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PubMed1977

Rapid spectrophotometric differentiation between glutathione-dependent and glutathione-independent gentisate and homogentisate pathways.

A total of four pathways are known for the catabolism by microorganisms of gentisate (2,5-dihydroxybenzoate) and homogentisate (2,5-dihydroxyphenylacetate). Both of these dihydric phenols can be degraded by either a glutathione-dependent or a glutathione-independent reaction sequence. We found that it is not always possible to unequivocally assign glutathione dependence or independence to a particular catabolic sequence by using the well-established spectrophotometric assays at 330 nm (gentisate pathway) or 320 nm (homogentisate pathway). This paper reports a modification of the classical spectrophotometric assays that allowed an unequivocal differentiation between glutathion-dependent and glutathione-independent pathways, even when crude cell extracts contained significant quantities of cell-derived, reduced glutathione. This was accomplished by performing assays in the presence of an approximately 10(-3) M solution of the sulfhydryl-binding agent N-ethylmaleimide.

Bacteria↗
PubMed2005

Replacement of tyrosine 181 by phenylalanine in gentisate 1,2-dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 enhances catalytic activities.

xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k(cat)/Km) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

Amino Acid Substitution↗
PubMed1998

Gentisate 1,2-dioxygenase from Haloferax sp. D1227.

Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42,000 +/- 1,000 Da subunits, with a native molecular weight of 174,000 +/- 6,000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45 degrees C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified.

Amino Acid Sequence↗
PubMed2001

Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867.

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.

Amino Acid Sequence↗
PubMed1980

High-pressure liquid chromatography analysis of salicylic acid, salicyluric acid, and gentisic acid in biological matrixes.

An assay is reported for salicylic acid and two of its metabolites, salicyluric acid and gentisic acid, in mouse and rat blood, urine, and feces and rat embryos. The procedure involves aqueous dilution of the biological sample, addition of methanol-1% acetic acid (in water) (40:60), centrifugation, and injection of the supernate onto a commerical high-pressure liquid chromatographic column. Detection is via a variable-wavelength UV detector set at 296 nm. Quick and easy sample preparation, good accuracy and precision, and sufficient sensitivity make analysis of large numbers of samples feasible.

Animals↗
PubMed1981

Stabilization of 99mTc-pyrophosphate injection with gentisic acid.

The in vitro oxidation of 99mTc-labelled bone-scanning agents with the release of free sodium 99mTc-pertechnetate results in poor skeletal images. Gentisic acid (2,5-dihydroxy-benzoic acid), which has been used as an antioxidant to stabilize HEDP and HMDP kits, has now been shown to be an effective stabilizer for 99mTc-Pyrophosphate Injection and doses not compete for 99mTc-label.

Animals↗
PubMed1982

Formation of 99mTc-plasmin in presence of gentisic acid using 99mTc pretreated with concentrated hydrochloric acid and vacuum evaporation.

The concentrated hydrochloric acid/vacuum evaporation method has been applied to form 99mTc-plasmin complexes free from radiocolloids for medical scintigraphy. The yield and radiochemical purity were insufficient for medical application when no antioxidant was present during the complex formation. With gentisic acid present the yield was increased by a factory of 2-3, and high radiochemical purity was obtained. No preparative chromatography was necessary. Presence of ascorbic acid inhibited the 99mTc-plasmin formation.

Fibrinolysin↗
PubMed1996

Direct analysis of salicylic acid, salicyl acyl glucuronide, salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography.

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C18 column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3-4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2-20 micrograms/ml and linearity was observed from 0.1-200 micrograms/ml and 5-2000 micrograms/ml for SA in plasma and urine, respectively. The method was validated to 0.2 microgram/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.

Chromatography, High Pressure Liquid↗
PubMed2003

In vitro evaluation of patch formulations for topical delivery of gentisic acid in rats.

Gentisic acid (GA) is used in cosmetics as a skin-whitening agent for the treatment of skin pigmentary disorders by influencing the synthesis of melanin through inhibition of melanosomal tyrosinase activity. In order to achieve effective topical delivery of GA to the active site in the skin, a matrix-type transdermal delivery system was developed. The in vitro skin permeation as well as skin deposition of GA was studied in rats. Among the five pressure-sensitive adhesives tested, DuroTak 87-2510 was the most effective to achieve the highest permeation rate of GA. Dodecylamine showed the most potent enhancement among the enhancers tested, and significantly increased the permeation rate of GA up to 112.99 (+/-30.12) microg/cm(2) per h at the concentration of 1%, when 6% GA was incorporated in DuroTak 87-2510. Moreover, a linear relationship was observed between the skin permeation rate of GA and the amount of the skin deposition after 12 h of permeation (r(2)=0.95). Thus, the in vitro skin permeation data may be useful to determine the amount of GA actually deposited in the skin.

Administration, Topical↗
PubMedDate not supplied

Stabilization of (90)y-labeled DOTA-biomolecule conjugates using gentisic acid and ascorbic acid.

Radiolytic degradation of radiolabeled compounds is a major challenge for the development of new therapeutic radiopharmaceuticals. The goal of this study is to explore the factors influencing the solution stability of a (90)Y-labeled DOTA-peptide conjugate (RP697), including the amount of total activity, the activity concentration, the stabilizer concentration, and the storage temperature. In general, the rate of radiolytic decomposition of RP697 is much slower at the lower activity concentration (<4 mCi/mL) than that at the higher concentration (>10 mCi/mL). RP697 remains relatively stable at the 20 mCi level and room temperature while it decomposes rapidly at the 100 mCi level under the same storage conditions. Radical scavengers, such as gentisic acid (GA) and ascorbic acid (AA), were used in combination with the low temperature (-78 degrees C) to prevent the radiolytic decomposition of RP697. It was found that RP697 remains stable for at least 2 half-lives of (90)Y when GA or AA (10 mg for 20 mCi of (90)Y) is used as a stabilizer when the radiopharmaceutical composition is stored at -78 degrees C. The stabilizer (GA and AA) can be added into the formulation either before or after radiolabeling. The post-labeling approach is particularly useful when the use of a large amount of the stabilizer interferes with the radiolabeling. The radiopharmaceutical composition developed in this study can also apply to other (90)Y-labeled DOTA-biomolecule conjugates. The amount of the stabilizer used in the radiopharmaceutical composition and storage temperature should be adjusted according to the sensitivity of the radiolabeled DOTA-biomolecule conjugate toward radiolytic decomposition.

Ascorbic Acid↗
PubMed1966

Inhibition of the secondary antibody response in vitro by salicylate and gentisate.

Salicylate inhibition of the secondary antibody response initiated in vitro on day 0 has been studied in cultures of rabbit lymph node fragments. Levels of 1.25 to 1.5 mM (0.20 to 0.24 mg/ml) sodium salicylate present in serum-free medium throughout an 18- or 21-day culture period completely inhibit the secondary response. This inhibition is largely accomplished by the drug's action during the first 9 days, which corresponds to the inductive phase for this culture system. Relatively little inhibition is produced by adding the drug only after day 9, although over 90% of the antibody produced during a 21-day experiment is synthesized after day 9. Studies with media of different pH's show that this inhibition is more correctly a function of the nonionized salicylic acid concentration in the medium than of the total salicylate concentration. Arguments are presented against the possibility that salicylate at the levels used here inhibits antibody synthesis by uncoupling oxidative phosphorylation. Acetylsalicylic acid (aspirin) produces the same degree of inhibition in vitro as do equimolar concentrations of sodium salicylate. Gentisate (5-hydroxysalicylate) is 15-fold more effective in producing 50% inhibition than salicylate; its temporal pattern of inhibition is similar to that of salicylate.

Animals↗
PubMed1992

Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4.

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.

Biodegradation, Environmental↗
PubMed1985

Gentisate, a salicylate metabolite with antioxidant properties.

Concentrations of gentisate found in plasma and synovial fluid were in excess of concentrations required to exert an antioxidant effect in vitro. Studies of diphenol/aminophenols suggested that an ortho or para configuration of functional groups was required for this in vitro antioxidant activity. Animal studies indicated some correlations between in vitro activity and pharmacology of these agents.

Animals↗
PubMed1992

Degradation of some phenols and hydroxybenzoates by the imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans: evidence for an operative gentisate pathway.

The imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans degraded 3-hydroxybenzoic acid via gentisate which was the cleavage substrate. 4-Hydroxybenzoic acid was metabolized via protocatechuate. No cleavage enzyme for the latter was detected. In stead of this NADH- and NADPH-dependent monooxygenases were present. In cells grown at the expense of hydroquinone and 4-hydroxybenzoic acid, enzymes of the hydroxyhydroquinone variant of the 3-oxoadipate pathway were demonstrated, which also took part in the degradation of 2,4-dihydroxybenzoic acid by C. parapsilosis.

Biodegradation, Environmental↗
PubMed2006

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

Benzoates↗
PubMed2005

Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867.

The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

Biodegradation, Environmental↗