Purification and Characterization of Kynurenine Aminotransferase I from Human Brain
Article first published online: 23 NOV 2002
Journal of Neurochemistry
Volume 62, Issue 2, pages 730–738, February 1994
How to Cite
Baran, H., Okuno, E., Kido, R. and Schwarcz, R. (1994), Purification and Characterization of Kynurenine Aminotransferase I from Human Brain. Journal of Neurochemistry, 62: 730–738. doi: 10.1046/j.1471-4159.1994.62020730.x
- Issue published online: 23 NOV 2002
- Article first published online: 23 NOV 2002
- Received February 9, 1993; revised manuscript received June 11, 1993; accepted June 14, 1993.
- Excitatory amino acids;
- Kynurenic acid;
Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l-kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute Km of 2.0 mM and 10.0 mM for l-kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l-glutamine, l-phenylalanine, and l-tryptophan, using either pyruvate (1 mM) or 2-oxoisocaproate (1 mM) as a cosubstrate. l-Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate (Ki = 480 µM) and competitively with regard to l-kynurenine (Ki = 200 µM). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.