Addition Of L-cysteine Research Articles

L-Cysteine and glutathione restore the reduction of rat hippocampal Na+, K+-ATPase activity induced by aspartame metabolites

Studies have implicated aspartame (ASP) ingestion in neurological problems. The aim of this study was to evaluate hippocampal Na+,K+-ATPase and Mg2+-ATPase activities after incubation with ASP or each of ASP metabolites, phenylalanine (Phe), methanol (MeOH) and aspartic acid (asp) separately.Suckling rat hippocampal homogenates or pure Na+,K+-ATPase were incubated with ASP metabolites. Na+,K+-ATPase and Mg2+-ATPase activities were measured spectrophotometrically. Incubation of hippocampal or pure Na+,K+-ATPase with ASP concentrations (expected in the cerebrospinal fluid (CSF)) after ASP consumption of 34, 150 or 200mg/kg resulted in hippocampal enzyme activity reduction of 26%, 50% or 59%, respectively, whereas pure enzyme was remarkably stimulated. Moreover, incubation with hippocampal homogenate of each one of the corresponding in the CSF ASP metabolites related to the intake of common, high/abuse doses of the sweetener, inhibited Na+,K+-ATPase, while pure enzyme was activated. Hippocampal Mg2+-ATPase remained unaltered. Addition of l-cysteine (cys) or reduced glutathione (GSH) in ASP mixtures, related with high/toxic doses of the sweetener, completely or partially restored the inactivated membrane Na+,K+-ATPase, whereas the activated pure enzyme activity returned to normal.CSF concentrations of ASP metabolites related to common, abuse/toxic doses of the additive significantly reduced rat hippocampal Na+,K+-ATPase activity, whereas pure enzyme was activated. Cys or GSH completely or partially restored both enzyme activities.

Functionalization of a styrene/butadiene random copolymer by radical addition of l-cysteine derivatives

The functionalization of a styrene/butadiene (20/80) random copolymer (SBR) is performed by radical-mediated addition of l-cysteine derivatives to the macromolecules' double bonds. The reaction carried out in solution and in the melt leads to SBR chain bearing amino and carboxylate functionalities through thiol addition to the vinyl double bonds of the 1,2-butadiene units with anti-Markovnikov regioselectivity. The addition yield (up to 5wt%) and the occurrence of the crosslinking side reaction are investigated with reference to feed conditions and process parameters. The optical rotation of the reaction products confirms the addition of l-cysteine to SBR chain and provides a simple route to prepare optically-active polymers containing pendant amino acid residues.

Interplay of IscA and IscU in Biogenesis of Iron-Sulfur Clusters

Increasing evidence suggests that sulfur in ubiquitous iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. In Escherichia coli, the major cysteine desulfurase activity for biogenesis of iron-sulfur clusters has been attributed to IscS. The gene that encodes IscS is a member of an operon iscSUA, which also encodes two highly conserved proteins: IscU and IscA. Previous studies suggested that both IscU and IscA may act as the iron-sulfur cluster assembly scaffold proteins. However, recent evidence indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU (Ding, H., Harrison, K., and Lu, J. (2005) J. Biol. Chem. 280, 30432-30437). To further elucidate the function of IscA in biogenesis of iron-sulfur clusters, we evaluate the iron-sulfur cluster binding activity of IscA and IscU under physiologically relevant conditions. When equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS, L-cysteine and dithiothreitol, iron-sulfur clusters are assembled in IscU, but not in IscA, suggesting that IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contrast, when equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS and dithiothreitol but without L-cysteine, nearly all iron is bound to IscA. The iron binding in IscA appears to prevent the formation of the biologically inaccessible ferric hydroxide under aerobic conditions. Subsequent addition of L-cysteine efficiently mobilizes the iron center in IscA and transfers the iron for the iron-sulfur cluster assembly in IscU. The results suggest an intriguing interplay between IscA and IscU in which IscA acts as an iron chaperon that recruits "free" iron and delivers the iron for biogenesis of iron-sulfur clusters in IscU under aerobic conditions.

A ≥ 100000-MW Soybean Protein Fraction that Inhibits the Formation of Methanethiol and Hydrogen Sulfide in Aqueous Slurries of Isolated Soy Proteins with Added L-Cysteine

The addition of L-cysteine to aqueous slurries of commercial isolated soy proteins (ISP) increased methanethiol headspace levels by 17- to 36-fold over the control. Corresponding levels of hydrogen sulfide were about 10 to 19 times greater than methanethiol. Neither methanethiol nor hydrogen sulfide were detected when L-cysteine was added to aqueous slurries of hexane-defatted soy flour. The production of hydrogen sulfide and methanethiol in aqueous slurries of commercial ISP was inhibited by the addition of a component(s) recovered from the pH 4.6 supernatant obtained during laboratory preparation of ISP by isoelectric precipitation. The inhibitory component had a molecular weight (MW) of ≥ 100000 and an isoelectric point of about 5.9. This component was not serine acetyl transferase. Its inhibitory properties were inactivated at 70°C and diminished with elevated levels of methionine. Adding the ≥ 100000-MW soluble-proteins (from the isoelectric precipitation step) back to a nonheat-treated laboratory ISP during processing reduced the methanethiol level by 88%.

Elimination efficiency of different reagents for the memory effect of mercury using ICP-MS

The present study was carried out to solve the problems of long washout time and non-linear calibration curves encountered in mercury analysis using inductively coupled plasma mass spectrometry. Comparisons of the washing efficiency for different reagents to eliminate the mercury memory effect were made. Of all the selected washing reagents, mercapto reagents such as 2-mercaptoethanol and L-cysteine could efficiently clean the instrument. Taking the toxicity and odor of mercaptoalkanol into account, L-cysteine was preferred as the most suitable washing reagent and was added into the standard and sample solutions. A good linear calibration curve was obtained with the correlation coefficient of 0.9999. The detetion limit for addition of L-cysteine was 0.024 μg l−1. Addition of 0.18% L-cysteine into the sample solution also facilitates the washout of mercury even using deionized water. The recoveries of mercury in certified reference materials, i.e. human hair and dogfish muscle, were 97.2% and 98.3%, respectively, when using 0.18% L-cysteine in the sample solutions.

Prevention of the deterioration of polyphenol-rich beverages

Only a small amount of H 2O 2 was detected in beverages, such as tea or coffee, immediately after opening caps of bottles, but H 2O 2 was gradually produced in the beverages after opening the caps, i.e. exposure to air. The beverages with high concentrations of polyphenols showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activities, but produced relatively high concentrations of H 2O 2 when they were exposed to air, possibly due to oxygen. The production of H 2O 2 increased both with duration of the exposure to air and rise in temperature. Since H 2O 2 is toxic, ways to prevent the deterioration of catechin-enriched green tea, i.e. H 2O 2 production, were studied. The addition of catalase, which is an enzyme decomposing H 2O 2, reduced the H 2O 2 concentration, but it was inactivated at a high temperature. The addition of l-cysteine or glutathione (reduced form), with a thiol residue, reduced the H 2O 2 concentration. Addition of citric acid, malic acid, succinic acid, fumaric acid, l-ascorbic acid, l-glutamic acid and l-aspartic acid also reduced it, possibly because they lower the pH of the tea.

Enhancing the Production of γ-Glutamylcysteine by a Mutant Strain Derived from Saccharomyces cerevisiae BCRC 21727

γ-glutamylcysteine (γ-GC) is an essential precursor for the biosynthesis of glutathione (GSH). The formation of γ-GC has been considered as the rate-limiting intermediate. The purpose of this study was to enhance the γ-GC production by a mutant strain derived from Saccharomyces cerevisiae BCRC 21727. The mutant strains were screened by the strategy including selection of methylglyoxal (MG) sensitive mutants through ethyl methanesulfonate (EMS) mutation and population analysis. The mutant’s characteristics and the effects of media compositions on the cell growth and γ-GC production were then studied. The results showed that a significantly high γ-GC-producing mutant, SY-35679, was isolated from the MG-sensitive mutants. The intracellular γ-GC content in the mutant SY-35679 rose to 4.77mg•g^(-1) DCW and had around eight folds increase, compared with that of the parent strain (0.59mg•g^(-1) DCW), while GSH content was at a similar level inside the mutant cell. The biosynthetic enzymes, GSH Ⅰ and GSH Ⅱ, were compared between the mutant and parent strain. The specific activity of GSH Ⅰ in the mutant SY-35679 was found to be significantly higher than that of the parent strain, however, the GSH Ⅱ specific activity maintained at a similar level. In additions, the intracellular γ-GC and GSH contents were highly affected by different concentrations of glucose and L-cysteine supplement. The medium containing glucose of 20 g. L^(-1) caused the significant increase in both γ-GC and GSH production. The proper addition of L-cysteine was also proved to have a positive effect on the levels of γ-GC and GSH.

Plasmodium berghei: Effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 μM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1 mM), and exflagellation center numbers were restored by the addition of Zn 2+. A reduction of ookinete production was observed when TPCK/TLCK (100 μM) was added at 2 h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1 mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.

Sodium Nitroprusside Potentiates Hydrogen-Sulfide-Induced Contractions in Body Wall Muscle From a Marine Worm

Hydrogen sulfide (H2S) at concentrations of about 0.05 to 1 mmol.l(-1) appears to function as a gasotransmitter in vertebrates, analogous to nitric oxide (NO) and carbon monoxide, but the actions of H2S in invertebrate tissue have not been well studied. In this study, we investigated the role of H2S in modulating body wall muscle tone in the marine echiuran worm Urechis caupo (Echiuridae). We first determined that U. caupo body wall homogenates produce H2S upon addition of L-cysteine and pyridoxal-5'-phosphate (PLP), and that the rate is increased by addition of 2-mercaptoethanol, suggesting the presence of an activated L-serine sulfhydrase pathway. We then measured the contractile response of U. caupo body wall circular muscle strips to sodium hydrosulfide (NaHS)--which produces H2S in solution--and the NO donor sodium nitroprusside (SNP), both with and without subsequent application of acetylcholine (ACh). We found that NaHS alone stimulated contraction in muscle strips equivalent to about one-third the force of ACh alone, whereas SNP alone had no effect on muscle tone. However, simultaneous addition of NaHS with SNP elicited a much stronger contraction, reaching more than twice that of ACh alone, which could be increased further by subsequent application of ACh.

Xanthine Oxidase Catalyzes Anaerobic Transformation of Organic Nitrates to Nitric Oxide and Nitrosothiols: CHARACTERIZATION OF THIS MECHANISM AND THE LINK BETWEEN ORGANIC NITRATE AND GUANYLYL CYCLASE ACTIVATION

Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century. Recently, xanthine oxidase (XO) has been reported to catalyze organic nitrate reduction under anaerobic conditions, but questions remain regarding the initial precursor of nitric oxide (NO) and the link of organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of XO-mediated biotransformation of organic nitrate, studies using electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, NO electrode, and immunoassay were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered the reduction of organic nitrate to nitrite anion (NO2-). Studies of the pH dependence of nitrite formation indicated that XO-mediated organic nitrate reduction occurred via an acid-catalyzed mechanism. In the absence of thiols or ascorbate, no NO generation was detected from XO-mediated organic nitrate reduction; however, addition of L-cysteine or ascorbate triggered prominent NO generation. Studies suggested that organic nitrite (R-O-NO) is produced from XO-mediated organic nitrate reduction. Further reaction of organic nitrite with thiols or ascorbate leads to the generation of NO or nitrosothiols and thus stimulates the activation of sGC. Only flavin site XO inhibitors such as diphenyleneiodonium inhibited XO-mediated organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. Thus, organic nitrite is the initial product in the process of XO-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

On-line cysteine modification for protein analysis: new probes for electrochemical tagging nanospray mass spectrometry

A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.

Residual Sulfur Metabolites in Isolated Soy Proteins: Sulfite to Cysteine

ABSTRACT: Mean methanethiol headspace concentrations above aqueous slurries of isolated soy proteins (ISP) increased 17‐to 36‐fold over the controls with the addition of L‐cysteine. Corresponding hydrogen sulfide levels were also greatly increased. Dithiothreitol, sodium sulfite, and glutathione increased headspace methanethiol from aqueous ISPs 23‐to 44‐fold, 8‐to 9‐fold, and 5‐fold, respectively, but did not elevate hydrogen sulfide. These observations, along with the effects from the addition of dithiothreitol/O‐acetyl‐serine, the addition of a pyridoxial phosphate inhibitor and the intrinsic sulfite content of ISP samples (22 to 31 ppm), indicate that methanethiol from soy proteins is formed by way of components of a sulfite‐to‐cysteine pathway.

Dynamic viscoelastic and tensile properties of gluten and glutenin gels of common wheats of different strength.

Dynamic viscoelastic properties at 25 degrees C of gluten and glutenin gels were obtained from Canadian common wheats of different strengths. The relaxation spectra showed a maximum intensity at a characteristic relaxation time (tau). The relaxation modulus associated with this maximum was taken as the strength of the glutenin or gluten gel transient network (G(tau)). The ratio of G(tau) for glutenin and gluten gels from the same cultivar ranged from 5.6 for an extra strong cultivar to 51.1 for a soft wheat. This gives indirect evidence that the gliadin fraction weakens the glutenin gel network more in weaker cultivars. In addition, the fact that both glutenin and gluten gels showed extensive stress relaxation coupled with the fact that addition of l-cysteine to a gluten gel eliminated the network structure at 25 degrees C and resulted in a power law stress relaxation spectrum suggests that the transient network in gluten is a reversible network. This power law relaxation pattern was not seen here for an entangled polymer melt (poly(dimethylsiloxane)). It was also found here that the viscosity of the gluten gel (G(tau) x tau) trended best with the tensile stress build-up in a uniaxial tensile test of gluten gels. Together, these results indicate that both network strength and relaxation times should be considered in characterizing the linear viscoelastic properties of hydrated cereal proteins.

Mechanistic aspects of on-line electrochemical tagging of free L-cysteine residues during electrospray ionisation for mass spectrometry in protein analysis.

The mechanistic details behind an electrochemically induced tagging of L-cysteine residues in peptides and proteins have been unravelled using cyclic voltammetry. It was found that when hydroquinone is oxidised in the medium used in electrospray ionisation mass spectrometry (ESI-MS) a protonated form of benzoquinone is produced that acts as an efficient electrophile for free L-cysteine residues. Upon substitution of L-cysteine the reduced form of the adduct is formed, which may be further oxidised leading to further substitution of L-cysteine. Digital simulations of the cyclic voltammograms corroborated the mechanism and allowed a determination of the homogeneous second order rate constant corresponding to the addition of L-cysteine onto the protonated form of benzoquinone. The selectivity of the tagging process was confirmed using ESI-MS, which showed that a protein without L-cysteine residues does not react with benzoquinone dissolved in the medium. Finally, the kinetic information obtained in this investigation is used to discuss the optimal parameters for a nanospray capable of quantitative tagging of L-cysteine residues.

Enzymatic hydrogen sulfide production in marine invertebrate tissues

At least some mammalian tissues produce H 2S in vitro from l-cysteine at rates sufficient to have physiological effects. To determine whether tissues of macrofaunal invertebrates have the same capacity, we measured H 2S production in tissue homogenates of the Manila clam Tapes philippinarum and the lugworm Arenicola marina. Tissue homogenates from both animals produced significant quantities of H 2S gas upon addition of l-cysteine and the enzyme cofactor pyridoxal -5′-phosphate (10 mmol l −1 and 2 mmol l −1, respectively), while only tissues from T. philippinarum produced measurable H 2S in the absence of added substrate or cofactor. In T. philippinarum tissues, H 2S production was completely inhibited by the cystathionine β-synthase (CBS) inhibitor aminooxyacetic acid (AOAA), suggesting that the majority of H 2S production was via CBS pathways, while in A. marina body wall, AOAA inhibited only half of the total H 2S production, indicating that the CBS pathway was not the only major source of H 2S production. H 2S production in tissues of T. philippinarum but not A. marina was doubled by the addition of a second thiol substrate (2.5 mmol l −1 2-mercaptoethanol), suggesting the presence of an ‘activated serine sulfhydrase pathway’, which had previously been demonstrated only in some microfauna.

Improving the analytical performance of hydride generation non-dispersive atomic fluorescence spectrometry. Combined effect of additives and optical filters

The effects of tetrahydroborate and acid concentration and the presence of l-cysteine and thiourea were investigated in the determination of As, Bi and Sn using continuous flow hydride generation atomic fluorescence spectrometry (HG AFS). The aim was to find conditions allowing the control of those effects exerting negative influence on the analytical performance of the HG AFS apparatus. The effects taken into account were: (i) the radiation scattering generated by carryover of solution from the gas–liquid separator to the atomizer; (ii) the introduction of molecular species generated by tetrahydroborate decomposition into the atomizer; and (iii) interference effects arising from other elements in the sample matrix and from different acids. The effects (i) and (ii) could be controlled using mild reaction conditions in the HG stage. The effect of HG conditions on carryover was studied by radiation scattering experiments without hydride atomization. Compromised HG conditions were found by studying the effects of tetrahydroborate (0.1–20 g l −1) and acid (0.01–7 mol l −1) concentration, and the addition of l-cysteine (10 g l −1) and thiourea (0.1 mol l −1) on the HG AFS signals. The effect of optical filters was investigated with the aim of improving the signal-to-noise ratio. Optical filters with peak wavelengths of 190 and 220 nm provided an improvement of detection limits by factors of approximately 4 and 2 for As and Te, respectively. Under optimized conditions the detection limits were 6, 5, 3, 2, 2 and 9 ng l −1 for As, Sb, Bi, Sn, Se and Te, respectively. Good tolerance to various acid compositions and sample matrices was obtained by using l-cysteine or thiourea as masking agents. Determination of arsenic in sediment and copper certified reference materials, and of bismuth in steel, sediment, soil and ore certified reference material is reported.

Sulfur-to-nitrogen transnitrosation: transfer of nitric oxide from S-nitroso compounds to diethanolamine and the role of intermediate sulfur-to-sulfur transnitrosation

S-Nitrosothiols are formed in vivo and are involved in NO signaling. We investigated the sulfur-to-nitrogen transnitrosation activity of S-nitrosocysteine, S-nitrosoglutathione, S-nitrosohomocysteine, S-nitrosocysteinylglycine and S-nitroso- N-acetylcysteine in their reaction with the secondary amine diethanolamine in vitro. The resulting N-nitrosodiethanolamine, a strong carcinogen, was formed in yields of up to 11% from S-nitrosocysteine and S-nitrosocysteinylglycine, whereas the transnitrosation activity of the other S-nitroso compounds was weak. However, the addition of l-cysteine to a solution of S-nitrosohomocysteine and diethanolamine accelerated the decomposition of S-nitrosohomocysteine and resulted in a significant formation of N-nitrosodiethanolamine accompanied by the intermediate generation of S-nitrosocysteine. Thus, reactive nitrosothiols can be formed from less reactive analogs via sulfur-to-sulfur transnitrosation. We suggest that this affects regulation of NO trafficking in vivo. The reaction provides an alternative mechanism for the generation of carcinogenic N-nitroso derivatives.

Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration.

Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH. There are conflicting data on these limitations, however, and systematic studies to assess the importance of these factors for growing bacterial cultures are lacking. In the present study, homogeneous expression of the gfpmut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of Streptococcus gordonii DL1. A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 0.1 p.p.m. dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0.025 p.p.m. dissolved oxygen (obtained by addition of L-cysteine as reducing agent). When an anaerobically grown (non-fluorescent) >50 microm thick biofilm was shifted to aerobic conditions, fluorescence could be detected within 4 min, reaching a maximum over the next 16 min. It was not possible to detect any fluorescence gradients (lateral or vertical) within the >50 microm thick biofilm, and fluorescence development after the shift to aerobic conditions occurred throughout the biofilm (even at the substratum). This suggests that oxygen gradients, which might result in reduced GFP fluorescence, did not exist in the >50 microm thick biofilm of this organism. Production of lactic acid and the subsequent acidification in batch cultures of S. gordonii DL1 led to a decrease in fluorescence intensity. However, severe pH reduction was prevented when the bacterium was grown as a biofilm in a flowcell, and a homogeneous distribution of a strong fluorescence signal was observed. These findings show that GFP can be applied to studies of oxygen-tolerant anaerobic bacteria, that densely packed, flowcell-grown biofilms of S. gordonii do not develop oxygen gradients inhibitory to GFP fluorescence development, and that the often transient nature of GFP fluorescence in acid-producing bacteria can be overcome in flowcells, probably by the elimination of metabolic by-product accumulation.

Comparison of Different Reducing Agents for Enhanced Detection of Heat-Injured Listeria monocytogenes

The effect of different reducing agents (l-cysteine, Oxyrase, and Enterococcus faecium) and their combinations on the detection of heat-injured (62.8°C, 7.5 min or 10 min) Listeria monocytogenes was examined. The incorporation of l-cysteine (0.5 g/liter) yielded higher percentage detection than any of the other reducing agents (P < 0.05). The combination of Oxyrase (10 U/ml) and E. faecium (103 CFU/ml) synergistically enhanced the detection of L. monocytogenes heat-injured for 10 min at 62.8°C(P < 0.05). Simultaneous addition of l-cysteine (0.5 g/liter) and Oxyrase (10 U/ml) significantly lowered the recovery of heat-injured L. monocytogenes (P < 0.05). Higher activities of Oxyrase (50 U/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). The rates of depletion of relative percentage O2 were in the order: l-cysteine (0.5 g/liter; 6.63%/min) > Oxyrase (10 U/ml; 5.00%/min) >E. faecium (108 CFU/ml; 1.66%/min) >E. faecium (103 CFU/ml; 0.20%/min). The final levels of redox potential (Eh) achieved were −110.5 mV, −100 mV, −83.5 mV, and −25 mV for E. faecium (l08 CFU/ml), l-cysteine, Oxyrase, and E. faecium (103 CFU/ml), respectively.

Purification and characterization of a NAD +-dependent sorbitol dehydrogenase from Japanese pear fruit

NAD +-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The K m values for sorbitol and fructose were 96.4±8.60 and 4239±33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.