Nitric oxide affects salt-induced changes in free amino acid levels in maize

It was assumed that salt-induced redox changes affect amino acid metabolism in maize (Zea mays L.), and this influence may be modified by NO. The applied NaCl treatment reduced the fresh weight of shoots and roots. This decrease was smaller after the combined application of NaCl and an NO-donor ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, DETA/NO) in the shoots, while it was greater after simultaneous treatment with NaCl and nitro-l-arginine (l-NNA, inhibitor of NO synthesis) in the roots. The quantum yield efficiency of photosystem II was not influenced by the treatments. NaCl had a significant effect on the redox environment in the leaves as it was shown by the increase in the amount of glutathione disulphide and in the redox potential of the glutathione/glutathione disulphide redox pair. This influence of NaCl was modified by DETA/NO and l-NNA. Pharmacological modification of NO levels affected salt-induced changes in both the total free amino acid content and in the free amino acid composition. NaCl alone increased the concentration of almost all amino acids which effect was strengthened by DETA/NO in the case of Pro. l-NNA treatment resulted in a significant increase in the Ala, Val, Gly and Tyr contents. The Ile, Lys and Val concentrations rose considerably after the combined application of NaCl and DETA/NO compared to NaCl treatment alone in the recovery phase. NaCl also increased the expression of several genes related to the amino acid and antioxidant metabolism, and this effect was modified by DETA/NO. In conclusion, modification of NO levels affected salt-induced, glutathione-dependent redox changes and simultaneously the free amino acid composition and the level of several free amino acids. The observed much higher Pro content in plants treated with both NaCl and DETA/NO during recovery may contribute to the protective effect of NO against salt stress.     

Volatile constituents from cauliflower and other crucifers

Vapour emanating from intact turnip and radish plants contained small amounts of only one component, probably hexenyl acetate, distinguishable from background vapours. From disrupted leaf tissues of cauliflower, turnip, radish and wallflower at 30°, the predominant vapour component was cis-hex-3-enyl acetate, whereas allyl isothiocyanate was the chief component from Brassica juncea. Other minor components were also identified. Apart from isothiocyanates, the only sulphur-containing component identified from these crucifers was dimethyl disulphide which was detected only from cauliflower, particularly in vapour from disrupted curd tissue.     

l-Cystine inhibits aspartate-β-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-β-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by l- and d-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, β-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK_a of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [³⁵S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [³⁵S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)₂TVSL, thus demonstrating that the essential ¹³⁵Cys is the amino acid residue modified by the treatment with cystine.     

Toxicity of Glucosinolates and Their Enzymatic Decomposition Products to Caenorhabditis elegans

An aquatic 24-hour lethality test using Caenorhabditis elegans was used to assess toxicity of glucosinolates and their enzymatic breakdown products. In the absence of the enzyme thioglucosidase (myrosinase), allyl glucosinolate (sinigrin) was found to be nontoxic at all concentrations tested, while a freeze-dried, dialyzed water extract of Crambe abyssinica containing 26% 2-hydroxyl 3-butenyl glucosinolate (epi-progoitrin) had a 50% lethal concentration (LC₅₀) of 18.5 g/liter. Addition of the enzyme increased the toxicity (LC₅₀ value) of sinigrin to 0.5 g/liter, but the enzyme had no effect on the toxicity of the C. abyssinica extract. Allyl isothiocyanate and allyl cyanide, two possible breakdown products of sinigrin, had an LC₅₀ value of 0.04 g/liter and approximately 3 g/liter, respectively. Liquid chromatographic studies showed that a portion of the sinigrin decomposed into allyl isothiocyanate. The results indicated that allyl isothiocyanate is nearly three orders of magnitude more toxic to C. elegans than the corresponding glncosinolate, suggesting isothiocyanate formation would improve nematode control from application of glucosinolates.     

Glutathione reductase activity with an oxidized methylated glutathione analog

Abstract

The activity of glutathione reductase with an unnatural analog of oxidized glutathione was explored. The analog, L-γ-glutamyl-2-methyl-L-cysteinyl-glycine disulfide, places an additional methyl group on the alpha position of each of the central cysteine residues, which significantly increases steric bulk near the disulfide bond. Glutathione reductase was completely unable to catalyze the sulfur–sulfur bond reduction of the analog. Additionally, enzyme kinetics experiments indicated that the analog acts as a competitive inhibitor of glutathione reductase. Computational studies confirm that the methylated analog fits within the active site of the enzyme but its disulphide bond geometry is altered, preventing reduction by the enzyme. The substitution of (R)-2-methylcysteine in place of natural (R)-cysteine in peptides constitutes a new strategy for stabilizing disulphide bonds from enzyme-catalyzed degradation.     

Molecular effects of homocysteine on cbEGF domain structure: insights into the pathogenesis of homocystinuria

Homocystinuria is an inborn error of methionine metabolism that results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Homocystinurics often exhibit phenotypic abnormalities that are similar to those found in Marfan syndrome (MFS), a heritable connective tissue disorder that is caused by reduced levels of, or defects in, the cysteine-rich extracellular matrix (ECM) protein fibrillin-1. The phenotypic similarities between homocystinuria and MFS suggest that elevated homocysteine levels may result in an altered function of fibrillin-1. We have used recombinant calcium binding epidermal growth factor-like (cbEGF) domain fragments from fibrillin-1, and an unrelated protein Notch1, to analyse the effects of homocysteine on the native disulphide (cystine) bonds of these domains. We show using analytical reverse phase, high performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry (ESI-MS) and limited proteolysis that homocysteine attacks intramolecular disulphide bonds causing reduction of cystine and domain misfolding, and that the effects of homocysteine are dependent on its concentration. We also identify the importance of calcium binding to cbEGF domains for their stabilisation and protection against homocysteine attack. Collectively, these data suggest that reduction of intramolecular cbEGF domain disulphide bonds by homocysteine and the resulting disruption of this domain fold may contribute to the change in connective tissue function seen in homocystinuria. Furthermore, since we show that the effects of homocysteine are not unique to fibrillin-1, other cbEGF-containing proteins may be implicated in the pathogenic mechanisms underlying homocystinuria.     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

An Ion-channel Modulator from the Saliva of the Brown Ear Tick has a Highly Modified Kunitz/BPTI Structure

Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 Å resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by β-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the ”Kunitz head” by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a β-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.     

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. α-Aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.     

NADP-malate dehydrogenase from Zea mays leaves: Amino acid composition and thiol content of active and inactive forms

NADP-malate dehydrogenase (L-malate: NADP⁺ oxidoreductase, E.C. 1.1.1.82) was purified from the leaves of Zea mays L. and its subunit molecular weight, amino acid composition and the changes in number of thiol groups during activation were determined. The amino acid composition we found differed from that reported earlier for the Z. mays enzyme but was very similar to that reported for the enzyme isolated from pea leaves. The maize enzyme contains fewer methionine residues (3 compared to 5 in pea) but a greater total number of cysteine residues (6 compared to 3 in pea). In its inactive form (oxidised) the enzyme contained 2 thiols per subunit of which only 1 reacts with 5,5′-dithiobis(2-nitrobenzoic acid) when the enzyme is in its native form. During activation by dithiothreitol two disulphide bonds are reduced per subunit to give 4 new thiol groups. We conclude that NADP-malate dehydrogenase from leaves of the C₄ plant Z. mays is very similar to the enzyme from the C₃ plant pea. However, apparently two disulphide bonds are reduced during the reductive activation of the Z. mays enzyme in vitro compared with 1 disulphide bond for the pea enzyme.     

Expansion of the Genetic Code Through the Use of Modified Bacterial Ribosomes

Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, β-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.     

Interactions between cysteine residues as probes of protein conformation: the disulphide bond between Cys-14 and Cys-38 of the pancreatic trypsin inhibitor.

The rate constants for the reversible reduction by dithiothreitol of the disulphide bond linking eysteines 14 and 38 of the native pancreatic trypsin inhibitor have been measured. The results are consistent with this disulphide bond being formed as the last step in refolding of the fully reduced inhibitor.The rates of reduction of several model linear disulphides have been measured under the same conditions. A linear relationship was found between the rate of reduction and the ionization tendency of the thiol group generated.The apparent pK value of the thiol groups of cysteines 14 and 38 of the selectively reduced inhibitor were measured by the pH-dependence of their rate of alkylation with iodoacetamide. The rate of reduction of the disulphide bond between these two residues was very close to that predicted from the model compounds.The kinetics and thermodynamics of disulphide bond formation and breakage are shown to be useful for experimental determination of conformational transitions in proteins and model peptides.     

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells

Abstract. Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 µ m cysteine and/or 200–400 µ m cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 m m BSO treatment significantly increased cell proliferation as measured by ³H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G₁ phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G₁-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.     

Characterisation of the thiol–disulphide chemistry of desmopressin by LC, μ-LC, LC-ESI-MS and Maldi-Tof

Summary. To date, the majority of therapeutic peptides and proteins have to be administered via parenteral routes, which are painful and inconvenient. In order to gain sufficient high blood concentrations after oral application, various barriers in the gastrointestinal tract have to be overcome. Apart from a poor membrane uptake and intense enzymatic degradation, this study will demonstrate that thiol–disulphide reactions are an underestimated essential part of the presystemic metabolism. Glutathione, integrative part of the antioxidant defence system in the gastrointestinal tract, may play an important role in the inactivation of orally given peptides and proteins. In order to verify this hypothesis, desmopressin which bears a single disulphide bond was used as model peptide drug. Desmopressin was incubated with GSH in various concentrations, and the extent of thiol/disulphide exchange reactions between the peptide drug and GSH was investigated in dependence on pH and ratio of reactants determined as a function of time via HPLC, LC-MS and Maldi-Tof-MS analyses. Results showed that desmopressin is degraded by 1% reduced glutathione at pH 4 and pH 5.5. In presence of 0.01%, 0.1% and 1% of reduced glutathione 6.1%, 19.4% and 52.1% of desmopressin, respectively, were degraded. The masses of the conjugates after deconvolution measured by liquid chromatography and electrospray ionisation mass spectrometric detection were m/z 1069.67, m/z 1376.50, m/z 1683.40 and m/z 2138. These molecular masses, confirmed by Maldi-Tof-MS analysis, correspond with the masses of conjugates expected in theory. Under defined conditions, these results reveal that thiol–disulphide exchange reactions have a considerable impact on the alteration of peptide drugs and proteins.     

Adaptive enhancement of cystine and glutamate uptake in human diploid fibroblasts in culture

The effect of cystine starvation on the transport system of cystine and glutamate was examined in cultures of human diploid fibroblasts. The 2-min uptake of cystine and glutamate increased progressively after a lag of 6 h of cystine starvation. There was approx. 2–3-fold increase, and the increased rate of uptake was accompanied by an increase in the V_max and unchanged K_m. The cystine starvation-induced enhancement appeared specific for the uptake of cystine and glutamate. Actinomycin D or cycloheximide completely blocked the time-related increase in the uptake. Depletion of glutamate did not lead to the enhanced uptake, whereas depletion of glycine and serine caused as much increase in the uptake as depletion of cystine did. The intracellular pool of glutathione was extremely reduced by depletion of cystine, or of glycine and serine, but to a far less extent by depletion of glutamate. The results indicate that the transport system for cystine and glutamate appears to undergo adaptive regulation. It is suggested that glutathione may function as a regulatory signal to this transport system.     

A diagonal paper-electrophoretic technique for studying amino acid sequences around the cysteine and cystine residues of proteins

1. A diagonal electrophoretic technique for studying the amino acid sequence around cysteine and cystine residues in proteins is described. The residues are first converted into S-aminoethylcysteine, and the protein is then treated with S-ethyl trifluorothioacetate, which trifluoroacetylates all the protein amino groups. The modified protein is digested enzymically and the resulting peptides are separated by paper electrophoresis. After exposure of the peptides on the paper to ammonia vapour, the electrophoresis is repeated, this time at right angles to the original direction. Peptides from which a trifluoroacetyl group is removed by the ammonia treatment will vacate the 45° diagonal formed by all other unaffected peptides owing to the exposure of an additional amino group and consequent increased electrophoretic mobility towards the cathode. Peptides containing lysine or S-aminoethylcysteine are readily purified by this technique. 2. The successful application of the technique to bovine insulin is described. 3. Various methods for distinguishing peptides containing lysine from those containing S-aminoethylcysteine in more complicated proteins are suggested and discussed.     

Role of a disulphide bond in Helicobacter pylori arginase

Arginase is a binuclear Mn²⁺-metalloenzyme of urea cycle that hydrolyses arginine to ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co²⁺. Previous study reported that DTT strongly inhibits the H. pylori enzyme activity suggesting that a disulphide bond is critical for the catalysis. In this study, we have undertaken steady-state kinetics, circular dichroism and mutational analysis to examine the role of a disulphide bond in this protein. By mutational analysis, we show that the disulphide bond is not important for catalytic activity; rather it plays an important role for the stability of the protein as observed from thermal denaturation studies. The loss of catalytic activity in the wild-type protein with DTT is due to the interaction with Co²⁺. This is verified with the Mn²⁺-reconstituted proteins which showed a marginal loss in the activity with DTT.     

Differential effect of the amino acid cystine in cultured mammalian and bacterial cells exposed to oxidative stress.

The effect of cystine in the cytotoxic response of cultured Chinese hamster ovary and Escherichia coli cells to challenge with hydrogen peroxide has been investigated. It was found that this amino acid could either protect or sensitize cells, depending on the cellular system. In fact, although a reduction in the growth-inhibitory effect of hydrogen peroxide was observed in mammalian cells, a marked increase in the susceptibility to oxidative stress was induced by cystine in bacteria. None of the amino acid precursors of glutathione, e.g., glutamate, glycine or cysteine, afforded protection in the mammalian cell system, whereas cysteine, but not glycine or glutamate, markedly sensitized bacteria to hydrogen peroxide-induced cell killing. In mammalian cells, methionine, an amino acid which is converted to cysteine, was also unable to modify the oxidative response. The results presented indicate that cystine displays differential effects in oxidatively injured mammalian or bacterial cells and suggest that the mechanism whereby the amino acid modulates the lethal action of hydrogen peroxide differs in the two cellular systems.