 MGy, which corresponds to the expected total dose delivered by the decay of radionuclides to the organic molecules present in comets and asteroids in 4.6×109 years, the age of the Solar System. It is shown that all the amino acids studied can survive a radiation dose of 14 MGy although they are reduced to 1/4-1/5 of their original value they had at the beginning of the history of the Solar System. Consequently, the amount of alanine or leucine found today in the meteorites known as carbonaceous chondrites is just 1/4-1/5 of the amount originally present at the epoch of the formation of the Solar System 4.6×109 years ago. Among the amino acids studied, tyrosine shows the highest radiation resistance while tryptophan does not combine its relatively high radiation resistance with an elevated level of radioracemization resistance. Apart from the exception of tryptophan, it is shown that the radiolysis rate constants k of all the amino acids studied are in reasonable agreement with the radioracemization rate constant krac.

  8. Yeast phenylalanine transfer RNA: atomic coordinates and torsion angles.

    PubMed Central

    Quigley, G J; Seeman, N C; Wang, A H; Suddath, F L; Rich, A

    1975-01-01

    The atomic coordinates of yeast phenylalanine transfer RNA (tRNA) as well as the torsion angles of the polynucleotide chain are presented as derived from an x-ray diffraction analysis of orthorhombic crystals. A comparison is made between the coordinates obtained from analysis of monoclinic crystals of the same material. It is concluded that the molecule has substantially the same form in the orthorhombic and the monoclinic lattices, except for differences found between residues at the 3' end of the polynucleotides chain. A number of observations are made concerning hydrogen bonding interactions which may account for many of the residues conserved in all tRNA sequences. PMID:802512

  9. The Extended Family of Protein Tyrosine Phosphatases.

    PubMed

    Alonso, Andrés; Nunes-Xavier, Caroline E; Bayón, Yolanda; Pulido, Rafael

    2016-01-01

    In higher eukaryotes, the Tyr phosphorylation status of cellular proteins results from the coordinated action of Protein Tyrosine Kinases (PTKs) and Protein Tyrosine Phosphatases (PTPs). PTPs have emerged as highly regulated enzymes with diverse substrate specificity, and proteins with Tyr-dephosphorylation or Tyr-dephosphorylation-like properties can be clustered as the PTPome. This includes proteins from the PTP superfamily, which display a Cys-based catalytic mechanism, as well as enzymes from other gene families (Asp-based phosphatases, His-based phosphatases) that have converged in protein Tyr-dephosphorylation-related functions by using non-Cys-based catalytic mechanisms. Within the Cys-based members of the PTPome, classical PTPs dephosphorylate specific phosphoTyr (pTyr) residues from protein substrates, whereas VH1-like dual-specificity PTPs dephosphorylate pTyr, pSer, and pThr residues, as well as nonproteinaceous substrates, including phosphoinositides and phosphorylated carbohydrates. In addition, several PTPs have impaired catalytic activity as a result of amino acid substitutions at their active sites, but retain regulatory functions related with pTyr signaling. As a result of their relevant biological activity, many PTPs are linked to human disease, including cancer, neurodevelopmental, and metabolic diseases, making these proteins important drug targets and molecular markers in the clinic. Here, a brief overview on the biochemistry and physiology of the different groups of proteins that belong to the mammalian PTPome is presented. PMID:27514797

  10. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  11. Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor.

    PubMed

    Laha, Kurt T; Tran, Phu N

    2013-01-01

    The prevalence of aromatic residues in the ligand binding site of the GABA(A) receptor, as with other cys-loop ligand-gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β(2) Tyr97, β(2) Tyr157, and β(2) Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK-293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA(A) receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β(2) Tyr157 and β(2) Tyr205 are more detrimental than β(2) Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process. PMID:23121119

  12. Amino acid supply to individual cerebral structures in awake and anesthetized rats.

    PubMed

    Hawkins, R A; Mans, A M; Biebuyck, J F

    1982-01-01

    The movement of phenylalanine from plasma into various cerebral structures was examined in detail in normal alert rats as well as in rats anesthetized with nitrous oxide, halothane, and sodium pentobarbital. Radioactive phenylalanine was infused intravenously in such a way as to rapidly establish and maintain a trace concentration in arterial blood. The rat was decapitated after 1.5 min, the brain removed, frozen, and thin sections cut for quantitative autoradiography. The plasma concentrations of phenylalanine as well as other neutral amino acids that compete with phenylalanine for entry into brain were measured. Phenylalanine influx varied considerably between structures, ranging from 5 nmol.min-1.g-1 in the globus pallidus to 12 nmol.min-1.g-1 in the inferior colliculus. Anesthetics reduced phenylalanine influx, but this effect was primarily due to alterations in the circulating neutral amino acid pattern. The anesthetics had little, if any, direct effect on the neutral amino acid transport process itself. PMID:7058882

  13. Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

    PubMed

    Dhayal, Surender Kumar; Sforza, Stefano; Wierenga, Peter A; Gruppen, Harry

    2015-12-01

    Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates. PMID:26282909

  14. Amino Acid Synthesis in Photosynthesizing Spinach Cells 1

    PubMed Central

    Larsen, Peder Olesen; Cornwell, Karen L.; Gee, Sherry L.; Bassham, James A.

    1981-01-01

    Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO2 fixation for more than 60 hours. The incorporation of 14CO2 under saturating CO2 conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to 14C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids. Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulatory effects in incorporation of 14C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginine, and histidine. No effect or slight decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, 14C labeling decreased, but specific radioactivity increased. The production of labeled γ-aminobutyric acid was virtually stopped by ammonia. The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. The data on the effects of added ammonia on total labeling, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis. PMID:16661904

  15. The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating.

    PubMed

    Cui, Liying; Aleksandrov, Luba; Hou, Yue-Xian; Gentzsch, Martina; Chen, Jey-Hsin; Riordan, John R; Aleksandrov, Andrei A

    2006-04-15

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function. PMID:16484308

  16. The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating

    PubMed Central

    Cui, Liying; Aleksandrov, Luba; Hou, Yue-Xian; Gentzsch, Martina; Chen, Jey-Hsin; Riordan, John R; Aleksandrov, Andrei A

    2006-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel employing the ABC transporter structural motif. Deletion of a single residue (Phe508) in the first nucleotide-binding domain (NBD1), which occurs in most patients with cystic fibrosis, impairs both maturation and function of the protein. However, substitution of the Phe508 with small uncharged amino acids, including cysteine, is permissive for maturation. To explore the possible role of the phenylalanine aromatic side chain in channel gating we introduced a cysteine at this position in cysless CFTR, enabling its selective chemical modification by sulfhydryl reagents. Both cysless and wild-type CFTR ion channels have identical mean open times when activated by different nucleotide ligands. Moreover, both channels could be locked in an open state by introducing an ATPase inhibiting mutation (E1371S). However, the introduction of a single cysteine (F508C) prevented the cysless E1371S channel from maintaining the permanently open state, allowing closing to occur. Chemical modification of cysless E1371S/F508C by sulfhydryl reagents was used to probe the role of the side chain in ion channel function. Specifically, benzyl-methanethiosulphonate modification of this variant restored the gating behaviour to that of cysless E1371S containing the wild-type phenylalanine at position 508. This provides the first direct evidence that a specific interaction of the Phe508 aromatic side chain plays a role in determining the residency time in the closed state. Thus, despite the fact that this aromatic side chain is not essential for CFTR folding, it is important in the ion channel function. PMID:16484308

  17. First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

    PubMed

    Arturo, Emilia C; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J; Parker, Emily J; Loll, Patrick J; Jaffe, Eileen K

    2016-03-01

    Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

  18. Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde

    PubMed Central

    Tieman, Denise; Taylor, Mark; Schauer, Nicolas; Fernie, Alisdair R.; Hanson, Andrew D.; Klee, Harry J.

    2006-01-01

    An important phenylalanine-derived volatile compound produced by plants is 2-phenylethanol. It is a major contributor to flavor in many foods, including fresh fruits, such as tomato, and an insect-attracting scent in roses and many other flowers. Despite the centrality of 2-phenylethanol to flavor and fragrance, the plant genes responsible for its synthesis have not been identified. Here, we describe a biosynthetic pathway for 2-phenylethanol and other phenylalanine-derived volatiles in tomato fruits and a small family of decarboxylases (LeAADC1A, LeAADC1B, and LeAADC2) that can mediate that pathway's first step. These enzymes each catalyze conversion of phenylalanine to phenethylamine and tyrosine to tyramine. Although tyrosine is the preferred substrate in vitro, phenylalanine levels in tomato fruits far exceed those of tyrosine, indicating that phenylalanine is a physiological substrate. Consistent with this view, overexpression of either LeAADC1A or LeAADC2 in transgenic tomato plants resulted in fruits with up to 10-fold increased emissions of the products of the pathway, including 2-phenylacetaldehyde, 2-phenylethanol, and 1-nitro-2-phenylethane. Further, antisense reduction of LeAADC2 significantly reduced emissions of these volatiles. Besides establishing a biosynthetic route, these results show that it is possible to change phenylalanine-based flavor and aroma volatiles in plants by manipulating expression of a single gene. PMID:16698923

  19. Infrared and Raman spectra of N-acetyl- L-amino acid methylamides with aromatic side groups

    NASA Astrophysics Data System (ADS)

    Matsuura, Hiroatsu; Hasegawa, Kodo; Miyazawa, Tatsuo

    Infrared and Raman spectra of N-acetyl- L-phenylalanine methylamide, N-acetyl- L-tyrosine methylamide and N-acetyl- L-tryptophan methylamide, as model compounds of aromatic amino acid residues in proteins, were measured in the solid state and in methanol solutions. Vibrational assignments of the spectra were made by utilizing the deuteration effect and by comparison with the spectra of related compounds which include toluene, p-cresol and 3-methylindole. The amide I, III and IV bands were strong in Raman scattering, but other characteristic amide bands were ill-defined. In the Raman spectra of methanol solutions, only the bands due to the aromatic side group vibrations were markedly observed, but those due to the peptide backbone vibrations were very weak, suggesting the coexistence of various molecular conformations in solution.

  20. Partitioning of amino-acid analogues in a five-slab membrane model

    SciTech Connect

    Sengupta, D; Smith, Jeremy C; Ullmann, G. Matthias

    2008-09-01

    The positional preferences of the twenty amino-acid residues in a phospholipid bilayer are investigated by calculating the solvation free energy of the corresponding side chain analogues using a five-slab continuum electrostatic model. The side-chain analogues of the aromatic residues tryptophan and tyrosine are found to partition in the head-group region, due to compensation between the increase of the non-polar component of the solvation free energy at the boundary with the aqueous region and the decrease in the electrostatic component. The side chain analogue of phenylalanine differs from the other aromatic molecules by being able to partition in both the head-group region and the membrane core. This finding is consistent with experimental findings of the position of phenylalanine in membrane helices. Interestingly, the charged side-chain analogues of arginine and lysine are shown to prefer the head-group region in an orientation that allows the charged moiety to interact with the aqueous layer. The orientation adopted is similar to the 'snorkelling' effect seen in lysine and arginine residues in membrane helices. In contrast, the preference of the charged side-chain analogues of histidine (protonated) and aspartate (deprotonated) for the aqueous layer is shown to be due to a steep decrease in the electrostatic component of the solvation free energy at the boundary to the aqueous region. The calculations allow an understanding of the origins of side chain positioning in membranes and are thus useful in understanding membrane-protein:lipid thermodynamics.

  1. Dietary aspartame with protein on plasma and brain amino acids, brain monoamines and behavior in rats.

    PubMed

    Torii, K; Mimura, T; Takasaki, Y; Ichimura, M

    1986-01-01

    Aspartame (APM; L-aspartyl-L-phenylalanine methyl ester), was investigated for its ability to alter levels of the large neutral amino acids and monoamines in overnight fasted rats allowed to consume meals with or without protein for two hours. Additionally, the possible long term behavioral consequences of APM in 25% casein diets with or without 10% sucrose were determined. Acute APM ingestion increased both plasma and brain phenylalanine and tyrosine levels, but brain tryptophan levels were not altered regardless of dietary protein. Brain norepinephrine and dopamine levels were unaltered by any of the diet while serotonin levels were slightly increased when a protein-free diet was consumed. But APM and/or protein ingestion minimized this increase of brain serotonin levels as much as controls. Chronic APM ingestion failed to influence diurnal feeding patterns, meal size distributions, or diurnal patterns of spontaneous motor activity. The chronic ingestion of abuse doses of APM produced no significant chemical changes in brain capable of altering behavioral parameters believed to be controlled by monoamines in rats. PMID:3714850

  2. Human spermatozoa possess an IL4I1 l-amino acid oxidase with a potential role in sperm function.

    PubMed

    Houston, B; Curry, B; Aitken, R J

    2015-06-01

    Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l-amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis. PMID:25767141

  3. Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

    PubMed

    Prota, Andrea E; Magiera, Maria M; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C; Kammerer, Richard A; Janke, Carsten; Steinmetz, Michel O

    2013-02-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  4. Structural basis of tubulin tyrosination by tubulin tyrosine ligase

    PubMed Central

    Prota, Andrea E.; Magiera, Maria M.; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C.; Kammerer, Richard A.; Janke, Carsten

    2013-01-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL–tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  5. The effects of short intensive exercise on plasma free amino acids in standardbred trotters.

    PubMed

    Hackl, S; van den Hoven, R; Zickl, M; Spona, J; Zentek, J

    2009-04-01

    The aim of this study was to investigate the effect of short intense exercise on plasma amino acid concentrations in trotters and to test the repeatability of plasma amino acids concentration in samples obtained on two independent days under field conditions. Plasma amino acid concentrations were analysed in blood samples of 36 standardbred trotters before and after intense exercise over a distance of 2000 m. Sampling was repeated in 20 horses after 35 days. Exercise intensity was estimated from post-exercise lactate levels. Horses were divided in two groups according to a cut-off lactate concentration at 15 mmol/l. The plasma concentrations of alanine, aspartate, glutamate, isoleucine, leucine, lysine and taurine increased and arginine, asparagine, citrulline, glutamine, glycine, histidine, methionine, serine, tryptophan and 3-methylhistidine decreased after exercise. Ornithine, threonine, tyrosine, phenylalanine and valine concentrations remained constant. Higher intensity of exercise significantly decreased tryptophan and increased taurine concentrations. Sampling day had a significant effect on the absolute pre- and post-exercise amino acid concentrations. Exercise had a significant influence on the concentrations of most plasma amino acids in trotters. These changes could reflect shifts between the free amino acid compartments, but there were also some indications for muscle catabolism. The amino acid supply of sporting horses could be of specific significance for maintaining muscle integrity and for the improvement of post-exercise recovery of competition horses. PMID:19320929

  6. Metabolism of orally administered branched-chain alpha-keto acids.

    PubMed

    Dalton, R N; Chantler, C

    1983-11-01

    The changes in serum branched-chain alpha-keto acid (BCKA) and plasma amino acid concentrations, in response to a therapeutic oral dose of an essential amino acid/keto acid mixture, were studied in fasting healthy adults. Of the branched-chain amino acids (BCAA), only the plasma leucine concentration rose significantly despite increases in al three serum BCKA concentrations. The plasma valine concentration tended to rise, but plasma isoleucine concentrations fell. When KMVA (keto-isoleucine) alone was given, there followed an increase in plasma isoleucine concentration and a fall in valine and leucine. Similarly, when KIVA (keto-valine) was given, plasma valine rose and leucine and isoleucine fell. These results suggest some transamination of the keto acid with amino groups of the other BCAA. KICA (keto-leucine), however, produced larger falls in plasma valine and isoleucine than was expected from the rise in leucine. In addition, KICA caused significant, insulin-independent reductions in plasma threonine, serine, cystine, methionine, tyrosine, phenylalanine, and alanine. We conclude that although orally administered BCKA's will increase the BCAA supply, their value may not simply relate to the supply of essential amino acids for protein synthesis but to a direct effect of KICA on protein metabolism. PMID:6368946

  7. Effect of spermine on the uptake of amino acids in Micrococcus lysodeikticus.

    PubMed

    Ariel, M; Grossowicz, N

    1972-08-01

    Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the V(max) decreased, whereas the apparent K(m) remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10(-1)m to 2.5 x 10(-2)m) and dinitrophenol (10(-3)m). The intracellular concentration of valine was 100-fold higher than in the medium. PMID:5053465

  8. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  9. Postinjection L-phenylalanine increases basal ganglia contrast in PET scans of 6-18F-DOPA

    SciTech Connect

    Doudet, D.J.; McLellan, C.A.; Aigner, T.G.; Wyatt, R.; Adams, H.R.; Miyake, H.; Finn, R.T.; Cohen, R.M. )

    1991-07-01

    The sensitivity of 18F-DOPA positron emission tomography for imaging presynaptic dopamine systems is limited by the amount of specific-to-nonspecific accumulation of radioactivity in brain. In rhesus monkeys, we have been able to increase this ratio by taking advantage of the lag time between 18F-DOPA injection and the formation of its main metabolite, the amino acid 18F-fluoromethoxydopa, the entrance of which into brain is responsible for most of the brain's nonspecific radioactivity. By infusing an unlabeled amino acid, L-phenylalanine, starting 15 min after 18F-DOPA administration, we preferentially blocked the accumulation of 18F-fluoromethoxydopa by preventing its entrance into brain through competition at the large neutral amino acid transport system of the blood-brain barrier. This method appears as reliable as the original and more sensitive, as demonstrated by the comparison of normal and MPTP-treated animals under both conditions.

  10. Functional analysis of the effect of monoclonal antibodies on monkey liver phenylalanine hydroxylase.

    PubMed Central

    Jennings, I G; Russell, R G; Armarego, W L; Cotton, R G

    1986-01-01

    An analysis of the effect of eleven monoclonal antibodies on the functional characteristics of monkey liver phenylalanine hydroxylase is presented. These eleven antibodies have been found to react with eight distinct regions on the phenylalanine hydroxylase protein. PH1 antibody inhibits enzyme activity, is dependent on phenylalanine for its binding, and appears to be related to structural changes occurring during phenylalanine activation of the enzyme activity. PH2 and PH3 antibodies stimulate enzyme activity, their binding is inhibited by lysolecithin and this group apparently is recognizing structures involved in lysolecithin activation of the enzyme activity. PH5, PH10, PH12 and PH6 recognise sites on phenylalanine hydroxylase affected by lysolecithin activation. PMID:2427069

  11. XAFS of human tyrosine hydroxylase

    NASA Astrophysics Data System (ADS)

    Meyer, W.; Haavik, J.; Winkler, H.; Trautwein, A. X.; Nolting, H.-F.

    1995-02-01

    Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

  12. Effect of tyrosine administration on duodenal ulcer induced by cysteamine in the rat

    SciTech Connect

    Oishi, T.; Szabo, S.

    1987-03-01

    Duodenal ulcers were produced by administering cysteamine to rats. Pretreatment with the catecholamine precursor, L-tyrosine (40 mg/100 g i.p. for 5 days), decreased the intensity of duodenal ulcers induced by cysteamine. Equimolar doses of tyrosine methyl ester (51.2 mg/100 g i.p. or s.c.) were equally effective in reducing ulcer intensity. Other amino acids (i.e., alanine, aspartic acid, glutamic acid, glycine, leucine, lysine, tryptophan and valine) did not prevent experimental duodenal ulcers. Coadministration of other large neutral amino acids (e.g., leucine and valine) that compete with tyrosine for uptake into the brain did not inhibit the effect of tyrosine on duodenal ulcers induced by cysteamine. Gastric, duodenal and brain dopamine concentrations were increased 1 hr after the injection of tyrosine methyl ester (25.6 mg/100 g s.c.). These results suggest that the effect of tyrosine on duodenal ulcer induced by cysteamine may be mediated by changes in gastrointestinal dopamine metabolism.

  13. Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity.

    PubMed Central

    Petan, Toni; Krizaj, Igor; Gubensek, Franc; Pungercar, Joze

    2002-01-01

    Ammodytoxins (Atxs) are group II phospholipases A(2) (PLA(2)s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA(2) toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe(24), in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp(24) mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40-80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA(2) activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe(24) also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn(24) mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe(24) in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity. PMID:11931665

  14. Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthesized oxygen-evolving system

    SciTech Connect

    Debus, R.J.; Barry, B.A.; Babcock, G.T.; McIntosh, L.

    1988-01-01

    Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P/sub 680//sup +/, while D /center dot//sup +/ gives rise to the dark-stable EPR spectrum known as signal II. D/center dot//sup +/ has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and E2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, the authors have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D/center dot//sup +/. The authors conclude that D is Tyr-160 of the D2 polypeptide. They suggest that the C/sub 2/ symmetry in photosystem II extends beyond P/sub 680/ to its immediate electron donor and conclude that Z is Try-161 of the D1 polypeptide.

  15. Enantiomeric Interactions between Liquid Crystals and Organized Monolayers of Tyrosine-Containing Dipeptides

    PubMed Central

    Bai, Yiqun; Abbott, Nicholas L.

    2011-01-01

    We have examined the orientational ordering of nematic liquid crystals (LCs) supported on organized monolayers of dipeptides with the goal of understanding how peptide-based interfaces encode intermolecular interactions that are amplified into supramolecular ordering. By characterizing the orientations of nematic LCs (4-cyano-4′-pentylbiphenyl (5CB) and TL205 (a mixture of mesogens containing cyclohexane-fluorinated biphenyls and fluorinated terphenyls)) on monolayers of either L-cysteine-L-tyrosine, L-cysteine-L-phenylalanine or L-cysteine-L-phosphotyrosine formed on crystallographically textured films of gold, we conclude that patterns of hydrogen bonds generated by the organized monolayers of dipeptides are transduced via macroscopic orientational ordering of the LCs. This conclusion is supported by the observation that the ordering exhibited by the achiral LCs is specific to the enantiomers used to form the dipeptide-based monolayers. The dominate role of the –OH group of tyrosine in dictating the patterns of hydrogen bonds that orient the LCs was also evidenced by the effects of phosphorylation of the tyrosine on the ordering of the LCs. Overall, these results reveal that crystallographic texturing of gold films can direct the formation of monolayers of dipeptides with long-range order, thus unmasking the influence of hydrogen bonding, chirality and phosphorylation on the macroscopic orientational ordering of LCs supported on these surfaces. These results suggest new approaches based on supramolecular assembly for reporting the chemical functionality and stereochemistry of synthetic and biological peptide-based molecules displayed at surfaces. PMID:22091988

  16. Tyrosine Recombinase Retrotransposons and Transposons.

    PubMed

    Poulter, Russell T M; Butler, Margi I

    2015-04-01

    Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes. PMID:26104693

  17. Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor.

    PubMed

    El-Batal, Ahmed I

    2002-01-01

    Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to

  18. Aligning 3D nanofibrous networks from self-assembled phenylalanine nanofibers†

    PubMed Central

    Wang, Xianfeng; Chen, Yi Charlie

    2015-01-01

    Self-assembled synthetic materials are typically disordered, and controlling the alignment of such materials at the nanometer scale may be important for a variety of biological applications. In this study, we have applied directional freeze-drying, for the first time, to develop well aligned three dimensional (3D) nanofibrous materials using amino acid like L-phenylalanine (Phe). 3D free-standing Phe nanofibrous monoliths have been successfully prepared using directional freeze-drying, and have presented a unique hierarchical structure with well-aligned nanofibers at the nanometer scale and an ordered compartmental architecture at the micrometer scale. We have found that the physical properties (e.g. nanofiber density and alignment) of the nanofibrous materials could be tuned by controlling the concentration and pH of the Phe solution and the freezing temperature. Moreover, the same strategy (i.e. directional freeze-drying) has been successfully applied to assemble peptide nanofibrous materials using a dipeptide (i.e. diphenylalanine), and to assemble Phe-based nanofibrous composites using polyethylenimine and poly(vinyl alcohol). The tunability of the nanofibrous structures together with the biocompatibility of Phe may make these 3D nanofibrous materials suitable for a variety of applications, including biosensor templates, tissue scaffolds, filtration membranes, and absorbents. The strategy reported here is likely applicable to create aligned nanofibrous structures using other amino acids, peptides, and polymers. PMID:25621167

  19. Exploration and Pharmacokinetic Profiling of Phenylalanine Based Carbamates as Novel Substance P 1–7 Analogues

    PubMed Central

    2014-01-01

    The bioactive metabolite of Substance P, the heptapeptide SP1–7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1–7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1–7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1–7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide. PMID:25516784

  20. Exploration and pharmacokinetic profiling of phenylalanine based carbamates as novel substance p 1-7 analogues.

    PubMed

    Fransson, Rebecca; Nordvall, Gunnar; Bylund, Johan; Carlsson-Jonsson, Anna; Kratz, Jadel M; Svensson, Richard; Artursson, Per; Hallberg, Mathias; Sandström, Anja

    2014-12-11

    The bioactive metabolite of Substance P, the heptapeptide SP1-7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1-7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1-7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1-7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide. PMID:25516784

  1. Metabolome-wide association study of phenylalanine in plasma of common marmosets

    PubMed Central

    Go, Young-Mi; Walker, Douglas I.; Soltow, Quinlyn A.; Uppal, Karan; Wachtman, Lynn M.; Strobel, Fredrick H.; Pennell, Kurt; Promislow, Daniel E. L.; Jones, Dean P.

    2014-01-01

    Little systematic knowledge exists concerning the impacts of cumulative lifelong exposure, termed the exposome, on requirements for nutrients. Phenylalanine (Phe) is an essential dietary amino acid with an aromatic ring structure similar to endogenous metabolites, dietary compounds and environmental agents. Excess plasma Phe in genetic disease or nutritional deficiency of Phe has adverse health consequences. In principle, structurally similar chemicals interfering with Phe utilization could alter Phe requirement at an individual level. As a strategy to identify components of the exposome that could interfere with Phe utilization, we tested for metabolites correlating with Phe concentration in plasma of a non-human primate species, common marmosets (Callithrix jacchus). The results of tests for more than 5000 chemical features detected by high-resolution metabolomics showed 17 positive correlations with Phe metabolites and other amino acids. Positive and negative correlations were also observed for 33 other chemicals, which included matches to endogenous metabolites and dietary, microbial and environmental chemicals in database searches. Chemical similarity analysis showed many of the matches had high structural similarity to Phe. Together, the results show that chemicals in marmoset plasma could impact Phe utilization. Such chemicals could contribute to early lifecycle developmental disorders when neurological development is vulnerable to Phe levels. PMID:25526869

  2. Phenylalanine and LED lights enhance phenolic compound production in Tartary buckwheat sprouts.

    PubMed

    Seo, Jeong-Min; Arasu, Mariadhas Valan; Kim, Yeon-Bok; Park, Sang Un; Kim, Sun-Ju

    2015-06-15

    The present study aimed to investigate the effects of different l-phenylalanine (l-Phe) concentrations and various light-emitting diodes (LEDs) on the accumulation of phenolic compounds (chlorogenic acid, vitexin, rutin, quercetin, cyanidin 3-O-glucoside, and cyanidin 3-O-rutinoside) in Tartary buckwheat sprouts. We found that 5mM was the optimum l-Phe concentration for the synthesis of total and individual phenolic compounds. The highest rutin (53.09 mg/g DW) and chlorogenic acid (5.62 mg/g DW) content was observed with Red+Blue and white lights. Comprehensive differences in total and individual anthocyanin content were observed between different lights; however, the total anthocyanin content (9.12 mg/g DW) was 1.5-fold higher in blue light. The expression levels of regulatory genes, such as FtDFR and FtANS, were 7.1-fold higher with l-Phe treatment. Gene expression results showed that the phenolic compounds in Tartary buckwheat sprouts increased with the use of l-Phe and LED lights. PMID:25660878

  3. Metabolome-wide association study of phenylalanine in plasma of common marmosets.

    PubMed

    Go, Young-Mi; Walker, Douglas I; Soltow, Quinlyn A; Uppal, Karan; Wachtman, Lynn M; Strobel, Fredrick H; Pennell, Kurt; Promislow, Daniel E L; Jones, Dean P

    2015-03-01

    Little systematic knowledge exists concerning the impacts of cumulative lifelong exposure, termed the exposome, on requirements for nutrients. Phenylalanine (Phe) is an essential dietary amino acid with an aromatic ring structure similar to endogenous metabolites, dietary compounds and environmental agents. Excess plasma Phe in genetic disease or nutritional deficiency of Phe has adverse health consequences. In principle, structurally similar chemicals interfering with Phe utilization could alter Phe requirement at an individual level. As a strategy to identify components of the exposome that could interfere with Phe utilization, we tested for metabolites correlating with Phe concentration in plasma of a non-human primate species, common marmosets (Callithrix jacchus). The results of tests for more than 5,000 chemical features detected by high-resolution metabolomics showed 17 positive correlations with Phe metabolites and other amino acids. Positive and negative correlations were also observed for 33 other chemicals, which included matches to endogenous metabolites and dietary, microbial and environmental chemicals in database searches. Chemical similarity analysis showed many of the matches had high structural similarity to Phe. Together, the results show that chemicals in marmoset plasma could impact Phe utilization. Such chemicals could contribute to early lifecycle developmental disorders when neurological development is vulnerable to Phe levels. PMID:25526869

  4. Amino acids implicated in plant defense are higher in Candidatus Liberibacter asiaticus-tolerant citrus varieties.

    PubMed

    Killiny, Nabil; Hijaz, Faraj

    2016-04-01

    Citrus Huanglongbing (HLB), also known as citrus greening, has been threatening the citrus industry since the early 1900's and up to this date there are no effective cures for this disease. Field observations and greenhouse controlled studies demonstrated that some citrus genotypes are more tolerant to Candidatus Liberibacter asiaticus (CLas) pathogen than others. However, the mechanisms underpinning tolerance has not been determined yet. The phloem sap composition of CLas-tolerant and sensitive citrus varieties was studied to identify metabolites that could be responsible for their tolerance to CLas. The citrus phloem sap was collected by centrifugation and was analyzed with gas chromatography-mass spectrometry after methyl chloroformate derivatization. Thirty-three metabolites were detected in the phloem sap of the studied varieties: twenty 20 amino acids, eight 8 organic acids, and five 5 fatty acids. Interestingly, the levels of most amino acids, especially those implicated in plantdefense to pathogens such as phenylalanine, tyrosine, tryptophan, lysine, and asparagine were higher in tolerant varieties. Although the level of organic acids varied between cultivars, this variation was not correlated with citrus resistance to CLas and could be cultivar specific. The fatty acids were found in trace amounts and in most cases their levels were not significantly different among varieties. Better understanding of the mechanisms underpinning citrus tolerance to CLas will help in developing economically tolerant varieties. PMID:27057814

  5. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes

    SciTech Connect

    Roussel, M.F. ); Shurtleff, S.A.; Downing, J.R. ); Sherr, C.J. Saint Jude Children's Research Hospital, Memphis, TN )

    1990-09-01

    Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an activating mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.

  6. Cisplatin stimulates protein tyrosine phosphorylation in macrophages.

    PubMed

    Kumar, R; Shrivastava, A; Sodhi, A

    1995-03-01

    Cisplatin [cis-dichlorodiamine platinum (II)], a potent anti-tumor compound, stimulates immune responses by activating monocyte-macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event that mediates cellular responses, we examined whether cisplatin alters tyrosine phosphorylation in macrophages. We found that cisplatin increased tyrosine phosphorylation of several proteins in peritoneal macrophages and in P388D1 and IC-21 macrophage cell lines. Treatment of macrophages with tyrosine kinase inhibitors, genestein and lavendustin A, inhibited cisplatin-stimulated protein tyrosine phosphorylation in macrophages. Macrophages treated with cisplatin also exhibit increased fluorescence with anti-phosphotyrosine-FITC antibody. These data indicate that protein tyrosine phosphorylation plays a role in cisplatin-induced activation of macrophages. PMID:7539662

  7. Characterization of Phospho-(Tyrosine)-Mimetic Calmodulin Mutants

    PubMed Central

    Stateva, Silviya R.; Salas, Valentina; Benaim, Gustavo; Menéndez, Margarita; Solís, Dolores; Villalobo, Antonio

    2015-01-01

    Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro. PMID:25830911

  8. Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking.

    PubMed

    Tábi, Tamás; Magyar, Kálmán; Szöko, Eva

    2005-05-01

    A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM. PMID:15818575

  9. PDGF-BB-mediated activation of p42(MAPK) is independent of PDGF beta-receptor tyrosine phosphorylation.

    PubMed

    Cartel, N J; Liu, J; Wang, J; Post, M

    2001-10-01

    Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42(MAPK) activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF beta-receptor (PDGFR-beta) did not abrogate PDGF-BB-induced p42(MAPK) activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42(MAPK) activation nor did a construct expressing PDGFR-beta with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-beta lacking the cytoplasmic signaling domain abrogated p42(MAPK) activity. These results suggest that PDGF-BB-mediated activation of p42(MAPK) requires the PDGFR-beta but is independent of its tyrosine phosphorylation. PMID:11557582

  10. Cascade Biocatalysis for Sustainable Asymmetric Synthesis: From Biobased l-Phenylalanine to High-Value Chiral Chemicals.

    PubMed

    Zhou, Yi; Wu, Shuke; Li, Zhi

    2016-09-12

    Sustainable synthesis of useful and valuable chiral fine chemicals from renewable feedstocks is highly desirable but remains challenging. Reported herein is a designed and engineered set of unique non-natural biocatalytic cascades to achieve the asymmetric synthesis of chiral epoxide, diols, hydroxy acid, and amino acid in high yield and with excellent ee values from the easily available biobased l-phenylalanine. Each of the cascades was efficiently performed in one pot by using the cells of a single recombinant strain over-expressing 4-10 different enzymes. The cascade biocatalysis approach is promising for upgrading biobased bulk chemicals to high-value chiral chemicals. In addition, combining the non-natural enzyme cascades with the natural metabolic pathway of the host strain enabled the fermentative production of the chiral fine chemicals from glucose. PMID:27512928

  11. Amino Acid Metabolism of Thermoanaerobacter Strain AK90: The Role of Electron-Scavenging Systems in End Product Formation

    PubMed Central

    Scully, Sean Michael; Orlygsson, Johann

    2015-01-01

    The catabolism of the 20 amino acids by Thermoanaerobacter strain AK90 (KR007667) was investigated under three different conditions: as single amino acids without an electron-scavenging system, in the presence of thiosulfate, and in coculture with a hydrogenotrophic methanogen. The strain degraded only serine without an alternative electron acceptor but degraded 11 amino acids (alanine, cysteine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, and valine) under both of the electron-scavenging systems investigated. Acetate was the dominant end product from alanine, cysteine, lysine, serine, and threonine under electron-scavenging conditions. The branched-chain amino acids, isoleucine, leucine, and valine, were degraded to their corresponding fatty acids under methanogenic conditions and to a mixture of their corresponding fatty acids and alcohols in the presence of thiosulfate. The partial pressure of hydrogen seems to be of importance for the branched-chain alcohol formation. This was suggested by low but detectable hydrogen concentrations at the end of cultivation on the branched-chain amino acid in the presence of thiosulfate but not when cocultured with the methanogen. A more detailed examination of the role of thiosulfate as an electron acceptor was performed with Thermoanaerobacter ethanolicus (DSM 2246) and Thermoanaerobacter brockii (DSM 1457). PMID:26413318

  12. Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.

    PubMed

    Torrens-Spence, Michael P; Lazear, Michael; von Guggenberg, Renee; Ding, Haizhen; Li, Jianyong

    2014-10-01

    Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs. PMID:25107664

  13. Quantitative determination of carboxylic acids, amino acids, carbohydrates, ethanol and hydroxymethylfurfural in honey by (1)H NMR.

    PubMed

    del Campo, Gloria; Zuriarrain, Juan; Zuriarrain, Andoni; Berregi, Iñaki

    2016-04-01

    A method using (1)H NMR spectroscopy has been developed to quantify simultaneously thirteen analytes in honeys without previous separation or pre-concentration steps. The method has been successfully applied to determine carboxylic acids (acetic, formic, lactic, malic and succinic acids), amino acids (alanine, phenylalanine, proline and tyrosine), carbohydrates (α- and β-glucose and fructose), ethanol and hydroxymethylfurfural in eucalyptus, heather, lavender, orange blossom, thyme and rosemary honeys. Quantification was performed by using the area of the signal of each analyte in the honey spectra, together with external standards. The regression analysis of the signal area against concentration plots, used for the calibration of each analyte, indicates a good linearity over the concentration ranges found in honeys, with correlation coefficients higher than 0.985 for the thirteen quantified analytes. The recovery studies give values over the 93.7-105.4% range with relative standard deviations lower than 7.4%. Good precision, with relative standard deviations over the range of 0.78-5.21% is obtained. PMID:26593586

  14. Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar

    PubMed Central

    de Jong, Femke; Hanley, Steven J.; Beale, Michael H.; Karp, Angela

    2015-01-01

    Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow. PMID:26070140

  15. Interaction of Tryptophane and Phenylalanine with Cadmium and Molybdenum Ferrocyanides and Its Implications in Chemical Evolution and Origins of Life.

    NASA Astrophysics Data System (ADS)

    Tewari, Brij

    2016-07-01

    Insoluble metal hexacyanoferrate(II) complexes could have concentrated biomonomers from dilute prebiotic soup during course of chemical evolution and origin of life or primitive earth. In the light of above hypothesis, adsorption of tryptophane and phenylalanine was studied on cadmium and molybdenum ferrocyanides at neutral pH (7.0 ± 0.01) and at a temperature of 30 ± 1º C. Interaction of amino acids with metal ferrocyanides are found to be maximum at neutral pH. Neutral pH is chosen for the adsorption studies because most of the reactions in biological systems taken place at neutral pH range. Adsorption trend follow Langmuir isotherm model. The Langmuir constants b and Qo were calculated at neutral pH, tryptophane was found to more adsorbed than phenylalanine on both metal ferrocyanides studied. Molybdenum ferrocyanides studied. Molybdenum ferrocyanides was found to have more uptake capacity for both adsorbates than cadmium ferrocyanides. The present study suggests that metal ferrocyanides might have played a role in the stabilization of biomolecules through their surface activity during course of chemical solution and origins of life on primitive earth.

  16. Disruption of a global regulatory gene to enhance central carbon flux into phenylalanine biosynthesis in Escherichia coli.

    PubMed

    Tatarko, M; Romeo, T

    2001-07-01

    Genetic engineering of microbes for commercial metabolite production traditionally has sought to alter the levels and/or intrinsic activities of key enzymes in relevant biosynthetic pathway(s). Microorganisms exploit similar strategies for flux control, but also coordinate flux through sets of related pathways by using global regulatory circuits. We have engineered a global regulatory system of Escherichia coli, Csr (carbon storage regulator), to increase precursor for aromatic amino acid biosynthesis. Disruption of csrA increases gluconeogenesis, decreases glycolysis, and thus elevates phosphoenolpyruvate, a limiting precursor of aromatics. A strain in which the aromatic (shikimate) pathway had been optimized produced twofold more phenylalanine when csrA was disrupted. Overexpression of tktA (transketolase) to increase the other precursor, erythrose-4-phosphate, yielded approximately 1.4-fold enhancement, while both changes were additive. These effects of csrA were not mediated by increasing the regulatory enzymes of phenylalanine biosynthesis. This study introduces the concept of "global metabolic engineering" for second-generation strain improvement. PMID:11375660

  17. Genetic Incorporation of Twelve meta-Substituted Phenylalanine Derivatives Using A Single Pyrrolysyl-tRNA Synthetase

    PubMed Central

    Wang, Yane-Shih; Fang, Xinqiang; Chen, Hsueh-Ying; Wu, Bo; Wang, Zhiyong U.; Hilty, Christian; Liu, Wenshe R.

    2012-01-01

    When coexpressed with its cognate amber suppressing tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant N346A/C348A is able to genetically incorporate twelve meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro, ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone, alkyne, and azide moieties can be applied to site-specifically label proteins with florescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system. PMID:23138887

  18. Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar.

    PubMed

    de Jong, Femke; Hanley, Steven J; Beale, Michael H; Karp, Angela

    2015-09-01

    Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow. PMID:26070140

  19. Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4-Azidomethyl-L-Phenylalanine

    PubMed Central

    Bazewicz, Christopher G.; Liskov, Melanie T.; Hines, Kevin J.; Brewer, Scott H.

    2013-01-01

    We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-Lphenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photo-stability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe. PMID:23865850

  20. Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Trush, Viacheslav V; Kharchenko, Sergiy G; Tanchuk, Vsevolod Yu; Kalchenko, Vitaly I; Vovk, Andriy I

    2015-09-01

    Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin. PMID:26205135

  1. A Survey of Aspartate-Phenylalanine and Glutamate-Phenylalanine Interactions in the Protein Data Bank: Searching for Anion-pi Pairs

    SciTech Connect

    Philip, Vivek M; Harris, Jason B; Adams, Rachel M; Nguyen, Don; Spiers, Jeremy D; Baudry, Jerome Y; Howell, Elizabeth E; Hinde, Robert J

    2011-01-01

    Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion-{pi} pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion-quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242-8249]. To study the role of anion-{pi} interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification of Phe-Asp or -Glu pairs separated by less than 7 {angstrom} in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura-Morokuma energy calculations were performed on roughly 19000 benzene-formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (-2 to -7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion-{pi} pairs are found throughout protein structures, in helices as well as {beta} strands. Numerous pairs also had nearby cation-{pi} interactions as well as potential {pi}-{pi} stacking. While more than 1000 structures did not contain an anion-{pi} pair, the 3134 remaining structures contained approximately 2.6 anion-{pi} pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.

  2. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    SciTech Connect

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C. )

    1989-11-25

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with (35S) sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I.

  3. Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis.

    PubMed

    Ruokolainen, Miina; Ollikainen, Elisa; Sikanen, Tiina; Kotiaho, Tapio; Kostiainen, Risto

    2016-06-22

    Protein phosphorylation has a key role in cell regulation. Oxidation of proteins, in turn, is related to many diseases and to aging, but the effects of phosphorylation on the oxidation of proteins and peptides have been rarely studied. The aim of this study was to examine the mechanistic effect of phosphorylation on peptide oxidation induced by titanium dioxide photocatalysis. The effect of phosphorylation was compared between nonphosphorylated and tyrosine phosphorylated peptides using electrospray tandem mass spectrometry. We observed that tyrosine was the most preferentially oxidized amino acid, but the oxidation reaction was significantly inhibited by its phosphorylation. The study also shows that titanium dioxide photocatalysis provides a fast and easy method to study oxidation reactions of biomolecules, such as peptides. PMID:27268440

  4. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans.

    PubMed

    Calvo, Ana C; Pey, Angel L; Ying, Ming; Loer, Curtis M; Martinez, Aurora

    2008-08-01

    In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S(0.5)(L-Phe) approximately 150 microM; K(m) for tetrahydrobiopterin (BH(4)) approximately 35 microM and comparable V(max)], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution. PMID:18460651

  5. Pyomelanin production in Penicillium chrysogenum is stimulated by L-tyrosine.

    PubMed

    Vasanthakumar, Archana; DeAraujo, Alice; Mazurek, Joy; Schilling, Michael; Mitchell, Ralph

    2015-06-01

    From a tomb in Upper Egypt we isolated a strain of Penicillium chrysogenum that was capable of producing brown pigment in vitro when grown in a minimal salts medium containing tyrosine. We present evidence that this pigment is a pyomelanin, a compound that is known to assist in the survival of some micro-organisms in adverse environments. We tested type strains of Pe. chrysogenum, which were also able to produce this pigment under similar conditions. Inhibitors of the DHN and DOPA melanin pathways were unable to inhibit the formation of the pigment. Fourier transform IR analysis indicated that this brown pigment is similar to pyomelanin. Pyrolysis-GC/MS revealed the presence of phenolic compounds. Using LC/MS, homogentisic acid, the monomeric precursor of pyomelanin, was detected in supernatants of Pe. chrysogenum cultures growing in tyrosine medium but not in cultures lacking tyrosine. Partial regions of the genes encoding two enzymes in the homogentisic acid pathway of tyrosine degradation were amplified. Data from reverse-transcription PCR demonstrated that hmgA transcription was increased in cultures grown in tyrosine medium, suggesting that tyrosine induced the transcription. PMID:25568259

  6. Induction of Phenylalanine Ammonia Lyase and Pisatin by Photosensitive Psoralen Compounds 1

    PubMed Central

    Hadwiger, Lee A.

    1972-01-01

    The psoralen compounds, xanthotoxin and 4,5′, 8-trimethylpsoralen, when activated, increased phenylalanine ammonia lyase (PAL) activity and the synthesis of pisatin in excised pea pods. Pods presoaked 1 hr with 4,5′,8-trimethylpsoralen and then irradiated 4 minutes with 366 nanometer ultraviolet light had twice as much PAL activity 3 hours after irradiation and 12 times as much PAL activity 20 hours after irradiation as the pods of the water-treated control. Increases in PAL activity and pisatin synthesis were not obtained with 4,5′,8-trimethylpsoralen, xanthotoxin, or 366 nanometer light treatment alone. 4,5′,8-Trimethylpsoralen in combination with the irradiation treatment (366 nanometers) enhanced the rate at which l-leucine is incorporated into various fractions of soluble proteins in excised pods 8 hours after treatment. This treatment decreased the rate at which orotic acid is incorporated into RNA. The increase in PAL activity induced by irradiated psoralens was prevented when 6-methylpurine (0.5 milligram per milliliter) or cycloheximide (10 micrograms per milliliter) was applied immediately following the irradiation period. Possible functions of psoralen compounds in plants are discussed. PMID:16658047

  7. The Phenylalanine Ammonia-Lyase Gene Family in Raspberry. Structure, Expression, and Evolution1

    PubMed Central

    Kumar, Amrita; Ellis, Brian E.

    2001-01-01

    In raspberry (Rubus idaeus), development of fruit color and flavor are critically dependent on products of the phenylpropanoid pathway. To determine how these metabolic functions are integrated with the fruit ripening program, we are examining the properties and expression of key genes in the pathway. Here, we report that l- phenylalanine ammonia-lyase (PAL) is encoded in raspberry by a family of two genes (RiPAL1 and RiPAL2). RiPAL1 shares 88% amino acid sequence similarity to RiPAL2, but phylogenetic analysis places RiPAL1 and RiPAL2 in different clusters within the plant PAL gene family. The spatial and temporal expression patterns of the two genes were investigated in various vegetative and floral tissues using the reverse transcriptase competitor polymerase chain reaction assay. Although expression of both genes was detected in all tissues examined, RiPAL1 was associated with early fruit ripening events, whereas expression of RiPAL2 correlated more with later stages of flower and fruit development. Determination of the absolute levels of the two transcripts in various tissues showed that RiPAL1 transcripts were 3- to 10-fold more abundant than those of RiPAL2 in leaves, shoots, roots, young fruits, and ripe fruits. The two RiPAL genes therefore appear to be controlled by different regulatory mechanisms. PMID:11553751

  8. The phenylalanine ammonia lyase (PAL) gene family shows a gymnosperm-specific lineage

    PubMed Central

    2012-01-01

    Background Phenylalanine ammonia lyase (PAL) is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, a precursor for the lignin and flavonoid biosynthetic pathways. To date, PAL genes have been less extensively studied in gymnosperms than in angiosperms. Our interest in PAL genes stems from their potential role in the defense responses of Pinus taeda, especially with respect to lignification and production of low molecular weight phenolic compounds under various biotic and abiotic stimuli. In contrast to all angiosperms for which reference genome sequences are available, P. taeda has previously been characterized as having only a single PAL gene. Our objective was to re-evaluate this finding, assess the evolutionary history of PAL genes across major angiosperm and gymnosperm lineages, and characterize PAL gene expression patterns in Pinus taeda. Methods We compiled a large set of PAL genes from the largest transcript dataset available for P. taeda and other conifers. The transcript assemblies for P. taeda were validated through sequencing of PCR products amplified using gene-specific primers based on the putative PAL gene assemblies. Verified PAL gene sequences were aligned and a gene tree was estimated. The resulting gene tree was reconciled with a known species tree and the time points for gene duplication events were inferred relative to the divergence of major plant lineages. Results In contrast to angiosperms, gymnosperms have retained a diverse set of PAL genes distributed among three major clades that arose from gene duplication events predating the divergence of these two seed plant lineages. Whereas multiple PAL genes have been identified in sequenced angiosperm genomes, all characterized angiosperm PAL genes form a single clade in the gene PAL tree, suggesting they are derived from a single gene in an ancestral angiosperm genome. The five distinct PAL genes detected and verified in P. taeda

  9. DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster abelson proto-oncogene homolog

    SciTech Connect

    Henkemeyer, M.J.; Bennett, R.L.; Gertler, F.B.; Hoffmann, F.M.

    1988-02-01

    The authors report their molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type 1b 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

  10. Cadmium inhibits mouse sperm motility through inducing tyrosine phosphorylation in a specific subset of proteins.

    PubMed

    Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Zhao, Na; Yang, Qiangzhen; Li, Sisi; Li, Xinhong

    2016-08-01

    Cadmium (Cd) has been reported to impair male fertility, primarily by disrupting sperm motility, but the underlying molecular mechanism remains unclear. Here we investigated the effects of Cd on sperm motility, tyrosine phosphorylation, AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and ATP levels in vitro. Our results demonstrated that Cd inhibited sperm motility, GAPDH activity, AMPK activity and ATP production, and induced tyrosine phosphorylation of 55-57KDa proteins. Importantly, all the parameters affected by Cd were restored to normal levels when incubated with 10μM Cd in the presence of 30μM ethylene diamine tetraacetic acid (EDTA). Interestingly, changes of tyrosine phosphorylation levels of 55-57KDa proteins are completely contrary to that of other parameters. These results suggest that Cd-induced tyrosine phosphorylation of 55-57KDa proteins might act as an engine to block intracellular energy metabolism and thus decrease sperm motility. PMID:27233480

  11. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    PubMed

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

  12. A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.

    PubMed

    Mockus, S M; Kumer, S C; Vrana, K E

    1997-08-01

    The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain. PMID:9356925

  13. Phenylalanine-restricted diet should be life long. A case report on long-term follow-up of an adolescent with untreated phenylketonuria.

    PubMed

    Merrick, Joav; Aspler, Shoshana; Schwarz, Gerard

    2003-01-01

    The aim of this brief report is to stimulate a debate and to advocate life-long treatment with phenylalanine restricted diet to persons with phenylketonuria. We present a case of an adolescent male who today is a 65 year old with moderate mental retardation. He was previously observed as having severe challenging and self-injurious behaviour, sleeping disorder, hyperactivity, and masturbation. In the last year before reassessment he displayed loss of weight, muscle weakness resulting in wheelchair placement, upper respiratory infections, and fungal skin infections. After reassessment in 1998 the patient was started on treatment with a phenylalanine-restricted diet supplemented with vitamin B12, iron, and folic acid. At follow-up one year later the patient had regained his weight and was walking around freely. The challenging and self-injurious behaviour, and the masturbation, had ceased, and he was participating in social activities which he had previously not been able to participate in. At follow-up four years later he was still at this level of accomplishment. In general, this patient's quality of life had increased. We therefore recommend life-long treatment with phenylalanine restricted diet for persons with previously treated, and also for those with untreated, phenylketonuria. PMID:12955819

  14. Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP.

    PubMed

    Yoneyama, T; Hatakeyama, K

    1998-08-01

    The activity of GTP cyclohydrolase I is inhibited by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and stimulated by phenylalanine through complex formation with GTP cyclohydrolase I feedback regulatory protein (GFRP). Gel filtration experiments as well as enzyme activity measurements showed that the number of subunits of GFRP in both the inhibitory and stimulatory complexes is equal to that of GTP cyclohydrolase I. Because GFRP is a pentamer and GTP cyclohydrolase I was shown here by cross-linking experiments to be a decamer, the results indicate that two molecules of a pentameric GFRP associate with one molecule of GTP cyclohydrolase I. Gel filtration analysis suggested that the complex has a radius of gyration similar to that of the enzyme itself. These observations support our model that one molecule of GFRP binds to each of the two outer faces of the torus-shaped GTP cyclohydrolase I. For formation of the inhibitory protein complex, both BH4 and GTP were required; the median effective concentrations of BH4 and GTP were 2 and 26 microM, respectively. BH4 was the most potent of biopterins with different oxidative states. Among GTP analogues, dGTP as well as guanosine 5'-O-(3'-thiotriphosphate) exhibited similar inducibility compared with GTP, whereas other nucleotide triphosphates had no effect. On the other hand, phenylalanine alone was enough for formation of the stimulatory protein complex, and positive cooperativity was found for the phenylalanine-induced protein complex formation. Phenylalanine was the most potent of the aromatic amino acids. PMID:9685352

  15. Comparison of different mass spectrometry techniques in the measurement of L-[ring-(13)C6]phenylalanine incorporation into mixed muscle proteins.

    PubMed

    Zabielski, Piotr; Ford, G Charles; Persson, X Mai; Jaleel, Abdul; Dewey, Jerry D; Nair, K Sreekumaran

    2013-02-01

    Precise measurement of low enrichment of stable isotope labeled amino-acid tracers in tissue samples is a prerequisite in measuring tissue protein synthesis rates. The challenge of this analysis is augmented when small sample size is a critical factor. Muscle samples from human participants following an 8 h intravenous infusion of L-[ring-(13)C(6)]phenylalanine and a bolus dose of L-[ring-(13)C(6)]phenylalanine in a mouse were utilized. Liquid chromatography tandem mass spectrometry (LC/MS/MS), gas chromatography (GC) MS/MS and GC/MS were compared to the GC-combustion-isotope ratio MS (GC/C/IRMS), to measure mixed muscle protein enrichment of [ring-(13)C(6)]phenylalanine enrichment. The sample isotope enrichment ranged from 0.0091 to 0.1312 molar percent excess. As compared with GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS showed coefficients of determination of R(2)= 0.9962 and R(2) = 0.9942, and 0.9217 respectively. However, the precision of measurements (coefficients of variation) for intra-assay are 13.0%, 1.7%, 6.3% and 13.5% and for inter-assay are 9.2%, 3.2%, 10.2% and 25% for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. The muscle sample sizes required to obtain these results were 8 µg, 0.8 µg, 3 µg and 3 µg for GC/C/IRMS, LC/MS/MS, GC/MS/MS and GC/MS, respectively. We conclude that LC/MS/MS is optimally suited for precise measurements of L-[ring-(13)C(6)]phenylalanine tracer enrichment in low abundance and in small quantity samples. PMID:23378099

  16. The use of a food supplementation with D-phenylalanine, L-glutamine and L-5-hydroxytriptophan in the alleviation of alcohol withdrawal symptoms.

    PubMed

    Jukić, Tomislav; Rojc, Bojan; Boben-Bardutzky, Darja; Hafner, Mateja; Ihan, Alojz

    2011-12-01

    We described the use of a food supplementation with D-phenylalanine, L-glutamine and L-5-hydroxytriptophan in the alleviation of alcohol withdrawal symptoms in patients starting a detoxification therapy. Since abstinence from ethanol causes a hypodopaminergic and a hypoopioidergic environment in the reword system circuits, manifesting with withdrawal symptoms, food supplements that contains D-phenylalanine a peptidase inhibitor (of opioide inactivation) and L-amino-acids (for dopamine synthesis) were used to replenish a lack in neurotransmitters and alleviate the symptoms of alcohol withdrawal. 20 patients suffering from alcohol addictions starting a detoxification therapy have been included in a prospective, randomized, double blind study. The patients have been randomly devided in two groups. One group recieved for a period of 40 days a food supplement containing D-phenylalanine, L-glutamine and L-5-hydroxytriptophan (investigation group), and the control (placebo) group. On the first day of hospitalization the patients performed a SCL-90-R test, and blood samples were taken for measuring liver enzymes, total bilirubin, unbound cortisol and lymphocyte populations. The same was done on the 40th day of hospitalization. During the therapy a significant decrease in SCL-90-R psychiatric symptoms scores and a significant increase in CD4 lymphocyte count was observed in the investigation group. The cortisol values were significantly, but equally decreased in both groups, the same was with the liver enzymes and the total bilirubin values. We conclude that abstinence causes a major stress for the patients. The use of food supplement containing D-phenylalanine, L-glutamine and L-5-hydroxytriptophan alleviates the withdrawal symptoms and causes a rise in CD4 lymphocyte population, but it dose not affect the serum cortisol levels, which are probably more affected by liver inflammation and the liver restitution. PMID:22397264

  17. Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

    SciTech Connect

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro Univ. of Tokyo )

    1989-08-01

    A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M{sub r} of its subunit was 77,000. The cells converted ({sup 14}C)-L-phenylalanine into ({sup 14}C)-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M{sub r} 77,137), a 22-bp 5{prime}-noncoding region and a 207-bp 3{prime}-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.

  18. Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

    PubMed Central

    Hart, Kristen C.; Robertson, Scott C.; Donoghue, Daniel J.

    2001-01-01

    Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3. PMID:11294897

  19. Influence of the tyrosine environment on the second harmonic generation of iturinic antimicrobial lipopeptides at the air-water interface.

    PubMed

    Nasir, Mehmet Nail; Benichou, Emmanuel; Loison, Claire; Russier-Antoine, Isabelle; Besson, Françoise; Brevet, Pierre-François

    2013-12-01

    The second harmonic generation (SHG) response at the air-water interface from the tyrosine-containing natural iturinic cyclo-lipopeptides mycosubtilin, iturin A and bacillomycin D is reported. It is shown that this response is dominated by the single tyrosine residue present in these molecules owing to the large first hyperpolarizability arising from the non-centrosymmetric aromatic ring structure of this amino acid. The SHG response of these iturinic antibiotics is also compared to the response of surfactin, a cyclo-lipopeptide with a similar l,d-amino acid sequence but lacking a tyrosine residue, and PalmATA, a synthetic linear lipopeptide possessing a single tyrosine residue but lacking the amino acid sequence structuring the cycle of the iturinic antibiotics. From the light polarization analysis of the SHG response, it is shown that the tyrosine local environment is critical in defining the SHG response of these peptides at the air-water interface. Our results demonstrate that tyrosine, similar to tryptophan, can be used as an endogenous molecular probe of peptides and proteins for SHG at the air-water interface, paving the way for SHG studies of other tyrosine-containing bioactive molecules. PMID:24149982

  20. Structure of tyrosine aminotransferase from Leishmania infantum

    PubMed Central

    Moreno, M. A.; Abramov, A.; Abendroth, J.; Alonso, A.; Zhang, S.; Alcolea, P. J.; Edwards, T.; Lorimer, D.; Myler, P. J.; Larraga, V.

    2014-01-01

    The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding l-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structure of LiTAT at 2.35 Å resolution is reported, and it is confirmed that the presence of two Leishmania-specific residues (Gln55 and Asn58) explains, at least in part, this specific reactivity. The difference in substrate specificity between leishmanial and mammalian TAT and the importance of this enzyme in parasite metabolism suggest that it may be a useful target in the development of new drugs against leishmaniasis. PMID:24817714

  1. Classroom Demonstration of a Spot Test for Pbenylpyruvic Acid and Its Relationship to Phenylketonuria

    ERIC Educational Resources Information Center

    Halkides, Christopher J.

    2004-01-01

    Classical phenylketonuria (PKU) is caused by a lack activity in the enzyme phenylalanine hydroxylase, leading to elevated concentrations of phenylalanine in the blood. A simple demonstration and three advanced demonstrations of a spot test for phenylpyruvic acid and its relationship to phenylketonuria are given.

  2. Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

    SciTech Connect

    Evdokimov, Artem G.; Mekel, Marlene; Hutchings, Kim; Narasimhan, Lakshmi; Holler, Tod; McGrath, Teresa; Beattie, Bryan; Fauman, Eric; Yan, Chunhong; Heaslet, Holly; Walter, Richard; Finzel, Barry; Ohren, Jeffrey; McConnell, Patrick; Braden, Timothy; Sun, Fang; Spessard, Cindy; Banotai, Craig; Al-Kassim, Loola; Ma, Weijun; Wengender, Paul; Kole, Denis; Garceau, Norman; Toogood, Peter; Liu, Jia

    2008-07-08

    In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality -- they only diffracted X-rays to 3--5 {angstrom} resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 {angstrom} or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

  3. [Micro-droplet characterization and its application for amino acid detection in droplet microfluidic system].

    PubMed

    Yuan, Huiling; Dong, Libing; Tu, Ran; Du, Wenbin; Ji, Shiru; Wang, Qinhong

    2014-01-01

    Recently, the droplet microfluidic system attracts interests due to its high throughput and low cost to detect and screen. The picoliter micro-droplets from droplet microfluidics are uniform with respect to the size and shape, and could be used as monodispensed micro-reactors for encapsulation and detection of single cell or its metabolites. Therefore, it is indispensable to characterize micro-droplet and its application from droplet microfluidic system. We first constructed the custom-designed droplet microfluidic system for generating micro-droplets, and then used the micro-droplets to encapsulate important amino acids such as glutamic acid, phenylalanine, tryptophan or tyrosine to test the droplets' properties, including the stability, diffusivity and bio-compatibility for investigating its application for amino acid detection and sorting. The custom-designed droplet microfluidic system could generate the uniformed micro-droplets with a controllable size between 20 to 50 microm. The micro-droplets could be stable for more than 20 h without cross-contamination or fusion each other. The throughput of detection and sorting of the system is about 600 micro-droplets per minute. This study provides a high-throughput platform for the analysis and screening of amino acid-producing microorganisms. PMID:24818488

  4. Alterations in the levels of plasma amino acids in polycystic ovary syndrome- A pilot study

    PubMed Central

    Unni, C. Sumithra N.; Lakshman, Lakshmi R.; Vaidyanathan, Kannan; Subhakumari, K.N.; Menon, N. Leela

    2015-01-01

    Background & objectives: Plasma amino acid levels are known to be altered in conditions like sepsis and burns which are situations of metabolic stress. Polycystic ovary syndrome (PCOS), a condition which affects a woman throughout her life, is said to be associated with metabolic stress. This study was undertaken to assess if there were significant alterations in the levels of plasma amino acids in women with PCOS. Methods: Sixty five women with PCOS along with the similar number of age matched normal controls were included in this study. Levels of 14 amino acids were determined using reverse phase high performance liquid chromatography. Results: The levels of methionine, cystine, isoleucine, phenylalanine, valine, tyrosine, proline, glycine, lysine and histidine were found to be significantly (P<0.001) lower in cases than in controls. Arginine and alanine levels were found to be significantly (P<0.001) higher in cases compared with controls. Interpretation & conclusions: Our findings showed significant derangement in the levels of plasma amino acids in women with PCOS which might be due to the oxidative and metabolic stress associated with it. Further studies need to be done to confirm the findings. PMID:26658589

  5. Postprandial Levels of Branch Chained and Aromatic Amino Acids Associate with Fasting Glycaemia.

    PubMed

    Ottosson, Filip; Ericson, Ulrika; Almgren, Peter; Nilsson, Jeanette; Magnusson, Martin; Fernandez, Céline; Melander, Olle

    2016-01-01

    High fasting plasma concentrations of isoleucine, phenylalanine, and tyrosine have been associated with increased risk of hyperglycaemia and incidence of type 2 diabetes. Whether these associations are diet or metabolism driven is unknown. We examined how the dietary protein source affects the postprandial circulating profile of these three diabetes associated amino acids (DMAAs) and tested whether the postprandial DMAA profiles are associated with fasting glycaemia. We used a crossover design with twenty-one healthy individuals and four different isocaloric test meals, containing proteins from different dietary sources (dairy, fish, meat, and plants). Analysis of the postprandial DMAAs concentrations was performed using targeted mass spectrometry. A DMAA score was defined as the sum of all the three amino acid concentrations. The postprandial area under the curve (AUC) of all the three amino acids and the DMAA score was significantly greater after intake of the meal with dairy protein compared to intake of the three other meals. The postprandial AUC for the DMAA score and all the three amino acids strongly associated with fasting glucose level and insulin resistance. This indicates the importance of the postprandial kinetics and metabolism of DMAAs in understanding the overall association between DMAAs and glycaemia. PMID:27274867

  6. Postprandial Levels of Branch Chained and Aromatic Amino Acids Associate with Fasting Glycaemia

    PubMed Central

    Ottosson, Filip; Ericson, Ulrika; Almgren, Peter; Nilsson, Jeanette; Magnusson, Martin; Fernandez, Céline; Melander, Olle

    2016-01-01

    High fasting plasma concentrations of isoleucine, phenylalanine, and tyrosine have been associated with increased risk of hyperglycaemia and incidence of type 2 diabetes. Whether these associations are diet or metabolism driven is unknown. We examined how the dietary protein source affects the postprandial circulating profile of these three diabetes associated amino acids (DMAAs) and tested whether the postprandial DMAA profiles are associated with fasting glycaemia. We used a crossover design with twenty-one healthy individuals and four different isocaloric test meals, containing proteins from different dietary sources (dairy, fish, meat, and plants). Analysis of the postprandial DMAAs concentrations was performed using targeted mass spectrometry. A DMAA score was defined as the sum of all the three amino acid concentrations. The postprandial area under the curve (AUC) of all the three amino acids and the DMAA score was significantly greater after intake of the meal with dairy protein compared to intake of the three other meals. The postprandial AUC for the DMAA score and all the three amino acids strongly associated with fasting glucose level and insulin resistance. This indicates the importance of the postprandial kinetics and metabolism of DMAAs in understanding the overall association between DMAAs and glycaemia. PMID:27274867

  7. Molecularly imprinted photonic polymer based on β-cyclodextrin for amino acid sensing.

    PubMed

    Liu, Xiao-Yan; Fang, Hong-Xun; Yu, Li-Ping

    2013-11-15

    A novel molecularly imprinted photonic polymer (MIPP) using maleic anhydride modified β-cyclodextrin (β-CD) and acrylic acid as functional monomers has been presented for amino acid sensing. Reactive β-CD monomer carrying vinyl carboxylic acid functional groups was first synthesized. MIPP was fabricated by filling precursor solution into the interstitial spaces of polystyrene photonic crystal templates, followed by a thermal polymerization at 55 °C. Characterization showed that the MIPP possessed an opal photonic crystal structure. This β-CD-based MIPP could undergo a swelling change from 590 nm to 704 nm and still retain the molecular imprinting recognition ability during the sensing of L-phenylalanine (L-Phe). A function relationship was found between the diffraction wavelength shift and the logarithm of L-Phe concentration in the range of 10(-8)M to 10(-4)M at pH 6. A wavelength shift of 114 nm for L-Phe was observed within 30s, whereas there were no obvious shifts for d-Phe, L-tyrosine and L-tryptophan, indicating that the β-CD-based MIPP had high specificity and rapid response to L-Phe. The developed MIPP sensor has been applied to detect L-Phe in compound amino acid injection sample. PMID:24148405

  8. Capsaicin Synthesis Requires in Situ Phenylalanine and Valine Formation in in Vitro Maintained Placentas from Capsicum chinense.

    PubMed

    Baas-Espinola, Fray M; Castro-Concha, Lizbeth A; Vázquez-Flota, Felipe A; Miranda-Ham, María L

    2016-01-01

    Capsaicinoids (CAP) are nitrogenous metabolites formed from valine (Val) and phenylalanine (Phe) in the placentas of hot Capsicum genotypes. Placentas of Habanero peppers can incorporate inorganic nitrogen into amino acids and have the ability to secure the availability of the required amino acids for CAP biosynthesis. In order to determine the participation of the placental tissue as a supplier of these amino acids, the effects of blocking the synthesis of Val and Phe by using specific enzyme inhibitors were analyzed. Isolated placentas maintained in vitro were used to rule out external sources' participation. Blocking Phe synthesis, through the inhibition of arogenate dehydratase, significantly decreased CAP accumulation suggesting that at least part of Phe required in this process has to be produced in situ. Chlorsulfuron inhibition of acetolactate synthase, involved in Val synthesis, decreased not only Val accumulation but also that of CAP, pointing out that the requirement for this amino acid can also be fulfilled by this tissue. The presented data demonstrates that CAP accumulation in in vitro maintained placentas can be accomplished through the in situ availability of Val and Phe and suggests that the synthesis of the fatty acid chain moiety may be a limiting factor in the biosynthesis of these alkaloids. PMID:27338325

  9. A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin.

    PubMed

    Connor, Katherine L; Colabroy, Keri L; Gerratana, Barbara

    2011-10-18

    We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases. PMID:21919439

  10. Control of the Handedness of Self-assemblies of Dipeptides by the Chirality of Phenylalanine and Steric Hindrance of Phenylglycine.

    PubMed

    Lin, Shuwei; Li, Yi; Li, Baozong; Yang, Yonggang

    2016-07-26

    Eight dipeptides, composed of phenylalanine and phenylglycine, that are able to self-assemble into twisted nanoribbons in deionized water are synthesized. The handedness of the nanoribbons is controlled by the chirality of the phenylalanine and the steric hindrance owing to the phenyl group of the phenylglycine. When the phenylalanine is at the C-terminal, π-π stacking by the phenyl groups, hydrogen bonding by the NH group of the phenylalanine, and hydrophobic associations of the alkyl chains control the stacking of the molecules. When phenylglycine is at the C-terminal, the chiral π-π stacking by the phenyl groups of the phenylalanines is suppressed. The hydrogen bonds formed by the NH groups of the phenylalanines had a greater contribution on forming organic self-assemblies than those formed by the NH groups of the phenylglycines. PMID:27389603

  11. Phenylalanine flux and gastric emptying are not affected by replacement of casein with whey protein in the diet of adult cats consuming frequent small meals

    PubMed Central

    2014-01-01

    Background Decreasing the rate of protein emptying from the stomach may improve efficiency of utilization of dietary amino acids for protein deposition. Some studies in rats and humans have shown casein to be more slowly released from the stomach than whey protein. To test if casein induces a slower rate of gastric emptying in cats than whey protein, L-[1-13C]phenylalanine (Phe) was dosed orally into 9 adult cats to estimate gastric emptying and whole-body Phe flux. Results Concentrations of indispensable amino acids in plasma were not significantly affected by dietary protein source. First-pass splanchnic extraction of Phe was not different between diets and averaged 50% (SEM = 3.8%). The half-time for gastric emptying averaged 9.9 min with casein and 10.3 min with whey protein, and was not significantly different between diets (SEM = 1.7 min). Phenylalanine fluxes were 45.3 and 46.5 μmol/(min · kg) for casein- and whey-based diets, respectively (SEM = 4.7 μmol/(min · kg)). Conclusions In adult cats fed frequent small meals, the replacement of casein with whey protein in the diet does not affect supply or utilization of amino acids. These two milk proteins appear to be equally capable of meeting the dietary amino acid needs of cats. PMID:25266643

  12. Biodegradation of the Allelopathic Chemical m-Tyrosine by Bacillus aquimaris SSC5 Involves the Homogentisate Central Pathway

    PubMed Central

    Khan, Fazlurrahman; Kumari, Munesh; Cameotra, Swaranjit Singh

    2013-01-01

    m-Tyrosine is an amino acid analogue, exuded from the roots of fescue grasses, which acts as a potent allelopathic and a broad spectrum herbicidal chemical. Although the production and toxic effects of m-tyrosine are known, its microbial degradation has not been documented yet. A soil microcosm study showed efficient degradation of m-tyrosine by the inhabitant microorganisms. A bacterial strain designated SSC5, that was able to utilize m-tyrosine as the sole source of carbon, nitrogen, and energy, was isolated from the soil microcosm and was characterized as Bacillus aquimaris. Analytical methods such as HPLC, GC-MS, and 1H-NMR performed on the resting cell samples identified the formation of 3-hydroxyphenylpyruvate (3-OH-PPA), 3-hydroxyphenylacetate (3-OH-PhAc), and homogentisate (HMG) as major intermediates in the m-tyrosine degradation pathway. Enzymatic assays carried out on cell-free lysates of m-tyrosine-induced cells confirmed transamination reaction as the first step of m-tyrosine degradation. The intermediate 3-OH-PhAc thus obtained was further funneled into the HMG central pathway as revealed by a hydroxylase enzyme assay. Subsequent degradation of HMG occurred by ring cleavage catalyzed by the enzyme homogentisate 1, 2-dioxygenase. This study has significant implications in terms of understanding the environmental fate of m-tyrosine as well as regulation of its phytotoxic effect by soil microorganisms. PMID:24098407

  13. Coexpression of Tyrosine Hydroxylase, GTP Cyclohydrolase I, Aromatic Amino Acid Decarboxylase, and Vesicular Monoamine Transporter 2 from a Helper Virus-Free Herpes Simplex Virus Type 1 Vector Supports High-Level, Long-Term Biochemical and Behavioral Correction of a Rat Model of Parkinson’s Disease

    PubMed Central

    SUN, MEI; KONG, LINGXIN; WANG, XIAODAN; HOLMES, COURTNEY; GAO, QINGSHENG; ZHANG, GUO-RONG; PFEILSCHIFTER, JOSEF; GOLDSTEIN, DAVID S.; GELLER, ALFRED I.

    2006-01-01

    Parkinson’s disease is due to the selective loss of nigrostriatal dopaminergic neurons. Consequently, many therapeutic strategies have focused on restoring striatal dopamine levels, including direct gene transfer to striatal cells, using viral vectors that express specific dopamine biosynthetic enzymes. The central hypothesis of this study is that coexpression of four dopamine biosynthetic and transporter genes in striatal neurons can support the efficient production and regulated, vesicular release of dopamine: tyrosine hydroxylase (TH) converts tyrosine to l-3,4-dihydroxyphenylalanine (l -DOPA), GTP cyclohydrolase I (GTP CH I) is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, aromatic amino acid decarboxylase (AADC) converts l -DOPA to dopamine, and a vesicular monoamine transporter (VMAT-2) transports dopamine into synaptic vesicles, thereby supporting regulated, vesicular release of dopamine and relieving feedback inhibition of TH by dopamine. Helper virus-free herpes simplex virus type 1 vectors that coexpress the three dopamine biosynthetic enzymes (TH, GTP CH I, and AADC; 3-gene-vector) or these three dopamine biosynthetic enzymes and the vesicular monoamine transporter (TH, GTP CH I, AADC, and VMAT-2; 4-gene-vector) were compared. Both vectors supported production of dopamine in cultured fibroblasts. These vectors were microinjected into the striatum of 6-hydroxydopamine-lesioned rats. These vectors carry a modified neurofilament gene promoter, and γ-aminobutyric acid (GABA)-ergic neuron-specific gene expression was maintained for 14 months after gene transfer. The 4-gene-vector supported higher levels of correction of apomorphine-induced rotational behavior than did the 3-gene-vector, and this correction was maintained for 6 months. Proximal to the injection sites, the 4-gene-vector, but not the 3-gene-vector, supported extracellular levels of dopamine and dihydroxyphenylacetic acid (DOPAC) that were similar to those observed in

  14. Plasma and skeletal muscle amino acids following severe burn injury in patients and experimental animals.

    PubMed Central

    Stinnett, J D; Alexander, J W; Watanabe, C; MacMillan, B G; Fischer, J E; Morris, M J; Trocki, O; Miskell, P; Edwards, L; James, H

    1982-01-01

    This study describes and analyzes sequential changes in plasma and skeletal muscle free amino acids following severe burn injury. Plasma free amino acids were determined in children (n = 9) with burns averaging 60% total b