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Cloning and Characterization of a Soluble Kynurenine Aminotransferase from Rat Brain: Identity with Kidney Cysteine Conjugate β‐Lyase
Author(s) -
AlberatiGiani Daniela,
Malherbe Pari,
Köhler Christer,
Lang Gabrielle,
Kiefer Vivian,
Lahm HansWerner,
Cesura Andrea M.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.64041448.x
Subject(s) - microbiology and biotechnology , biology , complementary dna , biochemistry , lyase , peptide sequence , molecular cloning , oligonucleotide , amino acid , enzyme , gene
In this study, we describe the cloning and characterization of a soluble form of kynurenine aminotransferase (KAT, EC 2.6.1.7) present in rat brain. Soluble KAT was purified from rat kidney and the amino acid sequences of four tryptic peptides determined. These peptides were found to belong to the amino acid sequence reported for rat kidney soluble cysteine conjugate β‐lyase, indicating that rat kidney KAT and β‐lyase represent the same molecular entity. Oligonucleotide probes derived from the β‐lyase cDNA were then used as primers for PCR of reverse‐transcribed rat brain poly(A) + RNA. After subcloning of the resulting PCR fragment and sequencing of the isolated rat brain clone, its oligonucleotide sequence was found to be identical to that reported for the β‐lyase cDNA. Further evidence that the isolated rat brain clone encoded for KAT was obtained by transfecting HEK‐293 cells with a construct containing the coding sequence for the enzyme. The transfected cells exhibited KAT activity and, in the presence of 2 m M pyruvate and 2‐oxoglutarate, the K m values for l ‐kynurenine were 1.2 m M and 86.3 µ M , respectively. Northern blot analysis of rat kidney, liver, and brain RNA revealed a single species of KAT/β‐lyase mRNA of ∼2.1 kb.