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PubMed1994

Presence of X-AG carrier in frog (Rana esculenta) red blood cells.

Evidence is presented that the high levels of internal L-glutamic and L-aspartic acid in frog (Rana esculenta) red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity (Km = 3 microM) and low capacity (Vmax 0.4 mumol L-Glu.Kg-1 dry cell mass.10 min-1). It is Na+ dependent and the incorporation of L-glutamic acid can be inhibited by L- and D-aspartate and L-cysteic acid, while D-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X-AG family. Besides the incorporation through this system, L-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity (Km 39 microM) but high capacity (Vmax 1.8 mumol L-Glu.Kg-1 dry cell mass.10 min-1).

Amino Acid Transport System X-AG↗
PubMed1973

Ribosomes, polyribosomes, and deoxyribonucleic acid from thermophilic mesophilic, and psychrophilic clostridia.

Analysis of deoxyribonucleic acid (DNA) from four species of Clostridium, including two thermophiles, a mesophile, and a psychrophile, revealed no obvious relationship between growth temperature and DNA base composition. The melting temperatures (T(m)) of the DNA from the four species varied no more among the thermophilic, mesophilic, and psychrophilic species than among many related mesophilic species. Characterization of ribosomes from the clostridia by means of optical rotatory dispersion yielded similar spectra in common with other unrelated organisms. Only small differences were noted in the base composition of ribosomal ribonucleic acid (RNA) and in the amino acid composition of ribosomal proteins, including half-cystine content, as determined by cysteic acid analysis, and accessible sulfhydryl groups, as determined by titration with dithiobis (2-nitrobenzoic acid). Except for the two thermophiles, the ribosomal protein electrophoretic patterns were dissimilar. No unusual thermal stability was manifested in the T(m) values of thermophile ribosomal RNA. However, thermophile ribosome T(m) values (69 C) were higher than were mesophile and psychrophile T(m) values (64 C). Ribosomes from the four clostridial species were also examined in regard to the effect of heat on their functional integrity, measured by their activity in poly U-directed (14)C-phenylaline incorporation, and their gross physical integrity, measured by sucrose gradient analysis. The T(d, 5) values (temperature which produces 50% inactivation after 5 min) was found to be 70 and 72 C for the two thermophiles C. tartarivorum and C. thermosaccharolyticum, respectively; 57 C for a mesophile, C. pasteurianum; and 53 C for a psychrophile, Clostridium sp. strain 69. At 55 C, little effect was seen on the thermophile ribosomes, but the mesophile ribosomes lost 90% of their activity in 1 hr, and psychrophile ribosomes lost 100% of their activity within 10 min. According to sucrose gradient profiles, heating at 55 C results in dissociation of mesophile ribosomes and aggregation of psychrophile ribosomes. Thermophile S-100 fractions were also more thermostable than were mesophile or psychrophile S-100 fractions. The T(d, 5) values were 69 C for C. tartarivorum and C. thermosaccharolyticum S-100 and 41 C for C. pasteurianum and Clostridium sp. strain 69 S-100. The effect of heat on the endogenous incorporation of (14)C-valine by polysomes was also examined. In the case of thermophile polysomes, the extent of incorporation at 55 and 37 C was about equal. In the case of mesophile and psychrophile polysomes, the extent at 55 C was 44 and 39%, respectively, of the value at 37 C. The initial rates of incorporation in all four cases were greater at 55 C than at 37 C.

Amino Acids↗
PubMed1977

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.

Acetates↗
PubMed1992

Alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding to human cerebral cortical membranes: minimal changes in postmortem brains of chronic schizophrenics.

The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗
PubMed1999

Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for determining cysteine and cystine in cereal and legume seeds.

A new colorimetric method has been developed for the determination of chemically available cysteine and half-cystine in maize and legume seeds. The method is based on the reduction of cystine with aqueous NaBH(4)/urea/EDTA solution and the reaction of cysteine with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 0.2 M sodium acetate/HCl buffer (pH 2.0) to form a greenish product showing maximum absorbance at 410 nm. The method is simple, accurate, and highly specific for cysteine, in the presence of other naturally occurring amino acids. The method was applied to the determination of cysteine and of cysteine plus half-cystine in some seed meals. There was a good correlation between the results obtained using this method and those obtained using Ellman's reagent [5, 5-dithiobis(2-nitrobenzoic acid)]. There was also a good correlation between the results obtained using this method and cysteic acid values calculated from amino acid analysis of the samples.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Correction for amino acid loss during acid hydrolysis of a purified protein.

Hydrolyzing a protein in acid for a single hydrolysis interval, normally 24 h, will lead to inaccurate estimates of the amino acid composition of that protein due to an effect of the time of hydrolysis on peptide bond cleavage and amino acid degradation. The simultaneous yield and decay of amino acids during the hydrolysis of a protein can be described by a compartmental model with parameters for the hydrolysis and loss rates specific to each amino acid in a protein. The amino acid composition of the protein prior to hydrolysis can be determined by nonlinear regression of data derived from multiple hydrolysis intervals. In the present study egg-white lysozyme was hydrolyzed in 6 M HCl using 18 hydrolysis intervals (range, 2-141 h) using the conventional duplicate hydrolyses/interval system. Hydrolysis and loss rates were determined for each amino acid. Increasing the number of hydrolysis intervals prior to the maximum point on the hydrolysis curve, and including an hydrolysis interval greater than 100 h increased the accuracy with which the hydrolysis and loss rates were estimated. Most of the amino acids underwent some degree of loss during hydrolysis. Of particular note was the loss rate for cysteic acid, which was greater than that found for serine which is commonly regarded as an acid-labile amino acid. The determined amino acid composition of the protein, based on the nonlinear regression of the data from four different series of hydrolysis intervals, was compared with the known amino acid composition (sequencing). Using the routine duplicate sampling system, a nonlinear regression including 10 hydrolysis intervals (2, 6, 10, 14, 18, 22, 26, 30, 60, and 141 h) resulted in a mean amino acid recovery of 100% (range, 94-110%) and provided an acceptable compromise between accuracy and the cost of analysis.

Amino Acids↗
PubMed1989

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level.

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1968

Selective purification of the thiol peptides of myosin.

1. A method for selective purification of thiol peptides is described. Thiol groups in a protein are treated with radioactive cystine by disulphide-thiol interchange. The labelled cystine peptides in a digest can then be fractionated for peptide ;maps'. Performic acid oxidation of paper strips containing the radioactive peptides followed by further ionophoresis yields the purified cysteic acid peptides. 2. The thiol peptides in a peptic digest of cystine-exchanged myosin were purified in this way, and their amino acid sequences were determined. 3. The conclusion that myosin contains at least 16, and probably between 20 and 22, unique thiol sequences indicates that the molecule consists of two chemically equivalent components.

Amino Acid Sequence↗
PubMed1996

Study of the reaction mechanism of the D-glutamic acid-adding enzyme from Escherichia coli.

The D-glutamic acid-adding enzyme of Escherichia coli, or MurD, was purified from an overproducing strain and a few aspects of its reaction mechanism were studied. The existence of a reactive cysteinyl residue was shown by the following experiments: (1) two thiol-modifying reagents, (5,5'-dithiobis)2-nitrobenzoic acid and 2-nitro-5-thiocyanobenzoic acid, inactivated the enzyme; (2) incubation with tetranitromethane led to inactivation and to the appearance of cysteic acid (not to 3-nitrotyrosine); (3) in each case, ATP or UDP-MurNAc-L-Ala (but not D-glutamic acid) protected the enzyme from inactivation. The existence of a reactive lysyl residue was shown by the action of 2,4,6-trinitrobenzenesulfonic acid, a reagent specific for lysyl residues present in phosphate-binding sites. The formation of an acyl phosphate intermediate was consistent with three types of results: (1) the molecular isotope exchange reaction, which took place only in the presence of phosphate, but which was not strictly dependent on the presence of ADP; (2) a release of phosphate, measured by the molybdate assay, observed when the enzyme was incubated with ATP and UDP-MurNAc-L-Ala (without D-glutamic acid); (3) the appearance of a new radioactive compound (besides ATP and Pi) after incubation for a few minutes with UDP-MurNAc-L-Ala and [gamma-32P]ATP. Finally, the fact that phosphinate 1 was a good inhibitor of the enzyme (IC50 = 0.7 microM) strongly suggested that a tetrahedral transition state follows the acyl phosphate in the reaction pathway.

Chromatography, Thin Layer↗
PubMedDate not supplied

Quantitative analysis of cystine, methionine, lysine, and nine other amino acids by a single oxidation--4 hour hydrolysis method.

The sulfur-containing amino acids cystine and methionine play important roles in animal, especially avian, nutrition. Because these sulfur-containing amino acids are destroyed to varying extents by 6N HCl hydrolysis, oxidation and hydrolysis of cystine to cysteic acid and methionine to methionine sulfone have been widely used for determination of cystine and methionine. Lysine is considered the next limiting amino acid after the sulfur amino acids in poultry nutrition; therefore, determination of the amino acid content of rations focuses first on these 3 amino acids. The objective of this investigation was to establish whether lysine and other amino acids could be accurately determined in proteinaceous materials which had undergone performic acid oxidation. To perform this evaluation, lysine was determined in a variety of protein-containing materials both with and without performic acid oxidation. Performic acid oxidation followed by 6N HCl hydrolysis at 145 degrees C for 4 h allows accurate measurement of 3 amino acids especially important to poultry nutrition, cystine, methionine, and lysine, in a single preoxidized hydrolysate; this method can be extended to another 9 protein amino acids.

Amino Acids↗
PubMed1988

[Amino acid analysis of proteins and physiological fluids in a single system of sodium-citrate buffer].

Programmes for automatic analysis of protein and peptide hydrolysates and physiological fluids in a single system of sodium citrate buffers are proposed. Retention time values of 59 ninhydrin-positive substances, including nonprotein amino acids, amino sugars (muramic acid, glucosamine, galactosamine, mannosamine and fucosamine) and corresponding aminopolyols, as well as three cysteine derivatives, viz. cysteic acid, S-carboxymethylcysteine and S-2-(pyridyl-4)ethylcysteine, have been determined. Several examples illustrate an increase of the resolution simply by change of the temperature programme of the column and (or) elution time.

Amino Acids↗
PubMed1981

Uptake of 14C-labeled dicarboxylic amino acids in hepatocytes and hepatoma cells.

In previous studies, we observed decreased uptake of 14C-labeled L-aspartate and L-glutamate in s.c. transplants of several rapidly growing hepatomas relative to that in normal liver. The present report extends these observations to isolated cells and indicates that circulation differences cannot be the major factor. Mean net uptakes for the two dicarboxylic amino acids in cells from the rapidly growing Morris Hepatomas 7288ctc and 7777 were 5 to 26% of corresponding values for normal hepatocytes. Rates for net uptake in Hepatoma 7787 cells were intermediate between those of the rapidly growing hepatomas and hepatocytes, while the rates for Hepatoma 5123C cells and hepatocytes were similar. The contribution of sodium-dependent uptake to the mean total net uptake of [14C]aspartate and [14C]glutamate tended to be higher in hepatoma cells than in hepatocytes. Studies with isolated hepatocytes and Hepatoma 5123C cells showed no significant effect on uptake by 10 mM alpha-(methylamino)isobutyric acid and 10 mM 2-amino-2-carboxybicyclo[2.2.1]heptane. On the other hand, L-cysteic acid, L-alanosine, and N-phosphonacetyl-L-aspartic acid were shown to be effective inhibitors of sodium-dependent uptake in Hepatoma 5123C cells. The data suggest that the A and L systems are not major contributors to the uptake of dicarboxylic amino acids in hepatic cells. It was concluded that decreased uptake of dicarboxylic amino acids in rapidly growing hepatomas may accompany decreased metabolism of these dietary nonessential amino acids.

Alanine↗
PubMed1975

Isolation and characterization of sulfhydryl oxidase from bovine milk.

A method is described for purification of sulfhydryl oxidase from bovine milk which consistently yields preparations with greater than 3000-fold purification over skim milk. A concentration-dependent association-dissociation of the enzyme was adapted to the development of an isolation procedure. Purified preparations exhibited two zones, both of which displayed activity, upon polyacrylamide disc gel electrophoresis, but only one zone following disc gel electrophoresis in sodium dodecyl sulfate. Its mobility indicated a subunit weight of 89,000. Several lines of evidence suggest that iron is an integral part of the enzyme. Treatment of the enzyme with EDTA resulted in complete loss of activity which could be subsequently restored by dialysis against 1 muM ferrous sulfate. Furthermore, atomic absorption analysis and neutron activation analysis of separate enzyme preparations each indicated 0.5 atom of iron per subunit. Chemical analyses of sulfhydryl oxidase accounted for 97% of the sample weight, of which 89% could be attributed to amino acid residues and 11% to carbohydrate residues. Five half-cystine residues per subunit were indicated by cysteic acid analysis and by sulfhydryl group determination following reaction with sodium borohydride. Comparison of this value to the total sulfhydryl groups without reduction tentatively suggests the presence of one disulfide bond. Sulfhydryl oxidase was found to catalyze the oxidation of sulfhydryl groups in both small compounds and proteins, using O2 as oxidant and producing, in equimolar quantities, H2O2 and the corresponding disulfide. A Michaelis constant of 90 muM was obtained using reduced glutathione as substrate, under conditions of optimal pH and temperature, viz., pH 7.0 and 35 degrees. Substrate inhibition was apparent at GSH concentrations above 0.8 mM. In the presence of sulfhydryl oxidase, reductively denatured RNase was reoxidized and fully reactivated within 1 hour, whereas in the absence of the oxidase under otherwise identical conditions, full recovery of RNase activity required 24 hours. The presence of reducing agent was not required for this activity, nor was prior reduction of the sulfhydryl oxidase. Based on the observed activity, it appears that the enzyme could be involved in the biosynthesis of disulfide bonds in certain proteins.

Amino Acids↗
PubMed1979

EDTA soluble protein of human mature normal enamel.

Pure human mature enamel was prepared using a careful microdissection technique. After EDTA dissolution, the soluble proteins were recovered representing a concentration of 0.035% in the initial enamel. When the samples were analyzed with polyacrylamide gel electrophoresis, Coomassie Brilliant Blue staining revealed only one sharp fast migrating band, whereas o-toluidine blue, methylene blue, Amido Black 10B, and pyronine red G showed a thin double band at the same migration distance. Ultracentrifugation studies suggested that the proteins were of low molecular weight or of weak density. Absorption spectra showed a strong absorbance at 260 nm. After hydrolysis, amino acid analyses yielded a composition of 25% Gly, 13.5% Glu, 11% Ser, and 11% Pro. Cysteine measured as cysteic acid was present at 2%, and 2% hydroxyproline was found. A carbohydrate content of 15% was estimated by the anthrone method. Glucose, galactose, mannose, and fucose, identified through gas chromatography, were in a molar ratio of 9:4:3:1. Thus the organic matrix of adult human enamel consists of one or possibly two acidic glycoproteins.

Amino Acids↗
PubMed1999

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.

Buffers↗
PubMed1995

Mass spectrometric identification of amino acid transformations during oxidation of peptides and proteins: modifications of methionine and tyrosine.

Liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS), tandem mass spectrometry with on-line liquid chromatography (LC/ESI-MS/MS) and high-resolution mass spectrometry with liquid secondary ionization (LSI-MS) were utilized to identify the modified amino acids in peptides and proteins formed during oxidation with performic acid. The procedure of protein oxidation was chosen to assist in protein unfolding by oxidizing the cystines to cysteic acids to allow for more complete proteolytic digestion and to create additional cleavage sites for endoproteinase Asp-N. Investigation of the Asp-N peptide map of oxidized superoxide dismutase (SOD) by LC/ESI-MS revealed that an expected proteolytic fragment of the protein was missing. In its place, two peptides with molecular weights 66 and 100 higher than that calculated for the missing peptide were observed. To identify the modified amino acids in the unexpected peptides, a model peptide with some amino acid similarities (tyrosine, arginine, methionine, lysine) to the missing peptide was chosen and was subjected to similar oxidation and enzymatic digestion steps, conditions, and reactions. After oxidation and digestion, the model peptide (TAP; sequence, Ac-MDKVLNRY) showed three major peaks in LC/MS. The peptides in the three peaks were identified as the unmodified peptide and two peptides whose molecular weights were 66 and 100 higher than that of TAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1980

Amino acids in the amniotic fluid of diabetic mothers.

Subsequent to the spontaneous rupture of membranes in the 40th week of pregnancy, and in the course of labour inductions in cases of prolonged pregnancy (42nd week) and of diabetic mothers in the 40th week of pregnancy, the concentration of amino acids in the amniotic fluid was studied. In the group of diabetic mothers the concentrations of lysine, histidine, cysteic acid, taurine, serine, glutamic acid, glycine, alanine and phenylalanine are significantly higher. The arteriovenous difference found for some amino acids in the umbilical blood of physiological fetuses is absent in the fetuses of diabetic mothers.

Amino Acids↗
PubMed1999

Melain G, a cysteine protease from green fruits of the bead tree, Melia azedarach: a protease affected by specific amino acids at P3 position.

A protease (melain G) was isolated from the greenish fruits of the bead tree, Melia azedarach var. japonica Makino. Melain G shares 110 identical amino acid residues (50%) with papain, 112 (51%) with actinidain, and 91 (41%) with stem bromelain. From the sites cleaved in the oxidized insulin B-chain and synthetic oligopeptide substrates by melain G, the enzyme preferred small amino acid residues such as Gly or Ser at the P2 position and negatively charged residues such as glutamic or cysteic acid at the P3 position. This is clearly different from the specificity of papain, which prefers the large hydrophobic amino acid residues such as Phe, Val, and Leu at the P2 position. Accordingly, it is presumed that the bottom of the S2 pocket of melain G is shallow due to the presence of a Phe residue, and a bulky P2 substrate (for example Phe residue) is not preferred by the enzyme. Negatively charged residues at the P3 position of substrates well suited the S3 site of melain G for making a salt bridge. It is likely that Arg61 is the S3 position of melain G by analogy with papain.

Amino Acid Sequence↗