Kynurenine aminotransferase (KAT-II) activity

KAT-II activity was measured in brain regions of mice of 23 and 60 days old, which were homogenized (1:10, v/v) in homogenization buffer (containing 0.5 M tris-base buffer pH 8 (J. T. Baker, USA), 50 µM of pyridoxal-5-phosphate (P5P) and 10 mM 2-mercaptoethanol (cat. No. P9855, M6250 Sigma-Aldrich)). 100 µl of homogenized tissues were incubated with 100 µl of cocktail (containing 100 µM of L-kyn (cat. No. K8625, Sigma-Aldrich), 80 µM of P5P, 1 mM of sodium pyruvate (cat. No. P2256, Sigma-Aldrich) containing 150 mM tris-base buffer pH 7.4) and incubated at 37 °C for 1 h. The reaction was stopped with 20 µl of trichloroacetic acid 50% and 1 mL of HCl 0.1 M (J.T. Baker, USA). Samples were centrifuged 10 min at 14000 g. KYNA was detected by fluorescence at emission wavelength of 398 nm and 344 nm of excitation wavelength (Perkin Elmer fluorescence detector series 200a)⁶¹. The results were presented as pmoles of KYNA/h/mg of protein.