Effects of pyrogallol, hydroquinone and duroquinone on responses to nitrergic nerve stimulation and NO in the rat anococcygeus muscle.
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1. The hypothesis that endogenous superoxide dismutase (SOD) protects the nitrergic transmitter from inactivation by superoxide and that this explains the lack of sensitivity of the transmitter to superoxide generators was tested in the rat isolated anococcygeus muscle. 2. Responses to nitrergic nerve stimulation or to NO were not significantly affected by exogenous SOD or by the Cu/Zn SOD inhibitor diethyldithiocarbamic acid (DETCA). 3. Hydroquinone produced a concentration-dependent reduction of responses to NO with an IC50 of 27 microM, and higher concentrations reduced relaxant responses to nitrergic nerve stimulation with an IC50 of 612 microM. The effects of hydroquinone were only slightly reversed by SOD, so it does not appear to be acting as a superoxide generator. 4. Pyrogallol produced a concentration-dependent reduction in responses to NO with an IC50 value of 39 microM and this effect was reversed by SOD (100-1000 u ml(-1)). Pyrogallol did not affect responses to nitrergic nerve stimulation. Treatment with DETCA did not alter the differentiating action of pyrogallol. 5. Duroquinone produced a concentration-dependent reduction of relaxations to NO with an IC50 value of 240 microM and 100 microM slightly decreased nitrergic relaxations. After treatment with DETCA, duroquinone produced greater reductions of relaxant responses to NO and to nitrergic stimulation, the IC50 values being 8.5 microM for NO and 40 microM for nitrergic nerve stimulation: these reductions were reversed by SOD. 6. The findings do not support the hypothesis that the presence of Cu/Zn SOD explains the greater susceptibility of NO than the nitrergic transmitter to the superoxide generator pyrogallol, but suggest that it may play a role in the effects of duroquinone. (+info)
Towards the reaction mechanism of pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici.
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Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase of the strictly anaerobic fermenting bacterium Pelobacter acidigallici. Transhydroxylation experiments in H218O revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group from the cosubstrate 1,2,3, 5-tetrahydroxybenzene (tetrahydroxybenzene) to the acceptor pyrogallol, and simultaneously regenerates the cosubstrate. This concept requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and aromatic N-oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydroxyl groups; the oxides reacted as cosubstrates in a transhydroxylation reaction rather than as true oxidants in a net hydroxylation reaction. No oxidizing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phloroglucinol in the absence of tetrahydroxybenzene was achieved if little pyrogallol and a high amount of enzyme preparation was used which had been pre-exposed to air. Obviously, the enzyme was oxidized by air to form sufficient amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism is proposed which combines an oxidative hydroxylation with a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol without involvement of water. With this, the transhydroxylase differs basically from all other hydroxylating molybdenum enzymes which all use water as hydroxyl source. (+info)
Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle.
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1. A sustained tone was produced in rat isolated anococcygeus muscles with guanethidine and clonidine and relaxant responses were elicited by electrical stimulation of its nitrergic nerves and by the three redox forms of nitrogen monoxide. 2. The nitroxyl anion (NO ) was donated by dissociation of Angeli's salt; the free radical (NO*) was from an aqueous solution of nitric oxide gas; the nitrosonium cation (NO+) was donated by dissociation of nitrosonium tetrafluoroborate. 3. The concentrations producing approximately 50% relaxations of the anococcygeus muscle were 0.3 microM for Angeli's salt (nitroxyl), 0.5 microM for NO* and 100 microM for nitrosonium tetrafluoroborate. Nitrergic nerve stimulation at 1 Hz for 10 s produced equivalent relaxant responses. 4. The superoxide generator pyrogallol (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 5. The NO* scavenger carboxy-PTIO (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 6. Hydroxocobalamin (30 microM) had no significant effect on responses to the nitrergic transmitter, enhanced the response to Angeli's salt, and significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 7. The findings suggest that the nitroxyl anion donated by Angeli's salt is a better candidate than NO* to serve as the nitrergic transmitter in the rat anococcygeus muscle, although it still does not behave exactly like the transmitter. (+info)
Cloning, sequencing and heterologous expression of pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici.
(4/110)
A genomic lambda-library of Pelobacter acidigallici has been established. Proteolytic digestion of homogeneous pyrogallol-phloroglucinol transhydroxylase from the same microorganism afforded polypeptide fragments whose N-terminal sequences allowed the generation of oligonucleotide primers. Together with primers deduced from the known N-terminal sequences of the two intact subunits these were used in PCR experiments to obtain three amplificates. Screening the lambda-library with the three amplificates led eventually to clones containing the whole gene coding for the transhydroxylase. Sequencing the gene revealed two open reading frames coding for 875 and 275 amino acids which correspond to the alpha- and beta-subunits of THL, respectively. The two subunits are separated by a 48-bp noncoding region. Comparison of the sequence with those of other molybdopterin cofactor (MoCo)-enzymes places THL in the dimethylsulfoxide reductase family. Possible contact sites to the MoCo and to the iron-sulphur clusters were spotted. Using the expression vectors pQE 30 and pT 7-7 three constructs harbouring the THL gene were created. One of them carried a His6-tag at the N-terminus of the alpha-subunit, another at the C-terminus of the beta-subunit. Immunoblot analysis showed high expression of THL, but the inclusion bodies could not be refolded to active enzyme. (+info)
Total protein determination in urine: elimination of a differential response between the coomassie blue and pyrogallol red protein dye-binding assays.
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BACKGROUND: The total protein content of urine is a good index of renal function, but its determination is unreliable. Protein dye-binding assays are simple, but they characteristically lack a uniform response to different proteins. METHODS: We investigated a differential response of the Sigma Microprotein Coomassie Brilliant Blue (CBB) and Pyrogallol Red-molybdate (PRM) protein dye-binding assays to urine, using human albumin, albumin/globulin, or urinary protein as calibrator. RESULTS: The urine protein values (n = 60) obtained with the CBB assay were 110-13 500 mg/L (mean, 2390 mg/L) compared with 160-18 300 mg/L (mean, 3470 mg/L) obtained with the PRM assay (CBB:PRM protein concentration ratio, 0.46-0.88, mean, 0. 69 +/- 0.10). The differential response was highly reproducible as indicated by Sigma urine control Level 1 (within-day CBB:PRM ratio, 0.68 +/- 0.02; between-day CBB:PRM ratio, 0.67 +/- 0.04) and Sigma urine control Level 2 (within-day CBB:PRM ratio, 0.60 +/- 0.01; between-day CBB:PRM ratio, 0.59 +/- 0.02). The use of urinary protein as a calibrator (rather than human albumin) greatly improved the agreement between the assays when applied to urine (y(CBB) = 0. 972x(PRM) - 16 vs y(CBB) = 0.685x(PRM) + 17). In studies using urine controls, this calibrator also improved agreement between the CBB, PRM, trichloroacetic acid (TCA), and benzethonium chloride protein methods and, to a lesser extent, agreement with the TCA-Ponceau S method. CONCLUSION: The use of a urinary protein calibrator improves the agreement between different methods used to determine total protein in urine. (+info)
Effects of cadmium on manganese peroxidase competitive inhibition of MnII oxidation and thermal stabilization of the enzyme.
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Inhibition of manganese peroxidase by cadmium was studied under steady-state and transient-state kinetic conditions. CdII is a reversible competitive inhibitor of MnII in the steady state with Ki approximately 10 microM. CdII also inhibits enzyme-generated MnIII-chelate-mediated oxidation of 2,6-dimethoxyphenol with Ki approximately 4 microM. CdII does not inhibit direct oxidation of phenols such as 2,6-dimethoxyphenol or guaiacol (2-methoxyphenol) in the absence of MnII. CdII alters the heme Soret on binding manganese peroxidase and exhibits a Kd approximately 8 microM, similar to Mn (Kd approximately 10 microM). Under transient-state conditions, CdII inhibits reduction of compound I and compound II by MnII at pH 4.5. However, CdII does not inhibit formation of compound I nor does it inhibit reduction of the enzyme intermediates by phenols in the absence of MnII. Kinetic analysis suggests that CdII binds at the MnII-binding site, preventing oxidation of MnII, but does not impair oxidation of substrates, such as phenols, which do not bind at the MnII-binding site. Finally, at pH 4.5 and 55 degrees C, MnII and CdII both protect manganese peroxidase from thermal denaturation more efficiently than CaII, extending the half-life of the enzyme by more than twofold. Furthermore, the combination of half MnII and half CdII nearly quadruples the enzyme half-life over either metal alone or either metal in combination with CaII. (+info)
Identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in Rhodococcus opacus 1cp.
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The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and (19)F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The (19)F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2, 3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp. (+info)
Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide.
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The activity of ascorbate peroxidase (APX) has been studied with H(2)O(2) and various reducing substrates. The activity decreased in the order pyrogallol>ascorbate>guaiacol>2, 2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The inactivation of APX with H(2)O(2) as the sole substrate was studied. The number of H(2)O(2) molecules required for maximal inactivation of the enzyme was determined as approx. 2.5. Enzymic activity of approx. 20% of the original remained at the end of the inactivation process (i.e. approx. 20% resistance) when ascorbate or ABTS was used as the substrate in activity assays. With pyrogallol or guaiacol no resistance was seen. Inactivation by H(2)O(2) followed over time with ascorbate or pyrogallol assays exhibited single-exponential decreases in enzymic activity. Hyperbolic saturation kinetics were observed in both assay systems; a similar dissociation constant (0.8 microM) for H(2)O(2) was obtained in each case. However, the maximum rate constant (lambda(max)) obtained from the plots differed depending on the assay substrate. The presence of reducing substrate in addition to H(2)O(2) partly or completely protected the enzyme from inactivation, depending on how many molar equivalents of reducing substrate were added. An oxygen electrode system has been used to confirm that APX does not exhibit a catalase-like oxygen-releasing reaction. A kinetic model was developed to interpret the experimental results; both the results and the model are compared and contrasted with previously obtained results for horseradish peroxidase C. The kinetic model has led us to the conclusion that the inactivation of APX by H(2)O(2) represents an unusual situation in which no enzyme turnover occurs but there is a partition of the enzyme between two forms, one inactive and the other with activity towards reducing substrates such as ascorbate and ABTS only. The partition ratio is less than 1. (+info)