Etsuo Okuno

Characterization of rat brain kynurenine aminotransferases I and II

The endogenous neuroprotectant kynurenic acid (KYNA) is produced by irreversible transamination of L‐kynurenine (KYN). In the brain, two distinct kynurenine aminotransferases (KAT I and KAT II) are responsible for the formation of KYNA. The present experiments were designed to examine the respective roles of the two KATs in the normal rat brain. To this end, the two enzymes were partially purified, and their characteristics were examined. KAT I (identical with glutamine transaminase K) had an optimal pH of 9.5, preferred pyruvate as a cosubstrate and was potently inhibited by glutamine. KAT II (identical with L‐α‐aminoadipate transaminase) had a neutral optimal pH, showed no preference for pyruvate, and was essentially insensitive to inhibition by glutamine. KAT II was selectively inhibited by quisqualic acid (IC50: 520 μM). The endogenous substrate 3‐hydroxykynurenine had an approximately 10‐fold preference for KAT II. The distinct properties of the two enzymes made it possible to measure brain KAT I and KAT II in parallel by using dialyzed tissue homogenate (to remove interfering endogenous amino acids). Under these conditions, both enzymes presented essentially the same apparent Km values as the partially purified enzymes. In lesioned, neuron‐depleted brain tissue and in brain regions other than the cerebellum, KYNA derived primarily from KAT II at physiologic pH. In summary, the present study describes a simple methodology for the simultaneous determination of the two KYNA‐producing enzymes in small rat brain tissue samples and provides baseline values for future work in experimentally challenged animals. J. Neurosci. Res. 50:457–465, 1997.

Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain.

The tryptophan metabolite kynurenic acid (KYNA), which is produced enzymatically by the irreversible transamination of l‐kynurenine, is an antagonist of α7 nicotinic and NMDA receptors and may thus modulate cholinergic and glutamatergic neurotransmission. Two kynurenine aminotransferases (KAT I and II) are currently considered the major biosynthetic enzymes of KYNA in the brain. In this study, we report the existence of a third enzyme displaying KAT activity in the mammalian brain. The novel KAT had a pH optimum of 8.0 and a low capacity to transaminate glutamine or α‐aminoadipate (the classic substrates of KAT I and KAT II, respectively). The enzyme was inhibited by aspartate, glutamate, and quisqualate but was insensitive to blockade by glutamine or anti‐KAT II antibodies. After purification to homogeneity, the protein was sequenced and the enzyme was identified as mitochondrial aspartate aminotransferase (mitAAT). Finally, the relative contributions of KAT I, KAT II, and mitAAT to total KAT activity were determined in mouse, rat, and human brain at physiological pH using anti‐mitAAT antibodies. KAT II was most abundant in rat and human brain, while mitAAT played the major role in mouse brain. It remains to be seen if mitAAT participates in cerebral KYNA synthesis under physiological and/or pathological conditions in vivo.

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L‐kynurenine (KYN) and by determining the irreversible conversion of [3H] KYN to [3H] KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2‐oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT‐rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate‐specific. Thus, of the amino acids tested, only α‐aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3‐hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 μM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 μMfor KYN with 1 mM 2‐oxoglutarate, but a much higher Km(910 μM) with 1 mM pyruvate. Km values for 2‐oxoglutarate and pyruvate were 150 and 160 μM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate. At that time of almost complete neuronal destruction and pronounced astrocytic proliferation, enzyme activity in the lesioned striatum was almost twice as high as in controls, suggesting a preferential astroglial localization of brain KAT. The characteristics of KAT described here are compatible with a central role of the enzyme in brain KYNA function in vivo. Abnormal KAT activity, therefore, should be considered as an etiological factor in pathological phenomena related to dysfunction of excitatory amino acid receptors in the brain.

Purification and characterization of kynurenine-pyruvate aminotransferase from rat kidney and brain

Kynurenine-pyruvate aminotransferase (KPT), the enzyme responsible for the biosynthesis of the endogenous excitatory amino acid receptor antagonist kynurenic acid, was purified to homogeneity from rat kidney, as judged by polyacrylamide and sodium dodecyl sulfate electrophoresis. The protein appeared to consist of 2 identical subunits of approximately 48 kDa. Kinetic analysis showed Km values of 2.8 mM (kynurenine) and 3.8 mM (pyruvate), respectively. KPT was also partially purified from rat brain. Kidney and brain KPT were found to be identical when analyzed by a spectrum of biochemical, physico-chemical and, after production of anti-kidney KPT antibodies, immunological methods. Partially purified anti-KPT antiserum was used for first immunohistochemical studies, which revealed the presence of the enzyme in astrocyte-like cells throughout the brain. Less frequently, KPT was also found in discretely arranged neurons. The availability of pure KPT and specific anti-KPT antibodies can be expected to be of value for the further examination of the neurobiology of kynurenic acid.

Rat 3‐Hydroxyanthranilic Acid Oxygenase: Purification from the Liver and Immunocytochemical Localization in the Brain

Abstract: 3‐Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6). the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37–38.000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical Km of 3 μM for the substrate 3‐hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe2+) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double‐diffusion experiments. Immuhohistochemical studies using purified anti‐rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brains quinolinic acid system.

Kynurenic Acid: A Potential Pathogen in Brain Disorders

As evidenced by several contributions to this volume, it is now widely believed that excitatory amino acids (EAAs) of exogenous or endogenous origin play a major role in a host of neuropsychiatric diseases. Simply stated, excitotoxins can cause neuronal dysfunction and subsequent nerve cell death by turning physiological signals mediated by the NMDA or non-NMDA subtypes of EAA receptors into a pathologic process. Overstimulation of these receptors has been shown to have catastrophic consequences which have been carefully characterized in a spectrum of experimental systems in viva and in vim. Administration of excitotoxins to animals causes specific chemical, behavioral, and neuropathologic disturbances, which vary vastly with the nature of the toxin, its route of administration, and the age and species of the animal. It is therefore not surprising that excitotoxic insults, depending on the nature of the experimental paradigm, can provide models for diseases of the central nervous system as divergent as Huntington’s disease, epilepsy, Alzheimer‘s disease, cerebrovascular disease, and schizophrenia.l.2 The large majority of hypotheses considering an excitotoxic basis of a given brain disorder center around the idea that an overabundance of an edgpwus EAA underlies pathophysiology. Glutamate and aspartate, which present millimolar concentrations in brain tissue homogenates, are commonly believed to trigger the cascade of events that ultimately lead to the structural breakdown of nerve cells. Indeed, strong supportive evidence for an involvement of glutamate in hypoglycemic and hypoxiclischemic cell damage has been obtained in animal It appears, however, that the ubiquitous excitatory neurotransmitter glutamate can exert in vim excitotoxicity primarily (or only) in situations where its inactivation processes (i.e., metabolism or reuptake) are impaired. Thus, the intact brain appears to be able to withstand even massive glutamatergic assaults without enduring significant neuropathologic consequences.5.6 Of the known endogenous non-neurotnnsmitter substances that can serve as li-

Molecular cloning of rat kynurenine aminotransferase: Identity with glutamine transaminase K

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of l‐kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS‐1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.

Kynurenine pathway enzymes in a rat model of chronic epilepsy: Immunohistochemical study of activated glial cells

The kynurenine pathway metabolites quinolinic acid and kynurenic acid have been hypothetically linked to the occurrence of seizure phenomena. The present immunohistochemical study reports the activation of astrocytes containing three enzymes responsible for the metabolism of quinolinic acid and kynurenic acid in a rat model of chronic epilepsy. Rats received 90 min of patterned electrical stimulation through a bipolar electrode stereotaxically positioned in one hippocampus. This treatment induces non-convulsive limbic status epilepticus that leads to chronic, spontaneous, recurrent seizures. One month after the status epilepticus, the rats showed neuronal loss and gliosis in the piriform cortex, thalamus, and hippocampus, particularly on the side contralateral to the stimulation. Astrocytes containing the kynurenic acid biosynthetic enzyme (kynurenine aminotransferase) and the enzymes for the biosynthesis and degradation of quinolinic acid (3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase, respectively) became highly hypertrophied in brain areas where neurodegeneration occurred. Detailed qualitative and quantitative analyses were performed in the hippocampus. In CA1 and CA3 regions, the immunostained surface area of reactive astrocytes increased up to five-fold as compared to controls. Enlarged cells containing the three enzymes were mainly observed in the stratum radiatum, whereas the stratum pyramidale, in which neuronal somata degenerated, showed relatively fewer reactive glial cells. Hypertrophied kynurenine aminotransferase- and 3-hydroxyanthranilic acid oxygenase-immunoreactive cells were comparable in their morphology and distribution pattern. In contrast, reactive quinolinic acid phosphoribosyl transferase-positive glial cells displayed diversified sizes and shapes. Some very large quinolinic acid phosphoribosyl transferase-immunoreactive cells were noticed in the molecular layer of the dentate gyrus. In the hippocampus, the number of immunoreactive glial cells increased in parallel to the hypertrophic responses. In addition, pronounced increases in immunoreactivities, associated with hypertrophied astrocytes, occurred around lesioned sites in the thalamus and piriform cortex. These findings indicate that kynurenine metabolites derived from glial cells may play a role in chronic epileptogenesis.

2-Aminoadipate-2-oxoglutarate aminotransferase isoenzymes in human liver : a plausible physiological role in lysine and tryptophan metabolism

Two major 2-aminoadipate aminotransferase (AadAT) activities of human liver extract were separated by DEAE-Sepharose column chromatography. The faster eluting enzyme was designated AadAT-I and the other one AadAT-II. AadAT-I had a hgih Km value for aminoadipate, 20 mmol/l, and a low Km value for glutamate, 1.4 mmol/l. In contrast, AadAT-II had a low Km value for aminoadipate, 0.25 mmol/l, and a high Km value for glutamate, 12.5 mmol/l. AadAT-I and AadAT-II were mainly localized in the supernatant and mitochondrial fraction, respectively. AadAT-I demonstrated only glutamate-2-oxoadipate or 2-aminoadipate-2-oxoglutarate aminotransferase activities. AadAT-II further showed the activity of tryptophan and kynurenine. On the basis of Km values and subcellular localization of the isoenzymes, a plausible role was suggested for them involving the metabolism of lysine and tryptophan.

Localization of quinolinic acid metabolizing enzymes in the rat brain. immunohistochemical studies using antibodies to 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase

Specific antibodies raised in rabbits against 3-hydroxyanthranilic acid oxygenase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) were used in immunohistochemical studies to map the cellular localization of the quinolinic acid metabolizing enzymes in the adult male rat brain. 3-Hydroxyanthranilic acid oxygenase immunoreactivity was found to be present in glial cells of presumed astroglial identity, as judged by co-localization with glial fibrillary acidic protein. 3-Hydroxyanthranilic acid oxygenase-immunoreactive glial cells were present in all brain regions and within major fiber tracts. The density of 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells as well as the intensity of staining of these cells differed among brain regions. In general, telencephalic acid diencephalic areas harbored a larger number of 3-hydroxyanthranilic acid oxygenase-positive cells than did mesencephalic regions. In the former regions the caudate nucleus, septum, nucleus accumbens, neocortex and hippocampus were particularly enriched in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In the thalamus, regional differences were noted with regard to the intensity of staining among glial cells with high densities of 3-hydroxyanthranilic acid oxygenase cells in the anteroventral, reticular and ventromedial nuclei. In the inferior and superior colliculi, numerous 3-hydroxyanthranilic acid oxygenase-positive glial cells were found in all layers. In the hypothalamus, 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells were encountered in the zona incerta, the lateral hypothalamic area, the caudal preoptic region and in the dorsomedial nucleus. In the mesencephalon, the substantia nigra contained numerous, moderately stained cells. At caudal levels of the brain-stem, a relatively large number of cells was detected in the nucleus of the solitary tract, the pontine nucleus and in the fascial nerve nucleus, while other nuclei, such as the reticular formation and the area postrema were relatively poor in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In addition to staining of glial cells, neuronal cell bodies containing 3-hydroxyanthranilic acid oxygenase immunoreactivity were detected in the main and in the accessory olfactory bulb, as well as in the ventromedial nucleus of the hypothalamus. Quinolinic acid phosphoribosyltransferase immunoreactivity was observed within glial cells and in association with neuronal cell bodies. Some, but not all, quinolinic acid phosphoribosyltransferase positive glial cells contained glial fibrillary acidic protein (Köhl