Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

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Abstract
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 [H-3]KYN to [H-3]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 alpha-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 K(i) of 5-mu-M. Kinetic analyses with partially purified rat brain KAT revealed a K(m) of 17-mu-M for KYN with 1 mM 2-oxoglutarate, but a much higher K(m) (910-mu-M) with 1 mM pyruvate. K(m) values for 2-oxoglutarate and pyruvate were 150 and 160-mu-M, 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.