A peptide containing aspartic acid, glutamic acid and serine in calf brain synaptic vesicles

Free amino acids and other amino compounds in calf brain synaptic vesicles were identified and determined by thin-layer chromatography and ion-exchange chromatography. The vesicles contained ten identified amino acids with glutamic acid, aspartic acid, taurine and gamma-aminobutyric acid in the highest concentrations, and also cysteic acid (or cysteinesulfinic acid), glutamine, alanine, serine, glycine and lysine. The vesicles also contained certain unknown acid-labile, ninhydrin-positive compounds, one of which was a peptide yielding, after acid hydrolysis, about 40% aspartic acid, 30% serine, 15% glutamic acid, 10% glycine and possibly some alanine and lysine. The concentration of the peptide in the vesicles was as high as that of all the other amino compounds together.     

Molecular orbital studies on the conformation of GABA (γ-aminobutyric acid)

In distinction to Extended Hückel Theory which predicts as the most stable conformation of free zwitterionic GABA a totally extended form, PCILO and SCF ab initio studies show that the intrinsically preferred conformation of the isolated molecule is a highly folded one, resulting from strong interactions between the two charged ends. Computations are also carried out for hydrated GABA in the supermolecule approach allowing moreover for the flexibility of binding of some of the water molecules of the first hydration shell. They predict the coexistence in solution of a large number of conformations showing different degrees of folding (or extension), a result confirmed by recent NMR studies. This and a number of similar results show that we have to adapt our thinking on the role of conformations in pharmacological activity to this situation, which was frequently obscured by the more abundant results of X-ray crystallography yielding a single conformation.     

The enantiomers of syn-2,3-difluoro-4-aminobutyric acid elicit opposite responses at the GABA(C) receptor

The conformational behaviour and GABA receptor activity of the different stereoisomers of 2,3-difluoro-4-aminobutyric acid are described. Two enantiomeric GABA(C)-active ligands are identified, one of which is an agonist while the other is an antagonist. The results support an existing QSAR model of the bioactive geometry of GABA at GABA(C).     

Determination of 4-aminobutyric acid, aspartate, glutamate and glutamine and their 13C stable-isotopic enrichment in brain tissue by gas chromatography-mass spectrometry

A selected-ion monitoring method was developed for measuring 4-aminobutyric acid, aspartate, glutamate, and glutamine in brain tissue. Natural isotopes of these amino acids and their stable-isotopic enrichment following intravenous infusion of a precursor, [13C]glucose, were quantitated. Frozen mouse brain tissue was homogenized in cold 80% ethanol, and the supernatant, equivalent to 1 mg of wet weight brain tissue, was extracted using solid-phase bonded silica ion-exchange columns. Aspartate and glutamate (dicarboxylic acids) were isolated from strong anion-exchange columns, whereas 4-aminobutyric acid and glutamine (neutral amino acids) were isolated from strong-cation exchange columns. n-Butyl ester pentafluoropropionyl amide derivatives of these amino acids were analyzed by gas chromatography-mass spectrometry using a methane positive chemical ionization mode after gas chromatographic separation on a wide-bore, fused-silica capillary column. The method is applicable to determination of brain concentrations of these amino acids as well as their fluxes following administration of a stable-isotopic tracer.     

Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase. Its limitation in use for post-mortem brain.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.     

A colorimetric method for the estimation of glutamic acid in protein hydrolyzates

On heating with hydroxylamine and on subsequent treatment with ferric chloride, pyrrolidonecarboxylic acid gives a colour reaction. This reaction can be used for the quantitative estimation of glutamic acid in protein hydrolyzates since this acid is almost completely converted into pyrrohdonecarboxylic acid at 125° and pH 3–4.     

A rapid method for the estimation of glutamic acid with chloramine-T

Chloramine-T (added in excess) oxidizes glutamic acid in various solvent media and the reaction is rapid and stoichiometric with a 4-electron change in buffers of pH 1-6, in 0.01 M sulphuric and perchloric acids and in 0.1M hydrochloric acid. A back-titration procedure using a pH-4 buffer or 0.1M hydrochloric acid as reaction medium has been developed. rho-Toluenesulphonamide and a nitrile have been identified in the reaction products. The effect of other species on the oxidation has been investigated.     

Novel syntheses of chiral beta- and gamma-amino acid derivatives utilizing N-protected (aminoacyl)benzotriazoles from aspartic and glutamic acids

Friedel-Crafts reactions of N-protected (alpha-aminoacyl)benzotriazoles with hetero- and benzenoid- aromatics give alpha-amino ketones that can be reduced by either triethyl silane or sodium borohydride to form the corresponding beta- and gamma-amino acid derivatives. The preservation of chirality throughout this process is confirmed by chiral HPLC results.     

Fragmentation of the deprotonated ions of peptides containing cysteine, cysteine sulfinic acid, cysteine sulfonic acid, aspartic acid, and glutamic acid

We examined the fragmentation of the electrospray-produced [M-H]- and [M-2H]2- ions of a number of peptides containing two acidic amino acid residues, one being aspartic acid (Asp) or glutamic acid (Glu), and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)], on an ion-trap mass spectrometer. We observed facile neutral losses of H2S and H2SO2 from the side chains of cysteine and C(SO2H), respectively, whereas the corresponding elimination of H2SO3 from the side chain of C(SO3H) was undetectable for most peptides that we investigated. In addition, the collisional activation of the [M-H]- ions of the C(SO2H)-containing peptides resulted in the cleavage of the amide bond on the C-terminal side of the C(SO2H) residue. Moreover, collisional activation of the [M-2H]2- ions of the above Asp-containing peptides led to the cleavage of the backbone N-Calpha bond of the Asp residue to give cn and/or its complementary [zn-H2O] ions. Similar cleavage also occurred for the singly deprotonated ions of the otherwise identical peptides with a C-terminal amide functionality, but not for the [M-H]- ions of same peptides with a free C-terminal carboxylic acid. Furthermore, ab initio calculation results for model cleavage reactions are consistent with the selective cleavage of the backbone N-Calpha bond in the Asp residue.     

Partial poly(glutamic acid) <--> poly(aspartic acid) exchange in layer-by-layer polyelectrolyte films. Structural alterations in the three-component architectures

Layer-by-layer (LBL) polyelectrolyte films were constructed from poly(L-glutamic acid) (PGA) and poly(L-aspartic acid) (PAA) as polyanions, and from poly(L-lysine) (PLL) as the polycation. The terminating layer of the films was always PLL. According to attenuated total reflection Fourier transform infrared measurements, the PGA/PLL and PAA/PLL films, despite their chemical similarity, had largely different secondary structures. Extended beta-sheets dominated the PGA/PLL films, while alpha-helices and intramolecular beta-sheets dominated the PAA/PLL films. The secondary structure of the polyelectrolyte film affected the adsorption of human serum albumin (HSA) as well. HSA preserved its native secondary structure on the PGA/PLL film, but it became largely deformed on PAA/PLL films. Both PGA and PAA were able to extrude to a certain extent the other polyanion from the films, but the structural consequences were different. Adding PAA to a (PGA/PLL)5-PGA film resulted in a simple exchange and incorporation: PGA/PLL and PAA/PLL complexes coexisted with their unaltered secondary structures in the mixed film. The incorporation of PGA into a (PAA/PLL)5-PAA film was up to 50% and caused additional beta-structure increase in the secondary structure of the film. The proportions of the two polyanions were roughly the same on the surfaces and in the interiors of the films, indicating practically free diffusion for both polyanions. The abundance of PAA/PLL and PGA/PLL domains on the film surfaces was monitored by the analysis of the amide I region of the infrared spectrum of a reporter molecule, HSA, adsorbed onto the three-component polyelectrolyte films.     

Enzyme-based microtiter plate assay for γ-aminobutyric acid: Application to the screening of γ-aminobutyric acid-producing lactic acid bacteria

An enzyme-based microtiter plate assay for γ-aminobutyric acid (GABA) was developed. GABA was quantified using γ-aminobutyrate glutamate aminotransferase and succinic semialdehyde dehydrogenase in the presence of NADP⁺ and -ketoglutarate. The NADPH produced by the series of enzymatic reactions was measured spectrophotometrically at 340 nm. A linear relationship between absorbance and the concentration of GABA was obtained in the ranges from 5.0 × 10⁻⁴ to 1.0 × 10⁻² M. The relative standard deviation for 10 successive measurements was 0.9% at the 10 mM GABA level. This analytical method was applied to the screening of GABA-producing lactic acid bacteria in de Man–Rogosa–Sharpe (MRS) medium. The proposed method enables one to assay 96 samples for an hour without the pre-treatment of samples. The method is by far superior to the traditional HPLC method from the point of view of rapidity and simplicity.     

Further studies on the fragmentation of protonated ions of peptides containing aspartic acid, glutamic acid, cysteine sulfinic acid, and cysteine sulfonic acid

Here we examined the fragmentation, on a quadrupole ion-trap mass spectrometer, of the protonated ions of a group of peptides containing one arginine and two different acidic amino acids, one being aspartic acid (Asp) or glutamic acid (Glu) and the other being cysteine sulfinic acid [C(SO2H)] or cysteine sulfonic acid [C(SO3H)]. Our results showed that, upon collisional activation, the cleavage of the peptide bond C-terminal to C(SO2H) is much more facile than that of the peptide bond C-terminal to Asp, Glu, or C(SO3H). There is no significant difference, however, in susceptibility to cleavage of peptide bonds that are C-terminal to Asp, Glu, and C(SO3H). To understand these experimental observations, we carried out B3LYP/6-31G* density functional theory calculations for a model cleavage reaction of GXG --> b2 + Gly, in which X is Asp, Glu, C(SO2H), or C(SO3H). Our calculation results showed that the cleavage reaction is thermodynamically more favorable when X = C(SO2H) than when X = Asp or C(SO3H). We attributed the less facile cleavage of the amide bond after Glu to that the formation of a six-membered ring b ion for Glu-bearing peptides is kinetically not as favorable as the formation of a five-membered ring b ion for peptides containing the other three acidic amino acids. The results from this study may provide useful tools for peptide sequencing.     

Kinetics of the N-chlorination of 2-aminobutyric, 3-aminobutyric, 3-aminoisobutyric and 4-aminobutyric acids in aqueous solution

We studied the kinetics of the N-chlorination of 2-aminobutyric, 3-aminoisobutyric, and 4-aminobutyric acids by sodium hypoclorite in strongly alkaline aqueous solution. As in the case of other amines, the rate of formation of the four N-chloroamino acids was proportional to the concentrations of hypochlorite and amino acid, and inversely proportional to the concentration of hydroxyl ions. A reaction mechanism compatible with these results is proposed and discussed. © John Wiley & Sons, Inc.     

New catalytic systems for the manufacture of poly(aspartic acid)

Aspartic acid (I), when heated to a temperature in excess of 180 °C, undergoes a solid-state condensation polymerization to afford the useful polymeric intermediate known as poly(succinimide) (II). Treatment of poly(succinimide) with aqueous base, such as sodium hydroxide, affords sodium poly(α,β-DL-aspartate) (III) also known as thermal poly(aspartate) (TPA). Acid catalysts, such as phosphoric acid have been added to the aspartic acid to afford higher-molecular-weight poly(succinimide) than is obtained in the non-catalyzed polymerization. Recently, new sulfur-based catalysts have been disclosed for the polymerization of aspartic acid. The sulfur-containing catalysts provide a route to highly biodegradable, low-color poly(aspartate)s in the molecular weight range of 2 000 to 20 000. A comparison of biodegradability, molecular weight, and spectral characteristics of the poly(succinimide)s and poly(aspartate)s derived from the catalyzed and non-catalyzed polymerizations is presented.     

Experimental and theoretical studies of sodium cation complexes of the deamidation and dehydration products of asparagine, glutamine, aspartic acid, and glutamic acid

The deamidation and dehydration products of Na+(L), where L = asparagine (Asn), glutamine (Gln), aspartic acid (Asp), and glutamic acid (Glu), are examined in detail utilizing collision-induced dissociation (CID) with Xe in a guided ion beam tandem mass spectrometer (GIBMS). Results establish that the Na+(L) complexes decompose upon formation in our dc discharge/flow tube ion source to form a bis-ligand complex, Na+(L-HX)(HX), composed of a sodium cation, the (L-HX) decomposition product, and HX, where HX = NH3 for the amides and H2O for the acids. Analysis of the energy-dependent CID cross sections for the Na+(L-HX)(HX) complexes provides unambiguous identification of the (L-HX) fragmentation products as 3-amino succinic anhydride (a-SA) for Asx and oxo-proline (O-Pro) for Glx. Furthermore, these experiments establish the 0 K sodium cation affinities for these five-membered ring decomposition products and the H2O and NH3 binding affinities of the Na+(a-SA) and Na+(O-Pro) complexes after accounting for unimolecular decay rates, the internal energy of reactant ions, and multiple ion-molecule collisions. Quantum chemical calculations are determined for a number of geometric conformations of all reaction species as well as a number of candidate species for (L-HX) at the B3LYP/6-311+G(d,p) level with single-point energies calculated at MP2(full), B3LYP, and B3P86 levels using a 6-311+G(2d,2p) basis set. This coordinated examination of both the experimental work and quantum chemical calculations allows for a complete characterization of the products of deamidation and dehydration of Asx and Glx, as well as the details of Na+, H2O, and NH3 binding to the decomposition species.     

Rapid photometric method for the determination of taurine and cysteic acid

A method is described for the rapid determination of microgram quantities of taurine or cysteic acid in the presence of relatively large amounts of other amino-acids. Dinitrophenylation of the sample followed by chloroform extraction yields an aqueous solution containing only DNP-taurine or DNP-cysteic acid, the absorbance of which gives a direct measure of these components.