A new N-acyl derivative of (S)-cysteine for quantitative determination of enantiomers of amino compounds by HPLC method with a precolumn modification with o-phthalaldehyde

N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enantiomers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was compared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctionalized amines and amino alcohols. After the NAC modification, the resulting diastereomeric isoindoles were difficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols. The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition, allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the amino component and the thiol reagent favored the diastereomer separation. This method was used for determination of the absolute concentration of individual enantiomers of amines in the course of stereoselective enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of penicillin acylase. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.     

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

Simultaneous electrochemical determination of l-cysteine and l-cysteine disulfide at carbon ionic liquid electrode

A linear sweep voltammetric method is used for direct simultaneous determination of l-cysteine and l-cysteine disulfide (cystine) based on carbon ionic liquid electrode. With carbon ionic liquid electrode as a high performance electrode, two oxidation peaks for l-cysteine (0.62 V) and l-cysteine disulfide (1.3 V) were observed with a significant separation of about 680 mV (vs. Ag/AgCl) in phosphate buffer solution (pH 6.0). The linear ranges were obtained as 1.0–450 and 5.0–700 μM and detection limits were estimated to be 0.298 and 4.258 μM for l-cysteine and l-cysteine disulfide, respectively. This composite electrode was applied for simultaneous determination of l-cysteine and l-cysteine disulfide in two real samples, artificial urine and nutrient broth. Satisfactory results were obtained which clearly indicate the applicability of the proposed electrode for simultaneous determination of these compounds in complex matrices.     

Inhibition of sulfate excretion by (aminooxy)acetate induced stimulation of taurine excretion in rats

Summary l-Cysteine is mainly metabolized to sulfate and taurine through cysteinesulfinate pathway. Alternatively, sulfate is formed in rat liver mitochondria via 3-mercaptopyruvate pathway. Intraperitoneal administration of 5 mmol ofl-cysteine per kg of body weight resulted in the increase in sulfate and taurine (plus hypotaurine) excretion in the 24-h urine, which corresponded to 45.3 and 29.3%, respectively, ofl-cysteine administered. Subcutaneous injection of (aminooxy)acetate, a potent inhibitor of transaminases, together withl-cysteine halved the sulfate excretion and doubled the taurine excretion. In vitro sulfate formation froml-cysteine and froml-cysteinesulfinate in rat liver mitochondria was inhibited by (aminooxy)-acetate. The sulfate-forming activity of liver mitochondria obtained from rats injected with (aminooxy) acetate was also inhibited. These results indicate that the transamination reaction is crucial in sulfate formation and in the regulation of sulfur metabolism. Sulfur equilibrium in mammals was discussed.     

An investigation into possible xenobiotic–endobiotic inter-relationships involving the amino acid analogue drug, <Emphasis Type="Italic">S</Emphasis>-carboxymethyl-<Emphasis Type="SmallCaps">l</Emphasis>-cysteine and plasma amino acids in humans

The amino acid derivative, S-carboxymethyl-l-cysteine, is an anti-oxidant agent extensively employed as adjunctive therapy in the treatment of human pulmonary conditions. A major biotransformation route of this drug, which displays considerable variation in capacity in man, involves the oxidation of the sulfide moiety to the inactive S-oxide metabolite. Previous observations have indicated that fasted plasma l-cysteine concentrations and fasted plasma l-cysteine/free inorganic sulfate ratios were correlated with the degree of sulfoxidation of this drug and that these particular parameters may be used as endobiotic biomarkers for this xenobiotic metabolism. It has been proposed also that the enzyme, cysteine dioxygenase, was responsible for the drug sulfoxidation. Further in this theme, the degree of S-oxidation of S-carboxymethyl-l-cysteine in 100 human volunteers was investigated with respect to it potential correlation with fasted plasma amino acid concentrations. Extensive statistical analyses showed no significant associations or relationships between the degree of drug S-oxidation and fasted plasma amino acid concentrations, especially with respect to the sulfur-containing compounds, methionine, l-cysteine, l-cysteine sulfinic acid, taurine and free inorganic sulfate, also the derived ratios of l-cysteine/l-cysteine sulfinic acid and l-cysteine/free inorganic sulfate. It was concluded that plasma amino acid levels or derived ratios cannot be employed to predict the degree of S-oxidation of S-carboxymethyl-l-cysteine (or vice versa) and that it is doubtful if the enzyme, cysteine dioxygenase, has any involvement in the metabolism of this drug.     

Comparison of N-acetyl-L-cysteine and L-cysteine in respect to their transmembrane fluxes

The objective of the present study was to compare cysteine and N-acetyl-L-cysteine in respect to their transmembrane fluxes and find out which one is a better available precursor for the cells and thus better supports the intracellular glutathione synthesis. Cysteine can directly participate in glutathione synthesis, whereas N-acetyl-L-cysteine must be first deacetylated before its incorporation to glutathione. In the present study we investigated and compared the efficiencies of cysteine and N-acetyl-L-cysteine influx and efflux through the erythrocyte membrane. Erythrocytes transported both cysteine and N-acetyl-L-cysteine in a concentration-dependent manner. However, our results demonstrated that cysteine crosses the erythrocyte membranes more efficiently as compared to N-acetyl-L-cysteine. Treatment of erythrocytes with 5 mM of cysteine or N-acetyl-L-cysteine for 1 hr raised the intracellular free sulfhydryl group (free-SH) levels to 3.37 ± 0.006 or 2.23 ± 0.08 μ mol/ml erythrocyte, respectively. Cysteine more effectively than N-acetyl-L-cysteine restored the intracellular free-SH level depleted beforehand. In erythrocytes previously depleted of free-SH, 5 mM cysteine raised the free-SH level to 1.45 ± 0.075 μ mol/ml within 1 hr, whereas N-acetyl-L-cysteine at the same concentration raised this level to 0.377 ± 0.034 μmol/ml only. The results of our study also revealed that both cysteine and N-acetyl-L-cysteine influx and efflux processes are temperature dependent indicating that their transport requires biological activity. Our results demonstrate that cysteine is a better thiol precursor for the erythrocytes. Availability of cysteine for the cells is higher than that of N-acetyl-L-cysteine. The article is published in the original.     

Mechanisms underlying the cardioprotective effect of l-cysteine

In many tissues the availability of l-cysteine is a rate-limiting factor in glutathione production, though this has yet to be fully tested in heart. This study aimed to test the hypothesis that supplying hearts with 0.5 mM l-cysteine would preserve glutathione levels leading to an increased resistance to ischaemia reperfusion.Left ventricular function was measured in isolated perfused rat hearts before, during and after exposure to 45 min global normothermic ischaemia. Control hearts received Krebs throughout, whilst in treated hearts 0.5 mM l-cysteine was added to the perfusate 10 min before ischaemia, and was then present throughout ischaemia and for the first 10 min of reperfusion. Reperfusion injury was assessed from the appearance of lactate dehydrogenase (LDH) in the effluent. In two separate groups of control and treated hearts, ATP and glutathione (GSH) contents were measured at the beginning and end of ischaemia.Hearts treated with 0.5 mM l-cysteine showed a significantly higher recovery of rate pressure product (16,256± 1288 mmHg bpm vs. 10,324± 2102 mmHg bpm, p < 0.05) and a significantly lower release of LDH (0.54± 0.16 IU/g wet weight vs. 1.44± 0.31 IU/g wet weight, p < 0.05) compared to controls. Also, the l-cysteine treated group showed significantly better preservation of ATP and GSH during ischaemia in comparison to control.These results suggest that the mechanisms underlying the cardioprotective effects of 0.5 mM l-cysteine may include: increased anaerobic energy production either directly or through reduced degradation of adenine nucleotides; direct scavenging of free radicals; and/or improved antioxidant capacity through glutathione preservation.     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

The interconversion of ACC deaminase and <Emphasis Type="SmallCaps">d</Emphasis>-cysteine desulfhydrase by directed mutagenesis

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.     

l-Cysteine metabolism via 3-mercaptopyruvate pathway and sulfate formation in rat liver mitochondria

Summary We have studied the 3-mercaptopyruvate pathway (transamination pathway) ofl-cysteine metabolism in rat liver mitochondria.l-Cysteine and other substrates at 10 mM concentration were incubated with mitochondrial fraction at pH 8.4, and sulfate and thiosulfate were determined by ion chromatography. Whenl-cysteine alone was incubated, sulfate formed was 0.7µmol per mitochondria from one g of liver per 60 min. Addition of 2-oxoglutarate and GSH resulted in more than 3-fold increase in sulfate formation, and thiosulfate was formed besides sulfate. The sum (A + 2B) of sulfate (A) and thiosulfate (B) formed was approximately 7-times that withl-cysteine alone. Incubation with 3-mercaptopyruvate resulted in sulfate and thiosulfate formation, and sulfate was formed with thiosulfate. These reactions were stimulated with glutathione. Sulfate formation froml-cysteinesulfinate and 2-oxoglutarate was not enhanced by glutathione and thiosulfate was not formed. These findings indicate thatl-cysteine was metabolized and sulfate was formed through 3-mercaptopyruvate pathway in mitochondria.     

Functional cardiovascular action of l-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat

The endogenous sulfur-containing amino acid l-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to l-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to l-glutamate (10 mM, 34 nl), microinjections of l-cysteine increased ABP and HR dose dependently (3–100 mM, 34 nl). The cardiovascular responses to l-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to l-cysteine. The results indicate that l-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to l-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of l-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.     

The outer membrane TolC is involved in cysteine tolerance and overproduction in <Emphasis Type="Italic">Escherichia coli</Emphasis>

l-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of l-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in l-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in l-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to l-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for l-cysteine tolerance in E. coli cells. However, l-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent l-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of l-cysteine, the transformants exhibited more l-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in l-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for l-cysteine production in E. coli. Natthawut Wiriyathanawudhiwong and Iwao Ohtsu contributed equally to this work.     

Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.     

Inhibition of sulfate-forming activity in rat liver mitochondria by (aminooxy)acetate

Summary The effect of (aminooxy)acetate, an inhibitor of aminotransferases, on the sulfate formation froml-cysteine andl-cysteinesulfinate in rat liver mitochondria was studied. Incubation of 10 mMl-cysteine with rat liver mitochondria at 37°C in the presence of 10 mM 2-oxoglutarate and 10 mM glutathione resulted in the formation of 4.60 and 1.52µmol of sulfate and thiosulfate, respectively, per 60 min per mitochondria obtained from 1 g of liver. Under the same conditions sulfate formation froml-cysteinesulfinate was 24.96µmol, but thiosulfate was not formed. The addition of (aminooxy)acetate at 2 mM or more completely inhibited the sulfate and thiosulfate formation froml-cysteine and the sulfate formation froml-cysteinesulfinate. These findings support our previous conclusion that cysteine transamination and 3-mercaptopyruvate pathway (MP pathway) are involved in the sulfate formation froml-cysteine in rat liver mitochondria (Ubuka et al., 1992).     

Synthesis of the mitogenic S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylpentapeptide from Escherichia coli lipoprotein

The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid. The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids. S-[2,3-Bis(palmitoyloxy)propyl]-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid. The compounds were characterized unequivocally by 13C-NMR and mass spectra. The diastereomers of S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column.     

Development and evaluation of a new growth medium for Helicobacter pylori

The present study was aimed at modifying the original formulation of Commercial Eugon agar (CEA) to develop a new H. pylori growth medium. Initial studies were carried out to determine the number of H. pylori colonies recovered on in-house H. pylori agar (IHPA), IHPA without l -cysteine and sodium sulfite (IHPA-NC), IHPA without l -cysteine (IHPA-C), IHPA without sodium sulfite (IHPA-N) and CEA as the control. Significant differences ( P <0.001) in the number of colonies recovered were observed between IHPA-N, IHPA-NC and IHPA-C. Incorporation of sodium sulfite decreased the number of colonies recovered, indicating that sodium sulfite was inhibitory to H. pylori growth. Removal of l -cysteine reduced the number of colonies recovered, suggesting that l -cysteine is necessary for the growth of H. pylori . In the subsequent study, incorporation of K₂HPO₄ further increased the number of colonies recovered compared with IHPA-N ( P <0.001), and 0.25% (w/v) of K₂HPO₄ yielded the highest numbers of colonies ( P ≤0.04). Finally, thirty other H. pylori clinical isolates were evaluated for their growth in the IHPAP-N, a new medium consisting of 1.5% (w/v) pepticase, 0.5% (w/v) peptone, 0.4% (w/v) sodium chloride, 0.03% (w/v) l -cysteine, 0.55% (w/v) dextrose, 0.25% (w/v) K₂HPO₄ and 1.5% (w/v) agar. The number of colonies recovered in IHPAP-N was significantly ( P <0.005) higher than that of CEA. IHPAP-N with 0.25% K₂HPO₄ and without sodium sulfite were adequate solid media for the growth of H. pylori .     

A New N-Acyl Derivative of (S)-Cysteine for Quantitative Determination of Enantiomers of Amino Compounds by HPLC with a Precolumn Modification with o-Phthalaldehyde

N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enantiomers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was compared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctionalized amines and amino alcohols. After the NAC-modification, the resulting diastereomeric isoindoles were difficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols. The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition, allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the amino component and the thiol reagent favored the diastereomer separation. This method was used for determination of the absolute concentration of individual enantiomers of amines in the course of stereoselective enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of penicillin acylase.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

Conversion of L-Cystine and L-Cysteine to Taurine by the Enzyme Systems of Liver Cells

The kinetics of the conversion of the sulfur-containing amino acids L-cystine and L-cysteine to taurin by the enzyme system of cattle liver cells was studied and a mathematical model was developed. It was shown that the conversions of L-cystine and L-cysteine obeyed the Michaelis–Menten equations of serial–sequential conversions with regard to inhibition by the final product and inactivation. The yield of taurin under the optimized conditions of L-cystine and L-cysteine conversion (temperature, 40°C; pH 1.5 and 3.0, respectively; and the addition of enzyme preparations in five equal portions at 2-h intervals) was in the range of 80–85% of the substrate weight.