Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme.

Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A. Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium. Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine. Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids. Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon. Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed. These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.     

The effect of cysteine and 2,4-dinitrophenol on heme and nonheme absorption in a rat intestinal model

Previous studies have showed that purified heme iron forms insoluble polymers that are poorly absorbed. The presence of peptides and of amino acids maintaining heme iron in a soluble form could improve its bioavailability. The digestive uptake and transfer of a concentrated hydrolysate of heme peptides (HPH) and of iron gluconate (Gluc) at 100 μM were compared in vitro in a Ussing chamber. The effects of an enhancing amino acid (L-cysteine) on the uptake and transfer of both forms were assessed. An inhibitor of the oxidative phosphorylation (2,4-dinitrophenol; DNP) was used to differentiate the active and passive mechanisms of the absorption. The mucosal uptake (%Tot) and enterocyte transfer (%S) of the two sources of iron did not differ. DNP significantly reduced %Tot and %S of both forms. Cysteine significantly enhanced %Tot and %S of HPH and Gluc, partly corrected the inhibition exerted by DNP on %Tot of HPH and %S of both forms, and fully restored %Tot of Gluc. In presence of peptides produced by globin hydrolysis, the absorption of hemoglobin iron was efficient; it was mostly energy dependent and, therefore, should have occurred by a regulated transcellular pathway. Cysteine enhanced the passive uptake of iron and the passive processes involved in the enterocyte transfer of the common pool made of both sources (heme and nonheme) of iron. These results showed that heme iron can be purified and concentrated without impairing its digestive absorption, provided it remains in presence of peptides or amino acids.     

The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures

Cysteine availability is normally the rate-limiting factor in glutathione synthesis. How neurons obtain cysteine from extracellular space is not well established. Here we used mouse cortical neuron cultures to examine the role of the excitatory amino acid transporters (EAATs) in neuronal cysteine uptake. The cultured neurons expressed both EAAT2 and EAAT3. Cysteine uptake was predominantly (> 85%) Na+-dependent, with an apparent Km of 37 microm. Cysteine uptake was reduced by the EAAT substrates l-glutamate and l-aspartate and by synthetic EAAT inhibitors. The non-selective EAAT inhibitor threo-beta-hydroxyaspartate had a significantly greater maximal inhibitory effect than did the EAAT2-selective inhibitor, dihydrokainate, indicating uptake by both EAAT2 and EAAT3. Serine, a substrate of ASC uptake system, had negligible effects on cysteine uptake at 10-fold excess concentrations. To assess the functional importance of EAAT-mediated cysteine uptake in neuronal glutathione synthesis, cultures were treated with diethylmaleate to deplete glutathione, then incubated with cysteine in the presence or absence of EAAT inhibitors. Threo-beta-benzyloxyaspartate and the non-transportable inhibitor threo-beta-hydroxyaspartate both inhibited the cysteine-dependent glutathione synthesis. The findings suggest that neuronal EAAT activity can be a rate-limiting step for neuronal glutathione synthesis and that the primary function of EAATs expressed by neurons in vivo may be to transport cysteine.     

The regulation of sulphurated amino acid junctions: fact or fiction in the field of inflammation?

Summary.  The diet of industrialised countries is usually rich in amino acids, which are in part used as a source of calories. However, metabolic alterations are observed in diseased patients and a preferential retention of Sulphurated Amino Acids (SAA) occurs during the inflammatory response. Moreover, it has been demonstrated in a model of an acute sepsis phase of rats that the metabolism of Cysteine is modified. The liver converts Cysteine at a different ratio of Sulphate to Taurine (Tau) i.e. the sulphate production decreases while the Tau conversion increases. The Glutathione (GSH) concentration is greater in the liver, kidneys and other organs and the Cysteine incorporation into proteins is higher in the spleen, lungs and plasma (Acute Phase Proteins) while the Albumin level decreases. The pro-inflammatory cytokines such as Interleukin-1, Interleukin-6 and TNF-α are the main initiators that alter protein and amino acid metabolism. Another important phenomenon is the impairment of Methionine conversion to Cysteine during stress. For example, premature infants or AIDS patients are capable of synthesizing Cysteine from Methionine at a much lower rate. Thus, the metabolic flow through the trans-sulphuration path may be inadequate to meet the Cysteine demand under critical conditions. In this complex picture, an SAA supply may contribute to an immune system regulation. Received November 30, 2001 Accepted January 15, 2002 Published online August 30, 2002 Author's address: Francesco Santangelo, Zambon Group, via Lillo del Duca 10, I-20091 Bresso, Milan, Italy, Email: francesco.santangelo@zambongroup.com     

Physicochemical properties of a beta-lactamase from Streptomyces UCSM-104

A purified beta-lactamase from Streptomyces UCSM-104 shows the presence of three subforms when stained for protein and/or for activity after polyacrylamide gel electrophoresis or after electrofocusing. The pI values of the three subforms were 5.45, 5.30 and 5.10, respectively. The respective electrophoretic mobilities were 4.6 X 10(-5), 5.2 X 10(-5) and 5.9 X 10(-5)m2/sV. Relative molecular mass of 14,900 was determined. The amino acid composition was established. Cysteine was not detected. A fairly high proline content (8.3%) differentiates this enzyme from other beta-lactamases. Lysine was the only N-terminal amino acid detected after dansylation. The possible origin of the subforms is discussed.     

Incorporation of specifically labeled cysteine into Escherichia coli protein

Protein obtained from several strains of Escherichia coli grown in the presence of [3,3′-¹⁴C]cystine contained the radiolabel in nearly all the other amino acids, suggesting catabolism of cysteine to pyruvic acid. Utilization in amino acid synthesis of the pyruvate thus generated can be blocked by growing the bacteria in a medium specifically enriched with most of the naturally occurring amino acids. Cysteine that is incorporated intact is diluted by de novo synthesis at low cystine concentrations; also, it was found that E. coli can use the sulfur of methionine for cysteine biosynthesis. Both of these latter two processes can be prevented by supplying an excess of exogenous cystine. This regiment leads to protein that is highly specifically labeled in the cysteine residues, with a minor amount (20–25%) of the label also appearing in alanine residues. Although this strategy was developed expressly for cysteine, it may be useful for incorporating other labeled amino acids that are also readily catabolized.     

Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine

Cysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents. Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine. The uptake of cysteine was mediated mainly by the ASC system (Na+-dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine). On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities. Because of the instability of cysteine, the culture media usually contained cystine but not cysteine. Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine. The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bathocuproinedisulfonate, a nontoxic copper-specific chelator that inhibits autoxidation of cysteine. Cysteine added at 25-400 microM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes. In the lipopolysaccharide-activated cells, however, cysteine enhanced the cellular glutathione contents in a dose-dependent manner. These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.     

Cysteine, even in low concentrations, induces transient amino acid starvation in Escherichia coli.

Cysteine, in concentrations down to 0.04 micrograms/ml, induces transient amino acid starvation in Escherichia coli growing in minimal medium. The duration depends on the concentration and is 5 min at 2 micrograms of cysteine per ml. At low cysteine concentrations, threonine and isoleucine almost completely abolish the starvation.     

AN EXAMINATION OF THE CATALYTIC PROPERTIES OF SEVERAL AMINOTRANSFERASES IN BRAIN AND LIVER BY MEANS OF AN IMPROVED RADIOMETRIC ASSAY WITH DINITROPHENYLHYDRAZINE

Abstract— The assay of aminotransferases, performed by solvent extraction of keto acids formed from labelled amino acids, has been modified to enhance the recovery of both aliphatic and aromatic keto acid products. The keto acids are first converted to their respective dinitrophenylhydrazones which are more completely extracted into less polar organic solvents. By this manoeuvre, both keto acid extraction is increased and the extraction of the precursor amino acid is reduced. Employing this technique, the kinetics of brain-stem γ-aminobutyric acid (GABA), tryptophan, 3,4-dihydroxyphenylalanine (DOPA) aminotransferases and brain-stem and liver tyrosine aminotransferases were examined. Brain-stem aminotransferases, particularly the aromatic amino acid transferases, have a higher affinity for both the amino acid and the keto acid when the aromatic keto acid, phenylpyruvate (0·8 mM), is employed as amino group acceptor, whereas maximal velocities for aminotransferase reactions are much greater when α-ketoglutarate (0·8 m m ) is the amino group acceptor. Brain-stem tyrosine aminotransferase exhibits a much lower affinity for tyrosine in the presence of either 0·8m m -α-ketoglutarate or 0·8 m m -phenylpyruvate than does liver tyrosine aminotransferase. p -Chlorophenylpyruvate and phenylpyruvate exhibit similar properties as amino group acceptors for brain-stem tryptophan aminotransferase. Cysteine inhibits tryptophan aminotransferase when phenylpyruvate is the amino group acceptor, in a manner which is competitive with the amino acid. Benzoylformate inhibits both tryptophan and DOPA aminotransferases when phenylpyruvate is the amino group acceptor, but this inhibition does not appear to be competitive with phenylpyruvate.     

Cysteine and Methionine Regulation of Sulfate Uptake in Potato Tuber Discs (Solanum tuberosum)

Sulfate uptake in potato tuber discs is inhibited by cysteine and methionine with an 8 h lag period. Cysteine, but not methionine, inhibition can be reversed by washing the treated discs. During the experimental period cysteine is rapidly metabolized, while methionine persists as a free amino acid. Amino acid inhibition of sulfate uptake is overcome by increasing sulfate concentration. The kinetic parameters change suggesting a loss of flexibility of the sulfate uptake system caused by sulfur amino acids.     

A purified cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K

Cysteine conjugate beta-lyase has been purified from rat kidney cytosol. The enzyme is a 100,000-dalton dimer of two 55,000-dalton subunits and has an absorption maximum at 432 nm. The enzyme has phenylalanine alpha-keto-gamma-methiolbutyrate transaminase activity and appears to be identical to rat kidney cytosolic glutamine transaminase K. Metabolism of S-1,2-dichlorovinyl-L-cysteine (DCVC) by the purified enzyme was dependent on the presence of either alpha-keto-gamma-methiolbutyrate or a protein factor which is present in the cytosolic fraction of rat kidney cortex. The protein factor was identified as a flavin containing L-amino acid oxidase which oxidized DCVC to S-(1,2-dichlorovinyl)-3-mercapto-2-oxopropionic acid. S-(1,2-Dichlorovinyl)-3-mercapto-2-oxopropionic acid has not been previously reported as a metabolite of DCVC. The data also show that rat kidney cytosolic glutamine transaminase K catalyzes both a beta-elimination and a transamination reaction with DCVC when alpha-keto-gamma-methiolbutyrate is present and that amino acid oxidase and alpha-keto-gamma-methiolbutyrate stimulate the enzyme activity by providing amino acceptors. When incubations were done with DCVC as substrate in the presence of excess alpha-keto-gamma-methiolbutyrate, the beta-lyase catalyzed beta-elimination and transamination in a ratio of 1:1.3, respectively. Under conditions where most of the alpha-keto-gamma-methiolbutyrate was consumed, the beta-elimination predominated indicating that the S-1,2-dichlorovinyl-3-mercapto-2-oxopropionic acid pool was consumed by transamination after the alpha-keto-gamma-methiolbutyrate had been depleted. The data are discussed with regard to the importance of these pathways as regulators or participants in the toxicity of S-cysteine conjugates.     

Molecular cloning of the E1 beta subunit of the rat branched chain alpha-ketoacid dehydrogenase.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.     

Role of Amino Thiols in Luminol Chemiluminescence Coupled with Copper(II)-catalysed Oxidation of Cysteine and Glutathione

The Cu(II)-catalysed oxidation of the amino thiols cysteine and glutathione with oxygen was examined in the presence of luminol. Light emission was markedly suppressed when the concentration of amino thiols was greater than that of Cu(II). Cysteine caused a delay of chemiluminescence (CL) and no CL emission was observed at high concentrations of GSH. The suppression in CL could be explained in terms of the complexation of the Cu(II) catalyst by formation of a Cu(II)–dicysteine complex in case of cysteine, and of a Cu(II)–GSSG complex in case of GSH.     

Comparison of properties of cancer procoagulant and human amnion-chorion procoagulant

Cysteine proteinases that initiate coagulation in the absence of factor VII have been isolated from rabbit V2 carcinoma and from human amnion-chorion. Many of their biochemical properties, including a molecular weight of 68 000 and inhibition by iodoacetamide and mercury, are the same. In the paper we compare the isoelectric point, the amino acid composition and the carbohydrate content of human amnion-chorion procoagulant and cancer procoagulant. With the exception of minor differences in the amino acid composition, attributable in part to differences in species, the two proteins are closely homologous.     

Analysis of thiohydantoins from amino acids by gas-liquid chromatography on short glass capillary columns

A method for separation of amino acid methyl or phenyl thiohydantoins by GLC on a short glass capillary column is described. Calculations of required parameters of the capillary column are presented. By the described methods, nineteen of twenty silylated methylthiohydantoins were separated in one run. The last one (histidine) can be identified on the same column by starting the analysis at a higher temperature. Cysteine and arginine were analysed as S-methylcysteine and ornithine, respectively.     

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.     

Conformationally sensitive residues in transmembrane domain 9 of the Na+/dicarboxylate co-transporter

The Na(+)/dicarboxylate co-transporter, NaDC-1, couples the transport of sodium and Krebs cycle intermediates, such as succinate and citrate. Previous studies identified two functionally important amino acids, Glu-475 and Cys-476, located in transmembrane domain (TMD) 9 of NaDC-1. In the present study, each amino acid in TMD-9 was mutated to cysteine, one at a time, and the accessibility of the membrane-impermeant reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) to the replacement cysteines was determined. Cysteine substitution was tolerated at all but five of the sites: the A461C mutant was not present at the plasma membrane, whereas the F473C, T474C, E475C, and N479C mutants were inactive proteins located on the plasma membrane. Cysteine substitution of four residues found near the extracellular surface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in proteins that were sensitive to inhibition by MTSET. The accessibility of MTSET to the four substituted cysteines was highest in the presence of the transported cations, sodium or lithium, and low in choline. The four mutants also exhibited substrate protection of MTSET accessibility. The MTSET accessibility to S478C, A481C, and A480C was independent of voltage. In contrast, T482C was more accessible to MTSET in choline buffer at negative holding potentials, but there was no effect of voltage in sodium buffer. In conclusion, TMD-9 may be involved in transducing conformational changes between the cation-binding sites and the substrate-binding site in NaDC-1, and it may also form part of the translocation pathway through the transporter.     

Derivatization of cysteine and cystine for fluorescence amino acid analysis with the o-phthaldialdehyde/2-mercaptoethanol reagent.

Previous reports (Drescher, D.G., and Lee, K.S. (1978) Anal. Biochem. 84, 559-569; Lee, K.S., and Drescher, D.G. (1978) Int. J. Biochem. 9, 457-467) have shown that high performance liquid chromatographic analysis of amino acids with the o-phthaldialdehyde/2-mercaptoethanol reagent (OPA/2-ME) is one of the most sensitive procedures currently available for micro amino acid analysis. In the present paper, methods are presented for the modification of cysteine and cystine in proteins for micro amino acid analysis using OPA/2-ME. Cysteine and cystine, which both show low fluorescence with OPA/2-ME, are converted to cysteic acid with performic acid directly, or to S-3-sulfopropylcysteine with 1,3-propane sultone after reduction of cystine with tri-n-butylphosphine. Cysteic acid and S-3-sulfopropylcysteine form highly fluorescent adducts with OPA/2-ME. The formation of S-3-sulfopropylcysteine in proteins and the subsequent hydrolysis of the proteins with methanesulfonic acid are particularly useful for complete amino acid analysis at the picomole level using a single sample.     

Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from western flower thrips (Frankliniella occidentalis)

Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR approach with degenerate oligonucleotides designed on conserved cathepsin L domains. At the deduced amino acid level, the clones possessed all functional and structural characteristics of cathepsin L, and showed high mutual identity and strong similarity with cathepsin L-like cysteine proteases from other insects and arthropods. Southern analysis indicated that a family of four closely related and 10-12 less-related genes encode the cathepsin L-like cysteine proteases in the thrips genome. Partial sequencing of genomic DNA demonstrated the presence of three introns in the coding DNA.