• Animal cells and systems
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• v.15 no.2
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• pp.131-138
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• 2011

Redox regulation is one of the ubiquitous mechanisms to modulate ion channels. We here investigated how 5,5'-dithio-bis (2-nitrobenzoic acid), a cysteine specific oxidizing reagent, modulates $Ca_v3.1$ and $Ca_v3.2$ T-type $Ca^{2+}$ channels expressed in Xenopus oocytes. Application of the reagent inhibited $Ca_v3.1$ and $Ca_v3.2$ currents in a dose-dependent manner. The oxidizing reagent (1 mM) reduced the peak amplitude of $Ca_v3.1$ and $Ca_v3.2$ currents by ~50% over 2-3 minutes and the decreased currents were fully recovered upon washout of it. The reagent slowed the activation and inactivation kinetics of $Ca_v3.1$, $Ca_v3.2$, and $Ca_v3.3$ channel currents. Notably, the reagent positively shifted both activation and steady-state inactivation curves of $Ca_v3.1$, while it did not those of $Ca_v3.2$. Utilizing chimeric channels from $Ca_v3.1$ and $Ca_v3.2$, we localized the domains III and IV of $Ca_v3.1$ responsible for the positive shifts of channel activation and steady-state inactivation. These findings provide hints relevant to the electrophysiological and molecular mechanisms accounting for the oxidative regulation of T-type channels.

• Journal of Microbiology
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• v.37 no.3
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• pp.148-153
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• 1999

Laccase produced by Coriolus hirsutus was purified to electrophoretic homogeneity by acetone precipitation, Sephacryl S-2000 HR chromatography, DEAE Sepharose CL-6B chromatography, and Mono Q HR 5/5 chromatography. The purification of laccase was 46.6-fold with an overall yield of 23.7%. Laccase from this fungus was a monomeric glycoprotein with 16% carbohydrate content, and has an isoelectric point of 4.2, and molecular mass of 78 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed significant homology to hoste of laccases from Coriolus versicolor, Pycnoporus cinnabarius, and an unidentified basidiomycete, PM1. The highest rate of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation by laccase was reached at 45$^{\circ}C$, and te pH optima of the enzyme varied depending on the substrate in the range of 2.0 to 4.5. The enzyme was stable at 60$^{\circ}C$ for 5 h and lost 80% activity at 80$^{\circ}C$ in 30 min. The enzyme oxidized a variety of usual laccase substrates including lignin-related phenol, and had the highest affinity toward ABTS. Under standard assay conditions, the apparent Km value of the enzyme toward ABTS was 8.1 ${\mu}$M. The enzyme was completely inhibited by L-cysteine and sodium azide, but not by potassium cyanide, SDS, ad thiourea.

• Food Science and Biotechnology
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• v.18 no.4
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• pp.878-883
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• 2009

In Brazil canned 'Biuti' peach is a very popular form of this sub-tropical fruit. This production represents an important economic agro-activity in Minas Gerais, Brazil during the summer period, in preparation for the Christmas celebrations. The aim of this work was to characterize the 'Biuti' peach polyphenoloxidase (PPO), since peach products show enzymatic oxidation of the polyphenols by oxidative enzymes, which affects the products during their shelf life. Two different hypothesis for the browning problem in processed peaches were studied: the inadequacy of the blanching treatment and the presence of a latent phenolase in the peaches. The PPO was characterized: pH optimum (5.5) and stability (5.5-6.5); optimum temperature at $20^{\circ}C$ and 80% of the activity retained after 30 min at $15-40^{\circ}C$. The test for the presence of latent PPO in the processed and canned peaches was negative. Ascorbic acid, ${\beta}$-mercaptoethanol, sodium metabisulfite, and cysteine were efficient in inhibiting the PPO.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.35-42
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• 2000

Reaction characteristics of 4-methylcatechol 2,3-dioxygenase (4MC230) purified from Pseudomonas putida SU10 with a higher activity toward 4-methylcatechol than catechol or 3-cethylcatechol were studied by altering their physical and chemical properties. The enzyme exhibited a maximum activity at pH 7.5 and approximately 40% at pH 6.0 for 4-methylcatechol hydrolysis. The optimum temperature for the enzyme was around $35^{\circ}C$, since the enzyme was unstable at higher temperature. Acetone(10%) stabilized the 4MC230. The effects of solvent and other chemicals (inactivator or reactivator) for the reactivation of the 4MC230 were also investigated. Silver nitrate and hydrogen peroxid severely deactivated the enzyme and the deactivation by hydrogen peroxide severely deactivated the enzyme and the deactivation by hydrogen peroxide was mainly due to the oxidation of ferrous ion to ferric ion. Some solvents acted as an activator and protector for the enzyme from deactivation by hydrogen peroxide. Ascorbate, cysteine, or ferrous ion reactivated the deactivated enzyme by hydrogen peroxide. The addition of ferrous ion together with a reducing agent fully recovered the enzyme activity and increased its activity abut 2 times.

• Molecules and Cells
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• v.26 no.4
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• pp.329-337
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• 2008

Src-family kinases are critically involved in the control of cytoskeleton organization and in the generation of integrin-dependent signaling responses, inducing tyrosine phosphorylation of many signaling and cytoskeletal proteins. Activity of the Src family of tyrosine kinases is tightly controlled by inhibitory phosphorylation of a carboxy-terminal tyrosine residue, inducing an inactive conformation through binding with its SH2 domain. Dephosphorylation of C-ter tyrosine, as well as its deletion of substitution with phenylalanine in oncogenic Src kinases, leads to autophosphorylation at a tyrosine in the activation loop, thereby leading to enhanced Src activity. Beside this phophorylation/dephosphorylation circuitry, cysteine oxidation has been recently reported as a further mechanism of enzyme activation. Mounting evidence describes Src activation via its redox regulation as a key outcome in several circumstances, including growth factor and cytokines signaling, integrin-mediated cell adhesion and motility, membrane receptor cross-talk as well in cell transformation and tumor progression. Among the plethora of data involving Src kinase in physiological and pathophysiological processes, this review will give emphasis to the redox component of the regulation of this master kinase.

• Journal of Food Hygiene and Safety
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• v.27 no.4
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• pp.461-465
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• 2012

Broccoli has a functional substance, sulforaphane that has effects of anticancer, antioxidant, antimicrobial and anti-inflammatory. Sulforaphane, one of the hydrolysis products of glucoraphanin in broccoli, cabbage and kale, was contributed to the role of antioxidant. Broccoli contains a number of bioactive compounds including glucosinolates, S-methyl cysteine sulfoxide and many antioxidants. The ethanol extract (BE), hexane extract (BH), propylene glycol extract (BP) and butylenesglycol extract (BB) of broccoli were used to investigate the antioxidation and anti-inflammatory effects of sulforaphane extracts from broccoli. The high scavenging abilities of DPPH, $O_2{^-}$ were observed. Also sulforaphane extracts from broccoli showed the inhibition effect on NO rate. These results demonstrated that sulforaphane extracts from broccoli could be useful as an antioxidation and anti-inflammatory functional ingredient.

• Nano
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• v.13 no.10
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• pp.1850116.1-1850116.14
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• 2018

In this study, we report the mercury ions ($Hg^{2+}$) mediated phosphorus-containing carbon dots (PCDs) as a selective "off-on" fluorescence probe for glutathione (GSH), cysteine (Cys) and homocysteine (Hcys). PCDs obtained by hydrothermal reaction are sensitive to $Hg^{2+}$ ions and its fluorescence can be significantly quenched owing to the electron transfer from the lowest unoccupied molecular orbital (LUMO) of PCDs to $Hg^{2+}$. Interestingly, the weak fluorescence of $Hg^{2+}$-mediated PCDs could be gradually recovered with the addition of GSH, Cys and Hcys. This can be attributed to the formation of $Hg^{2+}-S$ complex due to the super affinity of $Hg^{2+}$-sulfydryl bond. The formation of $Hg^{2+}-S$ complex extremely reduces the oxidation ability of $Hg^{2+}$ that inhibits the electron transfer from LUMO of PCDs to $Hg^{2+}$ and re-opens the native electron transition from LUMO to the highest occupied molecular orbital (HOMO) of PCDs. Thus, the green fluorescence of PCDs is switched on. Furthermore, the present $Hg^{2+}$-mediated PCDs assay exhibits a high selectivity for GSH, Cys and Hcy and has been successfully used to detect the total biothiols content in human urine samples.

• BMB Reports
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• v.55 no.3
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• pp.154-159
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• 2022

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.

• Journal of the Korean Society of Food Science and Nutrition
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• v.12 no.4
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• pp.316-322
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• 1983

In order to obtain elementary data of enzymatic browning of apples and apple products and to examine effectively inhibitory method of browning, we extracted polyphenol oxidase (EC 1.10.3.1) from apple (Ralls Janet) and investigated its general properties. The optimum conditions for the enzyme reaction were pH 6.0 and temperature of $30^{\circ}C$. The enzyme was very stable at pH 4.0, and at the range of pH 5.0-9.0 its activity was above 80% compared with pH 4.0. The enzyme was very stable by heating at $40^{\circ}C$ for 1 hour, and almost 50% of enzyme activity was lost by heating at $60^{\circ}C$ for 30 minutes. The polyphenol oxidase activity was enhanced by the addition of $Cu^{2+}$ and $Mn^{2+}$, respectively, meanwhile $Na^+$, $Hg^{2+}$ and $Co^{2+}$ inhibited the enzyme activity. The enzyme activity was greatly decreased in the presence of inhibitors such as cysteine, sodium metabisulfite and ascorbic acid. The polyphenol oxidase greatly catalyzed the oxidation of o-diphenols such as chlorogenic acid and catechol, which suggests that main substrate of polyphenol oxidase is o-diphenol compounds.

• Molecules and Cells
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• v.37 no.10
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• pp.719-726
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• 2014

The ${\gamma}$-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers $NADP^+$ over $NAD^+$ as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with $NADP^+$ at $1.6{\AA}$ and $2.1{\AA}$ resolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2'-phosphate group of adenine-side ribose in $NADP^+$. Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.