Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon‐γ (IFN‐γ) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN‐γ induced the expression of indoleamine 2,3‐dioxygenase (IDO) activity. Whereas tumor necrosis factor‐α did not affect enzyme induction by IFN‐γ, lipopolysaccharide modulated IDO activity differently in the two IFN‐γ‐activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3‐hydroxylase, and 3‐hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3‐hydroxylase activity was stimulated by IFN‐γ. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN‐γ markedly stimulated the activity of this enzyme only in MT2 cells. IFN‐γ‐treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3‐hydroxyanthranilic acid quinolinic acids from l‐[5‐3H]tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.

Several aminotransferases with kynurenine aminotransferase (KAT) activity are able to convert L-kynurenine into kynurenic acid, a putative endogenous modulator of glutamatergic neurotransmission. In the rat, one of the described KAT isoforms has been found to correspond to glutamine transaminase K. In addition, rat kidney ␣-aminoadipate aminotransferase (AadAT) also shows KAT activity. In this report, we describe the isolation of a cDNA clone encoding the soluble form of this aminotransferase isoenzyme from rat (KAT/AadAT). Degenerate oligonucleotides were designed from the amino acid sequences of rat kidney KAT/AadAT tryptic peptides for use as primers for reverse transcriptionpolymerase chain reaction of rat kidney RNA. The resulting polymerase chain reaction fragment was used to screen a rat kidney cDNA library and to isolate a cDNA clone encoding KAT/AadAT. Analysis of the combined DNA sequences indicated the presence of a single 1275-base pair open reading frame coding for a soluble protein of 425 amino acid residues. KAT/AadAT appears to be structurally homologous to aspartate aminotransferase in the pyridoxal 5-phosphate binding domain. RNA blot analysis of rat tissues, including brain, revealed a single species of KAT/AadAT mRNA of ϳ2.1 kilobases. HEK-293 cells transfected with the KAT/AadAT cDNA exhibited both KAT and AadAT activities with enzymatic properties similar to those reported for the rat native protein.

Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyridoxal-P)-depe11dent enzyme, catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse-transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G,) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Sarcharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P-binding site consensus sequence assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an -2.0-kb mRNA species in all tissues tested. A second mRNA species (-2.6 kb) was also detected in some tissues. After transfection of HEK-293 cells with the cDNA coding for kynureninase, the K,,, values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 -C 37 pM and 13.2 t 2.0 pM, respectively.

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A soluble aminotransferase with kynurenine amiconjugate fl-lyase activity [5,7] and has also been referred to notransferase (KAT) activity has been recently isolated from rat under this nomenclature. Due to its fl-lyasic activity, this brain. This enzyme corresponds to a cytosolic form of glutamine enzyme may be involved in the renal and cerebral toxicity of transaminase K (GTK). In addition to the cytosolic enzyme, a mitoehondrial-associated form of this KATIGTK also exists. In several halogenated xenobiotics [8]. The human form of kidney the present work we have isolated a rat brain cDNA clone encodcytosolic cysteine-S-conjugate fl-lyase/GTK has been recently ing a KATIGTK enzyme identical to the soluble form but carrying cloned and shown to have 82% amino acid similarity to the rat an additional stretch of 32 amino acids at its NH2-terminus.form [9]. Several structural features of this sequence resemble those of In addition to the soluble cytosolic enzyme (S-KAT/GTK), leader peptides for mitochondrial import. Evidence that the isothe existence of a mitochondrial KAT/GTK located in the lated cDNA encoded for mitochondrial KATIGTK was obtained matrix of mitochondria [10] has also been reported. Interestafter transfection of HEK-293 cells with the cDNA coding for ingly, mitochondrial GTK activity appears to be predominantly this new KATIGTK isoenzyme. In fact, a significant enrichment in rat brain, whereas the opposite is true for peripheral organs of both KAT and GTK enzymatic activities was found in the crude such as kidney [11]. In the present work, we report the isolation mitochondrial fraction of the transfected cells.of a rat brain cDNA clone encoding a mitochondrial-associated