Antibacterial activity of S-methyl methanethiosulfinate and S-methyl 2-propene-1-thiosulfinate from Chinese chive toward Escherichia coli O157:H7.
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S-Methyl methanethiosufinate (1) and S-methyl 2-propene-1-thiosulfinate (2) were easily seperated from Chinese chive (Allium tuberosum L.) using simple column chromatography. Both compounds showed significant antibacterial activities against E. coli O-157:H7 including spoilage microorganism in food. Structural assignment was based on Mass and NMR-spectroscopic methods. (+info)
Modulation of the reactivity of the essential cysteine residue of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.
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Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible NAD(P)(+)-dependent oxidation of betaine aldehyde to glycine betaine. In the human opportunistic pathogen Pseudomonas aeruginosa this reaction is an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. As with every aldehyde dehydrogenase studied so far, BADH possesses an essential cysteine residue involved in the formation of the intermediate thiohemiacetal with the aldehyde substrate. We report here that the chemical modification of this residue is conveniently measured by the loss in enzyme activity, which allowed us to explore its reactivity in a pH range around neutrality. The pH dependence of the observed second-order rate constant of BADH inactivation by methyl methanethiosulphonate (MMTS) suggests that at low pH values the essential cysteine residue exists as thiolate by the formation of an ion pair with a positively charged residue. The estimated macroscopic pK values are 8.6 and 4.0 for the free and ion-pair-forming thiolate respectively. The reactivity towards MMTS of both thiolate forms is notably lower than that of model compounds of similar pK, suggesting a considerable steric inhibition by the structure of the protein. Binding of the dinucleotides rapidly induced a significant and transitory increment of thiolate reactivity, followed by a relatively slow change to an almost unreactive form. Thus it seems that to gain protection against oxidation without compromising catalytic efficiency, BADH from P. aeruginosa has evolved a complex and previously undescribed mechanism, involving several conformational rearrangements of the active site, to suit the reactivity of the essential thiol to the availability of coenzyme and substrate. (+info)
Relative importance of the two major pathways for the conversion of cysteine to glucose in the perfused rat liver.
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The effects of dietary treatments and substrate availability on the rate of gluconeogenesis from L-cysteine has been investigated in the perfused rat liver. At an optimal concentration (10 mM) of [U-14C]cysteine, after 40 minutes, 3.9% of the label appeared in glucose. This corresponded to 90% of the net glucose coming from cysteine. Cysteine was then shown to be converted to glucose at a physiological concentration of substrate (0.1 mM) as well as at the optimal concentration. After 40 minutes of perfusion with 0.1 mM [U-14C]cysteine as the substrate, livers of 72-hour starved rats incorporated 1.7% of the label into glucose, and livers of rats perfused without prior starvation incorporated 0.53% of the label into glucose. This suggested that cysteine was glucogenic at optimal and physiological concentrations of cysteine in both fed and starved rats. To determine which, if either, of the two suggested pathways for the conversion of cysteine to glucose was quantitatively more important, livers were perfused with [U-14C]cysteine alone or with [U-14C]cysteine plus cysteine sulfinic acid. The addition of cysteine sulfinate (10 mM) reduced the incorporation of 14C from cysteine into glucose from 3.9 to 2.7%. This suggested that one-third of the cysteine to glucose proceeded via the cysteine sulfinate-dependent pathway. (+info)
Garlic attenuates nitric oxide production in rat cardiac myocytes through inhibition of inducible nitric oxide synthase and the arginine transporter CAT-2 (cationic amino acid transporter-2).
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It is now accepted that allicin, the main biologically active compound in garlic, exhibits antioxidant activity. The present study was designed to test the hypothesis that the antioxidant activity of garlic can be partially attributed to the inhibition of nitric oxide (NO) production by cytokine-induced NO synthase (iNOS). Cardiac myocytes cultured from neonatal Wistar rats were stimulated by lipopolysaccharide (LPS) and incubated for 24 h with various concentrations of allicin. This resulted in marked inhibition of nitrite production. Interestingly, a low concentration of allicin (10 microM) was significantly more potent in abrogating the effect of LPS on nitrite production than a higher concentration (40 microM). Allicin decreased steady-state iNOS mRNA levels, and this effect was maximal when a lower concentration was used (10 microM compared with 40 microM). In order to explore additional effects of allicin on NO generation that might counteract the effect on iNOS, we assessed the effects of higher allicin concentrations on arginine transport. Allicin inhibited the uptake of 1 mM extracellular arginine in a concentration-dependent manner. The expression of the two arginine transporters that are expressed in cardiac myocytes [CAT-1 (cationic amino acid transporter-1) and CAT-2] was studied using reverse transcription-PCR. A concentration of 200 microM allicin abolished the expression of CAT-2 mRNA, 100 microM significantly attenuated it, whereas 50 microM had no effect. Allicin had no effect on steady-state CAT-1 mRNA levels. Our results suggest that allicin inhibits iNOS activity through two different mechanisms: at lower concentrations it decreases iNOS mRNA levels, whereas at higher concentrations it inhibits arginine transport through down-regulation of CAT-2 mRNA. (+info)
A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress.
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Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells. (+info)
An essential role of N-terminal arginylation in cardiovascular development.
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The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues-aspartate, glutamate, and cysteine-are substrates for arginylation. The mouse ATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found that ATE1-/- embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor. (+info)
Peroxiredoxin evolution and the regulation of hydrogen peroxide signaling.
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Eukaryotic 2-Cys peroxiredoxins (2-Cys Prxs) not only act as antioxidants, but also appear to regulate hydrogen peroxide-mediated signal transduction. We show that bacterial 2-Cys Prxs are much less sensitive to oxidative inactivation than are eukaryotic 2-Cys Prxs. By identifying two sequence motifs unique to the sensitive 2-Cys Prxs and comparing the crystal structure of a bacterial 2-Cys Prx at 2.2 angstrom resolution with other Prx structures, we define the structural origins of sensitivity. We suggest this adaptation allows 2-Cys Prxs to act as floodgates, keeping resting levels of hydrogen peroxide low, while permitting higher levels during signal transduction. (+info)
Reversing the inactivation of peroxiredoxins caused by cysteine sulfinic acid formation.
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The active-site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid during catalysis, which leads to inactivation of peroxidase activity. This oxidation was thought to be irreversible. However, by metabolic labeling of mammalian cells with 35S, we show that the sulfinic form of peroxiredoxin I, produced during the exposure of cells to H2O2, is rapidly reduced to the catalytically active thiol form. The mammalian cells' ability to reduce protein sulfinic acid might serve as a mechanism to repair oxidatively damaged proteins or represent a new type of cyclic modification by which the function of various proteins is regulated. (+info)