Inhibitory activities of aromatic amino acid esters and peptides against ovalbumin permeation through Caco-2 cell monolayers

Trp, Phe, and Tyr ethyl esters and their dipeptides with Gly at the C-terminals inhibited ovalbumin (OVA) permeation through Caco-2 monolayers. The inhibitory activity of Trp ethyl ester was the highest at near the concentration of 10(-6) M. It was suggested that Trp ethyl ester inhibited transcellular permeation of OVA.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Conjugates of 3-phenyllactic acid and tryptophan enhance root-promoting activity without adverse effects in Vigna angularis

3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure–activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.     

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe,Tyr and Trp

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C^/positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D₂O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62–79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.     

Development of short and highly potent self‐assembling elastin‐derived pentapeptide repeats containing aromatic amino acid residues

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self‐assembly of elastin into fibers, and the temperature‐mediated self‐assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio‐applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin‐derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe‐containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X¹P²G³V⁴G⁵) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp‐containing elastin‐derived peptide analog (WPGVG)₃ with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin‐derived peptide analogs. Generally, however, the production of long‐chain synthetic polypeptides in quantities sufficient for commercial use remain cost‐prohibitive. Therefore, the identification of (WPGVG)₃, which is a 15‐mer short peptide consisting simply of five natural amino acids and shows temperature‐dependent self‐assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Semisynthetic ‘acid-esterase’: Conformational modification of ribonuclease

Bovine pancreatic ribonuclease has been modified by exposure to acidic conditions, addition of indole propionic acid and crosslinking with glutaraldehyde. The ‘acid-esterase’ generated was purified up to 100-fold by ammonium sulphate fractionation and gel filtration on Biogel P-30. The partially purified acid-esterase hydrolysed tryptophan ethyl ester (TrEE) and $\text{N-}\text{benzoyl}\text{-}\text{l}\text{-}\text{arginine}$ ethyl ester (BAEE) effectively at pH 6.0–6.3, but it had very little activity towards glycine ethyl ester and lysine ethyl ester. Hydrolysis of TrEE was competitively inhibited by tryptophan. The acid-esterase exhibited amidase activity towards benzoyl-l-arginine p-nitroanilide.     

An active compound against allergen absorption in hypoallergenic wheat flour produced by enzymatic modification

Hypoallergenic wheat flour produced by modification with cellulase and actinase showed inhibitory activity against ovalbumin permeation in an in vitro model by using the Caco-2 cell monolayer. The activity was found in the cellulase preparation used for producing the flour. An active compound was isolated by HPLC and identified as Trp-Ser-Asn-Ser-Gly-Asn-Phe-Val-Gly-Gly-Lys by 1H-NMR data and Edman degradation. The undecapeptide, some oligopeptides with the N-terminal sequences and Trp ethyl ester showed activity at 10(-7) M, acetyl Trp being active at 10(-2) M. These data suggest that the Trp residue without a free carboxyl group would be required for the inhibitory activity of ovalbumin absorption through the intestinal tract.     

Enantioselective Photolysis and Quantitative Chiral Analysis of Tryptophan Complexed With Alkali‐Metalized L‐Serine in the Gas Phase

The enantioselective photolysis of a cold gas‐phase noncovalent complex of tryptophan with alkali‐metalized L‐serine, M⁺(L‐Ser)(Trp) (M = Na and Li), was examined using a tandem mass spectrometer containing a variable‐temperature ion trap. CO₂ loss from Trp in the clusters was enantiomerically selective in ultraviolet excitation with linearly polarized light. M⁺(L‐Ser) promoted the enantioselective photolysis of Trp as a chiral auxiliary. The enantioselective photolysis of the D‐enantiomer was applied to a quantitative chiral analysis, in which the optical purity of tryptophan could be determined by measuring the relative abundance ratio R of the enantioselective CO₂ loss to the chiral‐independent evaporation of L‐Ser in a single photodissociation mass spectrum of M⁺(L‐Ser)(Trp). Chirality 27:349–352, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis and Immunomodulating Activity of New Amino Acid Derivatives of Glycyrrhizic Acid and Its Methyl Ester

New amino acid derivatives of glycyrrhizic acid and its methyl ester were selectively synthesized using active N-succinimide esters. The compounds with residues of glycine ethyl ester and alanine methyl and butyl esters increased the level of agglutinins and hemolysins in blood serum of mice two- to threefold in comparison with the control upon parenteral administration at a dose of 2 mg/kg for 14 days.     

An Aminopeptidase from Mouse Brain Cytosol That Cleaves N-Terminal Acidic Amino Acid Residues

An aminopeptidase with specificity directed toward peptides with acidic N-terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion-exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl-phenylala-nine methyl ester at a rate of 13.2 μu,mol/min/mg protein at pH 7.5, an increase in specific activity of 1000-fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is ?450,000. Dipeptides with N-terminal aspartyl residues are cleaved preferentially to glutamic-containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For pep-tides of the form aspartyl-X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripep-tides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N-terminal aspartyl residue of angiotensin II, aspartyl-phenylalanine methyl ester or aspartyl-alanine, and the N-terminal glu-tamyl residue of adrenocorticotropin(5-10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o-phenanthroline and EDTA. The latter effect could be reversed completely by Zn²⁺ and partially by Mn²⁺ or Mg²⁺; Co²⁺ and Fe²⁺ had no effect; Ca²⁺ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.     

Purification and Partial Characterization of Human C1-Esterase

C1-Esterase was purified from the euglobulin fraction of human plasma by successive column chromatography on DEAE-cellulose, hydroxylapatite and TEAE-cellulose. The final product, purified 3500-fold with respect to serum, hydrolyzed 1,155 μmoles of N^α-acetyl-l-tyrosine ethyl ester per milligram of protein at pH 7.4 and 37°C in 15 min. The homogeneity of the purified C1-esterase was confirmed by ultracentrifugation and disc-electrophoresis. Its s_20,w value was 4.3 and its molecular weight was determined as 113,000 by gel filtration on Sephadex G–200.

Cl-Esterase possesses esterolytic activity for both N^α-acetyl-l-tyrosine ethyl ester and N^α-tosyl-l-arginine methyl ester, and acts on human kininogen I and II releasing kinin very slowly.     

An activated by cobalt alkaline aminopeptidase from <Emphasis Type="Italic">Bacillus mycoides</Emphasis>

An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co²⁺ ions activated the enzyme, while Zn²⁺, Cu²⁺, Hg²⁺ and Mn²⁺ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

Neisseria meningitidis expresses a single 3‐deoxy‐d‐arabino‐heptulosonate 7‐phosphate synthase that is inhibited primarily by phenylalanine

Neisseria meningitidis is the causative agent of meningitis and meningococcal septicemia is a major cause of disease worldwide, resulting in brain damage and hearing loss, and can be fatal in a large proportion of cases. The enzyme 3‐deoxy‐d ‐arabino‐heptulosonate 7‐phosphate synthase (DAH7PS) catalyzes the first reaction in the shikimate pathway leading to the biosynthesis of aromatic metabolites including the aromatic acids l ‐Trp, l ‐Phe, and l ‐Tyr. This pathway is absent in humans, meaning that enzymes of the pathway are considered as potential candidates for therapeutic intervention. As the entry point, feedback inhibition of DAH7PS by pathway end products is a key mechanism for the control of pathway flux. The structure of the single DAH7PS expressed by N. meningitidis was determined at 2.0 Å resolution. In contrast to the other DAH7PS enzymes, which are inhibited only by a single aromatic amino acid, the N. meningitidis DAH7PS was inhibited by all three aromatic amino acids, showing greatest sensitivity to l ‐Phe. An N. meningitidis enzyme variant, in which a single Ser residue at the bottom of the inhibitor‐binding cavity was substituted to Gly, altered inhibitor specificity from l ‐Phe to l ‐Tyr. Comparison of the crystal structures of both unbound and Tyr‐bound forms and the small angle X‐ray scattering profiles reveal that N. meningtidis DAH7PS undergoes no significant conformational change on inhibitor binding. These observations are consistent with an allosteric response arising from changes in protein motion rather than conformation, and suggest ligands that modulate protein dynamics may be effective inhibitors of this enzyme.     

Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

An Efficient Synthesis Of Acyclic N7- and N9-Adenine Nucleosides Via Alkylation With Secondary Carbon Electrophiles to Introduce Versatile Functional Groups At the C-1 Position of Acyclic Moiety

The introduction of versatile functional groups, allyl and ester, at the C-1 position of the acyclic chain in acyclic adenine nucleosides was achieved for the first time directly by alkylation of adenine and N⁶-protected adenine. Thus, the C-1′-substituted N⁹-adenine acyclic nucleoside, adenine-9-yl-pent-4-enoic acid ethyl ester (11), was prepared by direct alkylation of adenine with 2-bromopent-4-enoic acid ethyl ester (6), while the corresponding N⁷-regioisomer, 2-[6, (dimethylaminomethyleneamino)-purin-7-yl]-pent-4-enoic acid ethyl ester (10), was obtained in one step by the coupling of N,N-dimethyl-N′- (9H-purin-6-yl)-formamidine (9) with 2-bromopent-4-enoic acid ethyl ester (6). The functional groups, ester and allyl, were converted to the desired hydroxymethyl and hydroxyethyl groups, and subsequently to phosphonomethyl derivatives and corresponding pyrophosphorylphosphonates.     

Production,partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis

Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24 h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The K_m and V_max values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 μmoles/mg min. The metal ions Ca²⁺, Mg²⁺ and Mn²⁺ had stimulatory effect on lipase activity, whereas Cu²⁺, Fe²⁺ and Zn²⁺ strongly inhibited the lipase activity. EDTA and PMSF at 10 mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.     

Proteinogenic amino acids labelled with 15N and/or 13C for application in peptide synthesis: A short review with a comprehensive list of published derivatives

A review is given of the literature dealing with the most common protected derivatives of ¹⁵N- and/or ¹³C-labelled amino acids of interest in peptide synthesis. The list contains all such Boc-, Z- and Fmoc-amino acids as well as published methyl, ethyl, t-butyl and benzyl esters.     

Production of indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein⁻¹ in the presence of 250 μg of Trp ml⁻¹. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant. Responsible Editor: Peter A. H. Bakker.     

UV resonance Raman spectroscopy of DNA and protein constituents of viruses: Assignments and cross sections for excitations at 257, 244, 238, and 229 nm

Ultraviolet resonance Raman (UVRR) spectra of H₂O and D₂O solutions of the nucleoside (dA, dG, dC, dT) and aromatic amino acid (Phe, Trp, Tyr) constituents of DNA viruses have been obtained with laser excitation wavelengths of 257, 244, 238, and 229 nm. Using the 981 cm⁻¹ marker of Na₂SO₄ as an internal standard, Raman frequencies and scattering cross sections were evaluated for all prominent UVRR bands at each excitation wavelength. The results show that UVRR cross sections of both the nucleosides and amino acids are strongly dependent on excitation wavelength and constitute sensitive and selective probes of the residues. The results provide a library of UVRR marker bands for structural analysis of DNA viruses and other nucleoprotein assemblies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 247–256, 1998