Purification and characterization of kynurenine aminotransferase I from human brain.
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 approximately 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 approximately 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 microM) and competitively with regard to L-kynurenine (Ki = 200 microM). 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.
Baran
H
H
Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore.
Okuno
E
E
Kido
R
R
Schwarcz
R
R
D000328:Adult; D000596:Amino Acids / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007158:Immunologic Techniques; D007700:Kinetics; D008190:Lyases; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D013379:Substrate Specificity; D017186:Titrimetry; D000637:Transaminases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*
D000596:Amino Acids; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases