4-chloro-3-hydroxyanthranilate inhibits brain 3-hydroxyanthranate oxidase

Quinolinic acid is synthesized from 3-hydroxyanthranilic acid via 3-hydroxyanthranilic acid oxidase. In liver, 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase. To determine whether 4-chloro-3-hydroxyanthranilic acid also inhibits 3-hydroxyanthranilic acid oxidase in brain, 3-hydroxyanthranilic acid was injected into the cisterna magna of rats either with or without 4-chloro-3 hydroxyanthranilic acid. 3-Hydroxyanthranilic acid increased quinolinic acid concentrations throughout the brain. 4-Chloro-3-hydroxyanthranilic acid attenuated increases in brain quinolinic acid. These observations indicate that 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase in brain.Quinolinic acid is a well established systemic metabolite of l-tryptophan which has been shown to be present in brain (Wolfensberger et al., 1983; Heyes and Markey, 1988a). QUIN has proved to be a convulsant (Lapin, 1982), neurotoxin (Schwarcz et al., 1983) and agonist of N-methyl-D-aspartate receptors (Perkins and Stone, 1983) when injected directly into the central nervous system of experimental animals. Therefore increased concentrations of QUIN in brain may have neoropathologic consequences. l-Tryptophan is converted to QUIN via the kynurenine pathway. The precursor of QUIN, 2-amino-3-carboxymuconic semialdehyde is synthesized from 3-hydroxyanthranilic acid (3-HAA) by the action of 3-hydroxyanthranilic acid oxidase (3-HAA/OX) in liver and brain (Foster et al., 1986; Okuno et al., 1987). QUIN is then formed from 2-amino-3-carboxymuconic semialdehyde by a spontaneous, non-enzymatic reaction. In liver, 3-HAA/OX is inhibited by 4-chloro-3-hydroxyantranilic acid (CL-HAA; Parli et al., 1980). In the present study, rats were given an intracisternal injection of 3-HAA and the resultant increases in regional brain QUIN concentrations quantified by gas chromatography/mass spectrometry (Heyes and Markey, 1988a,b). To determine whether CL-HAA inhibit 3-hydroxyanthranilic acid oxidase in brain, CL-HAA was co-administered with 3-HAA to see whether increases in QUIN were attenuated.     

Effects of oxygen on 3-hydroxyanthranilate oxidase of the kynurenine pathway

Iron containing 3-Hydroxyanthranilate oxidase (3HAO) converts 3-hydroxyanthranilate (3HAA) and dioxygen into a precursor which spontaneously converts to quinolinic acid (QA). 3HAO participates in de novo biosynthesis of NAD in mammalian kidney and liver, and it is present in low concentrations in brain where its function is controversial. However, QA increases in spinal fluid and is associated with convulsions in AIDS dementia, Huntington’s disease, and CNS inflammation. QA is a known N-methyl, D-aspartate receptor agonist and excitotoxin that causes convulsions when injected into the brain. Hyperbaric oxygen (HBO) also causes convulsions and we investigated the interrelationships among the stimulating and toxic effects of oxygen and the role of iron in vitro using rat liver enzyme which is reported to be identical to brain enzyme and is more abundant. 3HAO requires dioxygen as a substrate but it was inactivated approximately 40% by 5.2 atm HBO in vitro in 15 min. The apparent K_m was 2.6 × 10⁻⁴ M for oxygen and 5 × 10⁻⁵ M for 3HAA, and these values did not change for enzyme that was half-inactivated by HBO oxygen. Thus, oxygen-inactivation appears to be all-or-none for individual enzyme molecules. Freshly prepared enzyme was activated about 3-fold by incubation with acidic iron. Iron-staining of 3HAO, separated by gel electrophoresis after partial purification by FPLC, showed that loss of iron and loss of enzyme activity during HBO exposure were correlated. The apparent oxygen K_m of 3HAO is far higher than the oxygen concentration in brain cells. Thus, 3HAO is capable of being stimulated initially in animals breathing HBO, and subsequently of being inactivated with potential significance for brain QA and convulsions.     

Studies on chemiluminescence in the enzymatic and autoxidative transformation of 3,4-dihydroxyphenylalanine into eumelanins

The catechol oxidase-catalysed and autoxidative transformation of 3,4-dihydroxyphenylalanine (DOPA) to eumelanin have been studied by oxygen consumption, energy transfer, absorption and fluorescence spectroscopy. Formation of transient dopachrome (λ_max = 480 nm) and dopalutin (λ_ex = 423 nm, λ_em = 491 nm) have been found in the enzymatic and autoxidative reaction. In the enzymatic reaction, neither a photon emission with quantum yield Φ > 10^?13 nor energy transfer to triplet and singlet energy acceptors (sensitizers such as anthracene derivatives, xanthene dyes and chlorophyll-a) in water and micellar solutions have been found. The autoxidative reaction is chemiluminescent (Φ = 10^?9), the emission occurring in the 400-600 nm range. The excitation energy is not transferred to sensitizers. The effect of various enzymes and traps of active oxygen species as well as the spectral distribution of chemiluminescence indicate that there is no emission from oxygen dimoles. Carbonates and active species of oxygen are shown to participate in the chemiexcitation reaction.     

Permeability of the liver cell membrane to quinolinate.

Quinolinate was taken up by both rat and guinea-pig liver cells. Equilibrium was reached after approx. 20 min with rat cells, but guinea-pig cells had not achieved a steady state after 60 min. There was no evidence to suggest that quinolinate is rapidly metabolized by either species. The concentrations of quinolinate attained in rat and guinea-pig cells after short periods of incubation with 0.5 mM-quinolinate did not inhibit gluconeogenesis. These results raise further doubts as to the mechanism of quinolinate action in liver.     

The mechanism of inactivation of 3-hydroxyanthranilate-3,4-dioxygenase by 4-chloro-3-hydroxyanthranilate

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-heme Fe(II) dependent enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to quinolinate, an intermediate in the biosynthesis of nicotinamide adenine dinucleotide. Quinolinate has also been implicated in important neurological disorders. Here, we describe the mechanism by which 4-chloro-3-hydroxyanthranilate inhibits the HAD catalyzed reaction. Using overexpressed and purified bacterial HAD, we demonstrate that 4-chloro-3-hydroxyanthranilate functions as a mechanism-based inactivating agent. The inactivation results in the consumption of 2 +/- 0.8 equiv of oxygen and the production of superoxide. EPR analysis of the inactivation reaction demonstrated that the inhibitor stimulated the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated enzyme can be reactivated by treatment with DTT and Fe(II). High resolution ESI-FTMS analysis of the inactivated enzyme demonstrated that the inhibitor did not form an adduct with the enzyme and that four conserved cysteines were oxidized to two disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the enzyme, complexed to the inhibitor and O2, generates superoxide which subsequently dissociates, leaving the inhibitor and the oxidized iron center at the active site.     

Excretion of anthranilate and 3-hydroxyanthranilate by Saccharomyces cerevisiae: relationship to iron metabolism.

Resting suspensions of cells of Saccharomyces cerevisiae grown in iron-rich or iron-deficient conditions were studied by following the fluorescence emission changes (lambda em. 400-460 nm, lambda exc. 300-340 nm) occurring in these suspensions upon addition of glucose and ferric iron. The results show that, in addition to NAD(P)H, metabolites of the aromatic amino acid pathway interfere with the fluorescence measurements, and that they could be involved in ferric iron reduction. Wild-type strains of S. cerevisiae are known to excreted anthranilic acid and 3-hydroxyanthranilic acid in response to glucose. The major fluorescing compound excreted by a chorismate-mutase-deficient mutant strain of S. cerevisiae was identified as anthranilic acid. The excretion of anthranilic and 3-hydroxyanthranilic acids was correlated with the ferric-reducing capacity of the extracellular medium. Excretion during growth was much greater by cells cultured in iron-rich medium than by cells grown in iron-deficient medium. The possibility was examined that a link could exist between the biosynthesis of aromatics and the ferri-reductase activity of the cells, via chorismate synthase and its putative diaphorase-associated activity. Two ferri-reductase-deficient mutants excreted much less 3-hydroxyanthranilate than did the parental wild-type strains. However, the ferri-reductase activity of a chorismate-synthase-deficient mutant was comparable to that of the parental strain.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

耦合固定化技术在天冬氨酸转氨酶反应体系中的应用

对卡拉胶与明胶形成的耦合固定化体系进行了优化,并探讨海藻糖和金属离子（Mg^2＋）等辅助因子在该体系中对酶转化过程的影响。结果表明将该法应用于L-苯丙氨酸转化体系中,保护了天冬氨酸转氨酶的活性,显著提高了酶活回收率,天冬氨酸转氨酶的活力回收达到了93．6％,较文献报道有明显提高。     

Use of water-organic systems in the enzymatic transformation of steroids

This review surveys existing data on the use of water-organic systems, one of the promising methods of enzymic transformation of steroids. The procedures for reducing the inhibitory effect of solvents on enzymes and microbial cells are discussed. It is shown that hydrophobic capacity of gels and substrates, polarity of solvents, and coefficients of the distribution of reaction components between phases are to be taken into consideration when performing steroid transformation in the presence of organic solvents in order to increase its efficiency.     

Effect of quinolinate on aminotransferases

Quinolinate inhibits several aminotransferases (ornithine, alanine, and aspartate). However, it is considerably more potent as an inhibitor of liver and heart cytoplasmic aspartate aminotransferase. It is a much less potent inhibitor of mitochondrial aspartate aminotransferases. Quinolinate is bound to the active site of cytoplasmic aspartate aminotransferase. It has a much greater affinity for the pyridoximine-P than the pyridoxal-P form of the enzyme. According to kinetic results, the inhibition or dissociation constant of quinolinate is 0.2 and 20 mm, respectively, for the pyridoxamine-P and the pyridoxal-P forms of the enzyme. Since quinolinate is mainly bound to the pyridoxamine-P form: (a) it is a potent competitive inhibitor of α-ketoglutarate but has little effect when α-ketoglutarate is saturating even if the level of aspartate is low; (b) it decreases the effect of α-ketoglutarate on the absorption spectrum of the pyridoxamine-P form; and (c) it enhances the effect of glutamate on the absorption spectrum of the pyridoxal-P form. Quinolinate is also apparently bound to the apoenzyme since it inhibits reconstitution by either pyridoxamine-P or pyridoxal-P. Since quinolinate is a competitive inhibitor of α-ketoglutarate, it is possible that part of the inhibitory effect of quinolinate on hepatic gluconeogenesis could result from quinolinate inhibiting the conversion of aspartate to oxalacetate by the cytoplasmic aspartate aminotransferase. Quinolinate has no effect on either rat or bovine liver glutamate dehydrogenase or on kidney glutamate dehydrogenase.