Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids -alanine, -glutamine, and -aspartic acid have been observed in cerebrospinal fluid, and -alanine and -glutamic acid in urine. To the best of our knowledge no measurements of either -alanine in cerebrospinal fluid or -glutamic acid in urine have been presented in the literature before.     

MOBILIZATION OF SYNAPTIC MEMBRANE-BOUND CALCIUM BY ACIDIC AMINO ACIDS1

A fluorescent chelate probe (chlorotetracycline) and radioactive ⁴⁵Ca were used to study the effects of amino acids on the calcium bound to external synaptosomal membranes isolated from guineapig brain. Acidic amino acids released some of the membrane-bound calcium. On the basis of ⁴⁵Ca studies, the order of mobilization potency-DL-homocysteic acid and l -cysteic acid > l -aspartic acid, l -glutamic acid, d -glutamic acid > N-methyl-dl -glutamic acid and dl -cyteic acid-is in general agreement with that found by fluorescent chelate method with the exception of N-methyl-dl -aspartic acid and N-methyl-dl -glutamic acid, which are at least as potent as dl -homocysteic acid. This order of potency is observed only with a fraction enriched in external synaptosomal membranes, but not with microsomes, myelin and mitochondria. Neutral and basic amino acids, including glutamine. glycine and γ-aminobutyric acid are ineffective. These results suggest that acidic amino acids have a specific ability to mobilize membranebound calcium; this is consistent with the proposed role of some of these compounds as excitatory transmitters in the central nervous system.     

ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l-GLUTAMIC ACID IN RAT BRAIN SLICES

Kainic acid is a linear competitive inhibitor (K_is 250 μm ) of the ‘high affinity’ uptake of l -glutamic acid into rat brain slices. Kainic acid inhibits the ‘high affinity’ uptake of l -glutamic, d -aspartic and l -aspartic acids to a similar extent. Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices. Kainic acid (300 μm ) does not influence the steady-state release or potassium-stimulated release of preloaded d -aspartic acid from rat brain slices. Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l -glutamic acid. Only quisqualic and l -glutamic acid inhibit kainic acid binding in a potent manner. The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l -glutamic acid transport carrier. Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l -glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection. Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l -glutamic acid uptake since it appears to have little action on excitatory receptors. N-Methyl-d -aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l -glutamic acid uptake. N-Methyl-d -aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l -glutamic acid uptake.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: l-ASPARTIC ACID-BINDING PROTEIN FROM THE RAT CEREBRAL CORTEX

Abstract— Lyophilized rat cerebral cortex was treated with chloroform-methanol (2:1, v/v), and the extracted hydrophobic proteins (i.e. proteolipids) were separated by column chromatography on Sephadex LH-20. The first peak of protein, eluting with chloroform in the void volume, had high affinity binding for l -[¹⁴C]aspartic acid. The saturation of the binding showed three saturable sites with apparent dissociation constants of 0.2 μ m , 10 μ m and 50 μ m . The binding capacities of the three sites were 2.8, 132 and 617 nmol/mg of protein, respectively. There were 8.0 nmol of high affinity binding sites for l -aspartic acid and 1.53 nmol for l -glutamic acid per g of fresh tissue in the cerebral cortex of the rat. Differentiation between binding of l -aspartic and l -glutamic acid was clearly established by cross-binding and competition experiments with agonists and antagonists.
It is suggested that the isolated protein fraction may correspond to a synaptic receptor and not to the transport system. It is concluded that in the cerebral cortex there is a separate receptor for l -aspartic acid. This is further support to the possible role of this amino acid as a central excitatory transmitter.     

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

The influence of amino acids and inorganic salts on feeding by larvae of Heteronychus arator (Coleoptera: Scarabaeidae)

Abstract

Twenty amino acids were offered at 2 concentrations to Heteronychus arator F. larvae in an agar/cellulose powder medium. In comparison with the plain medium, only 0.1M l-alanine, 0.01M l-aspartic acid, and 0.01M l-glutamic acid significantly stimulated feeding. A mixture of inorganic salts did not induce feeding when presented in the medium alone, nor did it affect the vigorous feeding response of larvae to 0.1M maltose.     

Isolation and Characterization of Acidic Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. The acidic subfractions with various acidities were further fractionated, using a preparative amino acid analyzer and by paper chromatography to separate the acidic peptide components.

Four dipeptides and sugar derivatives of ten dipeptides and two tripeptides were isolated and characterized as the major acidic peptides in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their contents and taste intensities.     

Effects of excitatory neurotransmitter amino acids on swelling of rat brain cortical slices.

Abstract— With the single rat brain cortical slice serving as an in vitro bio-assay system, the effects of neurotransmitter amino acids (1 mm ) on brain swelling, water, sodium and potassium content, inulin space, and lactate production were studied. The putative dicarboxylic amino acid neurotransmitters, l -glutamic acid and l -aspartic acids, greatly increased intracellular brain swelling with increased intracellular Na⁺, water content and lactate production, and decreased inulin space and intracellular K⁺. Equimolar GABA, taurine, glycine, the putative inhibitory neurotransmitter amino acids, and equimolar α-amino-isobutyric acid had no effect. Brain swelling and intracellular Na⁺/K⁺ ratios were greatly increased by l -glutamate and l -aspartate at a concentration of 10 mm . However, l -aspartate at these concentrations greatly depleted the K⁺ content and lactate production as compared to l -glutamate. Further studies indicated that only the structural analogs and isomers of the dicarboxylic amino acids possessing two acidic groups and an α-amino group had a similar effect on the induction of brain swelling. Among the analogs of glutamic acid, dl -homocysteic acid and kainic acid had a greater effect on brain swelling, as observed from the total adenosine 5′-triphosphate (ATP) levels and the time-course and dose-response. A biphasic response in lactate production was induced by dl -homocysteic acid and kainic acid, suggesting that these analogs had a neurotoxic effect on cellular metabolism at higher concentrations.     

The Determination of d-Glutamic Acid and d-Aspartic Acid by Means of a New Oxidase

The fractional determination of d-glutamic and d-aspartic acids using the enzyme preparation of Aspergillus ustus strain f. was studied. In the first part of this paper, the procedure of enzyme preparation, the effect of sodium chloride on enzyme activity, and a new device for the fractional determination of d-glutamic and d-aspartic acids are described. In the latter part, the contents of d-glutamic and d-aspartic acids of cancer and normal tissues are estimated. However, it was found that the cancer tissues are not characterized by the presence of d-glutamic acid in opposition to Kögl’s claim.     

五步蛇蛇毒磷脂酶A_2的纯化及部分性质

经Sephadex G-75和QAE-Sephadex A-50离子交换层析等方法,从湖南产五步蛇(Agkistrodon acutus)蛇毒中纯化一种均一的酸性磷脂酶A_2。SDS-PAGE测得分子量为15.8kD,按氨基酸残基计算其分子量为14.352kD,IEF-PAGE测得等电点为5.32。氨基酸组份分析表明磷脂酶A_2分子由128个氨基酸残基组成,富含Asp和Glu,不含中性糖。PLA_2酶活性的最适温度为45℃,最适pH为8.5左右,没有抗胰蛋白酶的活性,具一定的热稳定性。K~+、Ca~(++)和Na~+离子激活,而Cd~(++)、Sn~(++)、Cu~(++)、Li~+、Hg(++)、Zn~(++)、Fe~(++)和Co~(++)离子可抑制或完全丧失酶活力。手工微量顺序分析测得PLA_2分子N-末端氨基酸为Leu。此酶对小白鼠的LD_(50)至少大于10mg/kg(ip)。     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

Polyethylene Glycol–Modified Papain Catalyzed Oligopeptide Synthesis from the Esters of l-Aspartic and l-Glutamic Acids in Benzene

Comparative studies were made of the polymerization of l-aspartic and l-glutamic acid dialkyl esters using polyethylene glycol–modified papain as a catalyst in phosphate buffer (pH 7.5) and in benzene. Changes in the substrate specificity of papain and in the composition of oligomerized products were observed. In the buffer, the diethyl and di-n-propyl esters of l-glutamic acid were sufficiently converted to high molecular weight oligomers with the accumulation of dimer and trimer, but l-aspartic acid esters were very poor substrates. In benzene, l-aspartic acid esters became more reactive than L-glutamic acid esters. In particular, from l-aspartic acid dimethyl ester the product, which was mainly composed of heptamer to decamer, was obtained in a 90% yield. The reaction in benzene required desalted substrates and a small amount of water to proceed extensively.     

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats

Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.     

The effects of some amino acids on growth and lipid production in Inonotus obliquus grown in vitro

The effect of the distribution of free and esterified fatty acids, including triterpenes and sterols, on the mycelial growth of the basidiomycete Inonotus obliquus (Pers. ex Fr.) Pilat in malt extract and solid supplemented mineral media was investigated. The amino acids DL-alanine. DL-aspartic acid, L-glutamic acid, L-leucine, L-methionine, and L-serine were added to the cultures at nitrogen concentrations of 0.1, 0.5 and 0.9 g/l, respectively. Differences in the average mycelial growth were distinct at concentrations of 0.5 g/l and 0.9 g/l. L-Methionine had the highest inhibitory effect on growth. At all concentrations, the amino acids reduced the production of lanosterol. The other identified triterpenes were inotodiol, 3β,hydroxy-lanosta-8,24-dien-21-al, 3β,21-dihydroxy-lanosta-8,24-diene, trametenolic acid, and ergosterol peroxide. The main fatty acids were either C 16:0 or C 18:2 both free or in glycerides.     

Disorder–order transitions induced in anionic homopolypeptides by cationic detergents

CD spectra have been obtained for poly(L -glutamic acid) and poly(L -aspartic acid) as functions of temperature and concentration of cationic detergents. Dodecylammonium chloride induces a coil–helix transition in fully ionized poly(L -glutamic acid). The interaction of the monomeric detergent with the polypeptide is responsible for the conformational transition. The detergent concentration required to produce the transition is independent of temperature. The CD of fully ionized poly(L -aspartic acid) is nearly unaffected by dodecylammonium chloride, in marked contrast to the situation found with poly(L -glutamic acid). However, these results do not imply that dodecylammonium chloride interacts differently with aspartyl and glutamyl residues. The observed results can be accounted for by the well-known fact that the glutamyl residue has a higher helix-forming tendency that the aspartyl residue. Cetyltrimethylammonium chloride destabilizes the helical form of poly(L -glutamic acid). This detergent presents an exception to the usual ability of ionic detergents to promote formation of ordered structures in oppositely charged homopolypeptides.     

Protein turnover and amino acid transport kinetics in end-stage renal disease

Protein and amino acid metabolism is abnormal in end-stage renal disease (ESRD). Protein turnover is influenced by transmembrane amino acid transport. The effect of ESRD and hemodialysis (HD) on intracellular amino acid transport kinetics is unknown. We studied intracellular amino acid transport kinetics and protein turnover by use of stable isotopes of phenylalanine, leucine, lysine, alanine, and glutamine before and during HD in six ESRD patients. Data obtained from amino acid concentrations and enrichment in the artery, vein, and muscle compartments were used to calculate intracellular amino acid transport and muscle protein synthesis and catabolism. Fractional muscle protein synthesis (FSR) was estimated by the precursor product approach. Despite a significant decrease in the plasma concentrations of amino acids in the artery and vein during HD, the intracellular concentrations remained stable. Outward transport of the amino acids was significantly higher than the inward transport during HD. FSR increased during HD (0.0521 +/- 0.0043 vs. 0.0772 +/- 0.0055%/h, P < 0.01). Results derived from compartmental modeling indicated that both protein synthesis (118.3 +/- 20.6 vs. 146.5 +/- 20.6 nmol.min-1.100 ml leg-1, P < 0.01) and catabolism (119.8 +/- 18.0 vs. 174.0 +/- 14.2 nmol.min-1.100 ml leg-1, P < 0.01) increased during HD. However, the intradialytic increase in catabolism exceeded that of synthesis (57.8 +/- 13.8 vs. 28.0 +/- 8.5%, P < 0.05). Thus HD alters amino acid transport kinetics and increases protein turnover, with net increase in protein catabolism.     

Intracellular Proteases of Bacillus thuringiensis subsp. kurstaki and a Protease-Deficient Mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis (Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum. Received: 4 January 2002 / Accepted: 6 March 2002     

Purification of cysteine proteinases from adult Schistosoma mansoni

Proteolytic activity against hemoglobin and low molecular weight synthetic substrates has been previously found in homogenates and excretion/secretion products of adult Schistosoma mansoni worms. This activity is stimulated in the presence of thiol compounds and is maximally active at acidic pH. To characterize further this proteolytic activity, lyophilized adult worms were extracted, and proteinases were isolated and purified. From extracts prepared in 0.2 M citrate buffer, pH 4.9, two proteinase species were purified to homogeneity by centrifugation, gel filtration, dialysis, and chromatofocusing chromatography. The proteinases, designated SMw32 and SMw28, have apparent molecular weights (SDS-PAGE) of 31,700 +/- 1400 and 27,800 +/- 1700, respectively. Both are thiol-dependent, acidic endopeptidases that cleave hemoglobin and a synthetic substrate, CBZ-arg-arg-AFC. A statistical comparison of amino acid compositions reveals that the proteinases are highly related.