相思子属三种药材中的氨基酸分析比较

利用全自动氨基酸分析仪和高效液相色谱仪分析比较相思子属三种药材中氨基酸成分及含量。结果9个样品都检测出17种氨基酸和2种色氨酸衍生物。结论:不同样品中的氨基酸含量有所差异,但其中门冬氨酸、谷氨酸、亮氨酸和赖氨酸的含量相对较高。     

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids

Summary The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5–3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), a-ketoglutaric acid (a-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with -KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with -KGA than through reductive amination of -KGA.The experimental data proved that intact bacteroids posess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon.The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N₂-fixation, and sucrose available from leaves.     

Effect of coordinated addition of specific amino acids on the synthesis of recombinant glucose isomerase

The amplified expression of a recombinant protein is known to lead to an intracellular depletion of specific amino acid pools which in turn may affect the production of the desired protein. In order to counteract and overcome such a situation during the fermentation of the recombinant Escherichia coli (PMSG27) containing the glucose isomerase (GI) gene from Streptomyces sp. NCIM 2730, the effect of addition of different amino acids on the specific activity of GI was studied. The amino acid composition of GI from Streptomyces sp. NCIM 2730 reveals predominantly aspartic acid, glutamic acid, and glycine; therefore, in the present paper, the effect of coordinated addition of the assorted combinations of these three amino acids on the synthesis of recombinant GI was studied. The results were analyzed using a 2³ factorial design. The following conclusions were derived from the analysis of two-factor interactions of the three amino acids: (i) The interaction between the aspartic and glutamic acid is independent of aspartic acid concentration but is affected by the increasing concentrations of glutamic acid, (ii) The effect of aspartic acid concentration is more than that of glycine, and (iii) During the interaction of glutamic acid and glycine, the effect of glutamic acid is more prominent than that of glycine. The three-factor interaction analyses reveal that the effect of the three amino acids is in the order aspartic acid > glutamic acid > glycine.     

Free alanine,aspartic acid,or glutamic acid reduce the glycation of human lens proteins

The amino acids lysine and glycine are reported to react with glucose at physiological pH and temperature and undergo non-enzymic glycation. Three other amino acids present in relatively larger amounts in the lens i.e. alanine, aspartic acid and glutamic acid were also found to undergo non-enzymic glycation as found by incorporation of uniformly labelled (U-[¹⁴C]) glucose into the amino acids. The glucose incorporation was 1.6 to 2.5% for alanine, 35 to 50% for aspartic acid and 2.3 to 3.3% for glutamic acid. Each amino acid of varying concentrations lowered the extent ofin vitro glycation of lens proteins significantly in glucose-treated homogenates of normal lens from humans. The decrease in glycation for alanine was between 32 and 69%, that for aspartate was between 18 and 74%, and for glutamate was between 52 to 74%. Decreased glycation was greater for higher concentrations of glucose. Scavenging of intracellular glucose and decreasing the extent of glycation of lens proteins could be the mechanism of action by which the amino acids alanine, aspartic acid and glutamic acid could exercise a beneficial effect on cataract and diabetic retinopathy.     

A method for the separation of peptides and α-amino acids

1. Peptides and alpha-amino acids, occurring in mixtures from various sources, can be separated into one fraction containing the amino acids and several peptide fractions. This is achieved by chelation of the mixture with Cu(2+) ions and subsequent chromatography of these chelates over the acetate form of diethylaminoethylcellulose or triethylaminoethylcellulose. 2. The amino acid fraction is obtained by elution with 0.01m-collidine-acetate buffer, pH8.0. 3. Peptide fractions are eluted with 0.01m-collidine-acetate buffer, pH4.5, 0.17n-acetic acid and 0.1n-hydrochloric acid respectively. 4. With the exception of aspartic acid and glutamic acid, which are partly found in the acidic peptide fraction, the amino acids are completely separated from the peptides. 5. Contamination of the acidic peptide fraction with glutamic acid and aspartic acid can be largely avoided by previous addition of an excess of arginine. 6. Copper is removed from the eluates by extraction with 8-hydroxyquinoline in chloroform.     

Forms of Aspartic Acid in Human Urine

It was found by amino acid analysis before and after acid hydrolysis of human urine that most glutamic and aspartic acid was in bound form, while glycine, glutamic and aspartic acids accounted for about 70% of bound amino acids. Fractions rich in peptides containing aspartic acid were obtained by chromatography on various columns, and 7 peptides containing aspartic acid were isolated from these fractions. It may be inferred from these results and from the literatures that there are numerous oligopeptides containing aspartic acid in human urine.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

Synthesis and erythropoietin receptor binding affinities of N,N-disubstituted amino acids

N,N-Dicinnamyl, N-benzyl-N-cinnamyl, and N,N-dibenzyl amino acids were prepared and evaluated in an EPO binding assay. Several derivatives of aspartic acid, glutamic acid, and lysine exhibited moderate (10-50 microM) affinity for EBP; 'dimerization' of the most potent analogues by coupling with linear diamines led to EPO competitors having 1-2 microM binding affinities.     

Role of protein dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cell.

The pH profile for the uptake of L-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na+-dependent uptake by System A, and an increasing uptake by Na+-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na+-dependent component, (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicious exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na+-dependent component of uptake retained by L-glutamic acid but not by D-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports L-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic aic or of the norbornane amino acid. The dicarboxylic amino acids take the sequence, aspartic acid less than glutamic acid less than alpha-aminoadipic acid less than S-carboxymethylcysteine, in their rate of mediated, Na+-independent uptake at low pH. Diiodotyrosine and two dissimilas isomers of nitrotyrosine also show acceleration of uptake as the phenolate group on the sidechain is protonated, a result indicating that the acidic group need not be a carboxyl group and need not take a specific position in space to be accepted at the receptor site L. The presence of the carboxyl group does not upset the normal stereospecificity of System L until it falls on the beta-carbon in aspartic acid; even then it is the presence of the carbonyl group and not of the intact carboxyl group nor of its hydroxyl group that cancels out the stereospecificity, as was shown by the absence of normal stereospecificity for aspartic acid and asparagine and its presence in glutamic acid, homoserine and glutamine. In agreement, the uptak of aspartic acid is peculiarly sensitive to the presence of an alpha-methyl group or of other structures that modify the orientation of the sidechain.     

Determination of gamma-aminobutyric and glutamic acids in rat brain by liquid chromatography with electrochemical detection

The determination of glutamic and gamma-aminobutyric acids in rat brain regions by derivatization with o-phthaldialdehyde-thiol, isocratic separation by liquid chromatography, and quantification by electrochemical detection provides a simple and precise method for assessing changes in glutamatergic and GABAergic neuronal systems. Gamma-aminobutyric acid was eluted in 30-35 minutes followed by a washout step with 90% methanol to remove all amino acid derivatives with longer retention times. Homoserine was used as an internal standard. Significant increases in gamma-aminobutyric acid content in nucleus accumbens and substantia nigra could be detected 20 minutes after injection of 400 mg/kg valproic acid.     

Contents of amino acids in Tricholoma giganteum

Seventeen protein-constitutive and free amino acids were isolated and determined from the fruit-bodies of Tricholoma giganteum . Aspartic acid and alanine were the most abundant protein constitutive amino acids. Among the free amino acids, alanine, glutamic acid, serine, aspartic acid and glycine were present in the largest amounts, with aspartic acid and glutamic acids especially dominant.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.     

Tracer studies on the biosynthesis of amino acids from lactate by Peptostreptococcus elsdenii

Peptostreptococcus elsdenii, a strict anaerobe from the rumen, was grown on a medium containing yeast extract and [1-(14)C]- or [2-(14)C]-lactate. Radioisotope from lactate was found in all cell fractions, but mainly in the protein. The label in the protein fraction was largely confined to a few amino acids: alanine, serine, aspartic acid, glutamic acid and diaminopimelic acid. The alanine, serine, aspartic acid and glutamic acid were separated, purified and degraded to establish the distribution of (14)C from lactate within the amino acid molecules. The labelling patterns in alanine and serine suggested their formation from lactate without cleavage of the carbon chain. The pattern in aspartic acid suggested formation by condensation of a C(3) unit derived directly from lactate with a C(1) unit, probably carbon dioxide. The distribution in glutamic acid was consistent with two possible pathways of formation: (a) by the reactions of the tricarboxylic acid cycle leading from oxaloacetate to 2-oxoglutarate, followed by transamination; (b) by a pathway involving the reaction sequence 2 acetyl-CoA-->crotonyl-CoA-->glutaconate-->glutamate.     

Effect of L-Aspartic Acid and L-Glutamic Acid on Production of L-Proline

To elucidate the effect of aspartic acid on growth of Kurthia catenaforma during the proline fermentation, this organism was compared with other bacteria with respect to the rate of consumption of aspartic acid, and to the activities of enzymes concerned in the metabolism of aspartic acid. Although no marked difference in enzyme activities was observed, the aspartic acid consumption rate of K. catenaforma was markedly higher than that of other organisms. The consumption of glutamic acid by K. catenaforma was not detected at 24 hr of culture. The difference between the consumption of aspartic acid and glutamic acid in this strain might result from a difference in permeability to the amino acids. We considered that L-glutamic acid might substitute for L-aspartic acid if the uptake of glutamic acid could be increased. A number of detergents were screened for their effect on consumption of glutamic acid. Cetyltrimethylammonium bromide, sodium laurylphosphate, and polyoxyethylene sorbitan monolaurate were found to increase the transport rate of glutamic acid, but not of aspartic acid. A method of producing L-proline from glutamic acid was established with the aid of detergents.     

Amino acid transport in a water-mould: the possible regulatory roles of calcium and N6-(delta2-isopentenyl)adenine.

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different systems elucidated are: (i) methionine, (ii) cysteine. (iii) proline, (iv) serine-threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine-tyrosine-tryptophan and leucine-isoleucine-valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.     

Synthesis and in vitro hydroxyapatite binding of peptides conjugated to calcium-binding moieties

To confer bone-binding properties to proteins and other biological agents that lack specific targeting capacity, model peptide-based molecules were synthesized containing poly(aspartic acid), poly(glutamic acid), or a bisphosphonate (pamidronate). These motifs have well-documented affinities to hydroxyapatite, a property desirable for the targeting of molecules to bone for drug delivery and tissue engineering applications. Model peptides of increasing molecular mass (5-33 amino acids) were directly conjugated to eight aspartic acids (Asp8), eight glutamic acids (Glu8), or pamidronate, purified by high-performance liquid chromatography, and characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The modified peptides were incubated with hydroxyapatite in phosphate-buffered saline at physiological conditions over 24 h. This study revealed a significant amount (>90%) of conjugated peptides adsorbed to the hydroxyapatite as compared to unmodified peptides (<5%). It was found that while there were significant differences between the different hydroxyapatite-binding and control groups for all time points, the size of the peptide had no statistical effect on peptide-hydroxyapatite binding. These results demonstrate that bisphosphonate and oligopeptide conjugates hold great promise for the development of new bioactive molecules for bone-specific applications.     

Amino Acid Concentrations in Rumen Fluid

Methods using dialysis or ultrafiltration are described for the collection of extracellular fluid in rumen contents for analysis of amino acids. Marked differences in the concentration of aspartic acid, glutamic acid, and alanine were found in samples of either diffusate or ultrafiltrate and in clarified acidified rumen liquor. Concentrations are given for aspartic acid, glutamic acid, alanine, glycine, γ-aminobutyric acid, valine, δ-aminovaleric acid, and leucine.     

Utilization in vivo of glucose and volatile fatty acids by sheep brain for the synthesis of acidic amino acids

1. Free glutamic acid, aspartic acid, glutamic acid from glutamine and, in some instances, the glutamic acid from glutathione and the aspartic acid from N-acetyl-aspartic acid were isolated from the brains of sheep and assayed for radioactivity after intravenous injection of [2-¹⁴C]glucose, [1-¹⁴C]acetate, [1-¹⁴C]butyrate or [2-¹⁴C]propionate. These brain components were also isolated and analysed from rats that had been given [2-¹⁴C]propionate. The results indicate that, as in rat brain, glucose is by far the best precursor of the free amino acids of sheep brain. 2. Degradation of the glutamate of brain yielded labelling patterns consistent with the proposal that the major route of pyruvate metabolism in brain is via acetyl-CoA, and that the short-chain fatty acids enter the brain without prior metabolism by other tissue and are metabolized in brain via the tricarboxylic acid cycle. 3. When labelled glucose was used as a precursor, glutamate always had a higher specific activity than glutamine; when labelled fatty acids were used, the reverse was true. These findings add support and complexity to the concept of the metabolic `compartmentation' of the free amino acids of brain. 4. The results from experiments with labelled propionate strongly suggest that brain metabolizes propionate via succinate and that this metabolic route may be a limited but important source of dicarboxylic acids in the brain.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.