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 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 Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)
Okuno
E
E
Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore 21228.
Schmidt
W
W
Parks
D A
DA
Nakamura
M
M
Schwarcz
R
R
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002852:Chromatography, Ion Exchange; D005968:Glutamate Decarboxylase / Q000378:metabolism; D007700:Kinetics; D007737:Kynurenine / Q000502:physiology*; D008190:Lyases; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D009928:Organ Specificity; D051381:Rats; D011919:Rats, Inbred Strains; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D007737:Kynurenine; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases; D005968:Glutamate Decarboxylase