Presence Of Tryptophan Research Articles

Interaction of the trp repressor from Escherichia coli with a constitutive trp operator.

The interaction of the trp repressor from Escherichia coli with a 20 bp fragment of DNA (CGTACTGATT.AATCAGTACG) corresponding to a mutant trp operator was studied by c.d. in the presence and absence of the co-repressor, L-tryptophan, and as a function of the concentration of K+ and Na+ ions. The affinity of the repressor for the mutant operator in the presence of tryptophan is about three orders of magnitude lower than the wild-type sequence. Binding in the absence of tryptophan is about 100-fold weaker than to the wild-type. The dependence of the dissociation constant on the concentration of K+ or Na+ is weak [d(log Ks)/d(log[M+]) = 2.5], and independent of the cation, indicating that electrostatic interactions are not as important for this repressor as for others.

Photochemical-like behaviour of riboflavin in the dark promoted by enzyme-generated triplet acetone.

Abstract— Triplet acetone obtained enzymically by the isobutyraldehyde/peroxidase/O2 system transfers its energy to the riboflavin, which changes into a decomposition product of lumichrome type and produces aggregates. When riboflavin and tryptophan are added to this system, further products—formylkynurenine and an adduct between the riboflavin and the tryptophan—are formed. The adduct can be separated by gel filtration and ion exchange chromatography and is similar to that prepared by irradiating riboflavin in the presence of tryptophan.

Isolation of two neuropeptides in the AKH/RPCH-family from horseflies (diptera)

Two neuropeptides (DCCI and DCCII) in the adipokinetic/red pigment concentrating hormone-family have been isolated and purified from the corpora cardiaca of horseflies (Diptera : Tabanidae). Both peptides were purified by a sequence of three reversed phase-high performance liquid chromatographic steps. Amino acid analysis of the purified peptides indicated the following composition for DCCI: Glx(l), Gly(1), Leu(1), Phe(1), Pro(1), Thr(2) and for DCCII: Glx(1), Gly(2), Leu(1), Phe(1), Pro(1), Thr(2), and Tyr(1). Photodiode array ultraviolet spectroscopy indicated the presence of tryptophan in both DCCI and II. Both DCCI and II had red pigment concentrating hormone activity in the crab, Uca pugilator.

Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 ( S 0.5 = 188 μM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 μ m tryptophan to the assay resulted in maximum activation of the enzyme with a K a of 1.2 μ m. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0–8.0. Addition of tryptophan also decreased the K m for chorismate from 185 to 45 μ m. Tryptophan (0.1 m m) completely protected CM from inhibition by phenylalanine (1.8 m m) and tyrosine (1.8 m m). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 m m concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with K i values of 550 and 440 m m, respectively. Arogenate (1.0 m m) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.

Alkaloidbildung und Biosynthese aromatischer Aminosäuren in Zellkulturen von Ruta graveolens L.: Alkaloid Formation and Biosynthesis of Aromatic Amino Acids in Cell Suspension Cultures of Ruta graveolens L.

Summary Growth, alkaloid formation and enzyme activities of some shikimic acid pathway enzymes were examined in cell suspension cultures of Ruta graveolens. Aromatic amino acids inhibit slightly the activity of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase indicating a weak allosteric regulation. Feedback inhibition of chorismate mutase by phenylalanine and tyrosine as well of anthranilate synthase by tryptophan was observed. Tryptophan at 1mM stimulated the activity of chorismate mutase and reversed the Phe and/or Tyr inhibition of CM. Tissue cultures of Ruta graveolens were grown in liquid media containing L-tryptophan. The levels of DAHP-synthase and tryptophan synthase were elevated in these cells. A slight repression of anthranilate synthase was observed in 4 days old cells which were grown in media containing upto 1.5 mM tryptophan. A remarkable decrease of AA-Sase activity was found in 10 days old cells cultivated in the presence of tryptophan. A low alkaloid producing R. graveolens cell line was cultivated in the presence of exogenously added rutacridone and the influence of this particular acridone alkaloid on growth and shikimate pathway enzymes was investigated.

Photochemical coupling of 5-bromouracil to tryptophan, tyrosine and histidine, peptide-like derivatives in aqueous fluid solution.

AbstractDirect irradiation of 5‐bromouracil (BU) in aqueous fluid solution in the presence of tryptophan (trp), tyrosine (tyr) or histidine (his) derivatives using a XeCl excimer laser at 308 nm yielded photocoupling of BU to the aromatic ring of each amino acid. Irradiation of BU at 308 nm most likely results in excitation of the n‐φ* transition, intersystem crossing to the triplet manifold, and coupling via electron transfer from the aromatic amino acid. The coupling observed was regiospecific between the 5‐position of uracil (U) and the 2‐position of the indole and phenol rings and the 5‐position of the imidazole ring of the respective amino acids. Quantum yields of photocoupling to BU ranged from 1 × 10‐3 to 7 × 10‐3 and paralleled known rates of electron transfer and ionization potentials of the aromatic rings. The photocoupling between BU and some of the aromatic amino acid peptide‐like derivatives possibly mimics photocrosslinking of BU‐DNA to associated proteins, a potentially useful photoreaction for studying nucleic acid‐protein interactions. Formation of crosslinks of the type proposed here might be detected by the characteristic fluorescence emission of the uracil amino acid adducts.

Binding of ketoprofen with bovine serum albumin

The binding of Ketoprofen, i.e 2-(3-benzoylphenyl) propionic acid, with bovine serum albumin, BSA, was investigated by equilibrium dialysis. Limiting the studies to low drug (10 to 100 μM) concentrations, the binding data correspond to a single set of binding sites, namely, the high-affinity sites. Scatchard and Klotz methods of analysis have been employed to determine the binding parameters for the high-affinity sites. The binding constant does not vary significantly with [BSA] in the concentration range 7·25 to 21·5 μM. The molar ratio, [drug]/[protein] is found to be a critical factor in determining the nature and number of binding sites. The quenching of intrinsic fluorescence of the protein at the emission wavelength of 346 nm indicates the presence of tryptophan in the binding site.

The production of inflammatory compounds by Propionibacterium acnes and other skin organisms.

Culture supernatants from four species of skin microorganisms (P. acnes, P. avidum, P. granulosum and S. epidermidis) were assayed for smooth muscle contracting substances which are indicative of inflammatory activity. At least three types of smooth muscle contracting substances were detected. These were: first, a substance active on a rat fundic strip preparation and antagonized by N,N-DMT. Activity was enhanced when cultures were grown in the presence of tryptophan. This substance was probably tryptamine. Second, a substance active on a guinea-pig ileum preparation and antagonized by mepyramine. Activity was enhanced when cultures were grown in the presence of histidine. This substance was probably histamine. Third, a substance active on a rat fundic strip preparation but not antagonized by N,N-DMT, mepyramine, atropine or indomethacin. Activity was enhanced when cultures were grown in the presence of glucose. This activity was probably due to acetate, propionate or other short-chain fatty-acid salts. Chromatographic analysis confirmed the presence of histamine, tryptamine and short-chain fatty acids in the culture supernatants. These substances if produced in vivo may cause or contribute to inflammation and pain directly without the prior mediation of the immune system. Cell extracts of Propionibacterium species were analysed by bioassay for the presence of prostaglandin-like compounds. These could not be detected in any of the eight strains of organisms tested.

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.

Interaction between 7-anilino-4-methylcoumarin-3-acetic acid and human serum albumin.

The fluorescence characteristics of 7-anilino-4-methylcoumarin-3-acetic acid (A MCA) were examined in human serum albumin (HSA) solution. The fluorescence of AMCA was significantly enhanced in the presence of HSA. Scatchard plots and continuous variation plots indicated that one molecule of AMCA binds to HSA. The binding parameters of AMCA were also estimated. Specific binding sites on HSA were examined in connection with Sudlow's classification. The primary binding site of AMCA was found to be site III (digitoxin site) from the results of competitive displacement studies with several drugs. Furthermore, it was suggested from the fluorescence behavior in the presence of tryptophan-, tyrosine-, and lysine-modified HSAs that the binding site of AMCA lies in a region near a tryptophan residue on HSA. The present data indiate that AMCA is useful as a site III marker.

Precipitation of calcium oxalates from high ionic strength solutions IV. Testing of kinetic models

The following parameters characterizing the kinetics of precipitation from unseeded solutions are defined: the induction period ( t i), the maximum precipitation rate ( R max), the number of particles per cm 3 ( N), the order ( p) and rate constants ( K α , K s) of the crystal growth process, the critical time ( t aggr ∗), the critical supersaturation ( S aggr ∗) and the rate constant of aggregation ( k j) as well as the relative changes in the surface area ( ΔP/ P) and the degree of the reaction (Δα/α) due to aggregation. The information which these parameters yield on nucleation, crystal growth and aggregation is evaluated. The model is applied to the precipitation of calcium oxalate trihydrate (COT) from 0.3 molar sodium chloride solutions, with and without the presence of tryptophan or ornithine. At the given concentrations both aminoacids inhibit crystal growth and change the mode of aggregation of COT.

Analysis by HPLC – mass spectrometry of the indole compounds released by the ectomycorrhizal fungus Hebeloma hiemale in pure culture

Indole compounds released by Hebeloma hiemale Bres. in pure culture were studied by cultivating the fungus on a nutrient solution supplemented or not with 1 mM tryptophan. Analytical thin-layer chromatography revealed numerous indole compounds in 7-week-old culture filtrates obtained in the presence of tryptophan. Such compounds were isolated by preparative thin-layer chromatography and purified by high performance liquid chromatography. The most abundant of them, i.e., indolyl-3-acetic acid, indolyl-3-carboxylic acid, and indole-3-aldehyde, were identified by mass spectrometry. Indole-3-aldehyde and indolyl-3-carboxylic acid, which were detected in filtrates throughout the culture period, might result from breakdown of indolyl-3-acetic acid by fungal exocellular enzymes.

A calcium binding protein from Drosophila melanogaster which activates cAMP phosphodiesterase: Comparison of this protein with porcine brain calmodulin

A calcium binding protein from Drosophila melanogaster has been isolated and characterized. This protein shows several analogies with pig brain calmodulin in its molecular weight, isoelectric point, peptide maps, calcium binding properties, and ability to activate cyclic AMP phosphodiesterase. However, some differences were observed; the most remarkable one is the presence of tryptophan, an amino acid which is absent from all the calmodulins analyzed previously.

Further evidence for the heterogeneity of serum albumin

1. 1. Albumin samples from three species (avian, bovine and human) were eieetrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregrarn from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. 2. 2. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, α 1-glycoprotein, α 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. 3. 3. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. 4. 4. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. 5. 5. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.

Mammalian ribonucleases: The absence of a glycosylated Asn-Pro-Thr sequence in horse ribonuclease and the presence of tryptophan at position 39 in horse and dromedary ribonuclease

Parts of the amino acid sequences of horse and dromedary pancreatic ribonuclease were reinvestigated. The sequence of residues 21–25 in horse ribonuclease is Ser-Asn-Pro-Thr-Tyr or Ser-Asn-Ser-Thr-Tyr. The asparagine in the latter sequence is glycosylated. Horse ribonuclease possesses four additional amino acid residues at the C-terminus, like a number of other ribonucleases. Position 39 in horse and dromedary ribonuclease is not deleted but is occupied by tryptophan.

Characterization of a substance produced by Azospirillum which causes branching of wheat root hairs

Previous studies have shown that Azospirillum or its culture filtrates induce root hairs in wheat seedlings to branch; this phenomenon is influenced by the bacterial genome at the strain level and by the plant genome at the cultivar level. Strain Sp245 caused most branching of root hairs; between wheat cultivars frequency of branching increased in the following order: cv. Tobari, cv. Tonari, cv. BH1146, cv. Lagoa. Similarly, plant growth response to inoculation with Azospirillum, measured in mature plants, increased in the same order, suggesting that both phenomena (root hair branching and plant growth response) have a similar basis. In this study, the "branching substance" present in Azospirillum culture filtrate was found to be thermostable at 100 °C, removed by dialysis, present in the acidic nonpolar fraction of the cell-free extract, and co-chromatographed with authentic indole acetic acid on thin-layer chromatograms. Purified indole acetic acid caused branching of root hairs similar to that caused by the Azospirillum culture filtrates. Susceptibility of wheat cultivars to indole acetic acid induced branching increased in the order: cv. Tobari, cv. Tonari, cv. BH1146, cv. Lagoa. Of five strains examined, strain Sp245 produced the most indole acetic acid in culture in the presence of tryptophan. The presence of indole acetic acid was confirmed by gas chromatography – mass spectrometry.

The purification of kallikrein from human whole saliva

1. 1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%. 2. 2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography. 3. 3. The results indicate that another protein component binds to the enzyme at pH 8.0. 4. 4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate. 5. 5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments. 6. 6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700. 7. 7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point. 8. 8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas.

Adenosine (S)-5'-[alpha-17O, 18O2]triphosphate has been synthesized and used to investigate the stereochemical course of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas. It is shown that the reaction proceeds by displacement of Mg pyrophosphate from MgATP with inversion of configuration at P alpha. Tryptophanyl-tRNA synthetase catalyses positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the presence of tryptophan but not in its absence or in the presence of the competitive inhibitors tryptamine and tryptophanol. These observations eliminate a multi-adenylyl transfer or dissociative mechanism and in combination with the sterochemical study leave a direct associative 'in line' displacement at P alpha of MgATP as the only tenable mechanism for the activation of tryptophan by tryptophanyl-tRNA synthetase.

Absolute dependence of phenylalanine and tyrosine biosynthetic enzyme on tryptophan in Candida maltosa.

Candida maltosa synthesizes phenylalanine and tyrosine only via phenylpyruvate and p-hydroxyphenylpyruvate. Tryptophan is absolutely necessary for the enzymatic reaction of chorismate mutase and prephenate dehydrogenase; activity of prephenate dehydratase can be increased 2.5-fold in the presence of tryptophan. Activation of the chorismate mutase, prephenate dehydratase and prephenate dehydrogenase by tryptophan is competitive with respect to chorismate and prephenate with Ka 0.06mM, 0.56mM and 1.7mM. In addition tyrosine is a competitive inhibitor of chorismate mutase (Ki = 0.55mM) and prephenate dehydrogenase (Ki = 5.5mM).

Crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase containing enzymatically formed acyl transfer product tryptophanyl-ATP, an active site maker for the 3' CCA terminus of tryptophanyl-tRNATrp.

It has previously been shown that tryptophanyl-tRNA synthetase from Bacillus stearothermophilus crystallizes in different forms, depending on the substrates present during crystallization [Carter, C. W., Jr., & Carter, C. W. (1979) J. Biol. Chem. 254, 12219-12223]. Radiolabeling experiments show that the tetragonal crystals (type IV), grown in the presence of tryptophan and ATP, contain enzymatically formed 3'(2')-tryptophanyladenosine 5'-triphosphate (Trp-ATP). Trp-ATP is formed by acyl transfer of the tryptophanyl moiety of an acyladenylate intermediate, Trp-5'-AMP, to a second molecule of ATP bound in the site normally occupied by the 3' CCA terminus of tRNATrp. This compound is therefore a chemical marker in type IV crystals for that part of the tRNA binding site on the synthetase. Solution of this crystal structure, now in progress, may therefore provide useful information concerning the mechanism of aminoacylation of tRNATrp by this enzyme and may help locate its tRNA binding site.