Kynurenine aminotransferase 3/glutamine transaminase L/cysteine conjugate beta-lyase 2 is a major glutamine transaminase in the mouse kidney

Abstract

Background: Kynurenine aminotransferase 3 (KAT3) catalyzes the transamination of Kynurenine to kynurenic acid, and is identical to cysteine conjugate beta-lyase 2 (CCBL2) and glutamine transaminase L (GTL). GTL was previously purified from the rat liver and considered as a liver type glutamine transaminase. However, because of the substrate overlap and high sequence similarity of KAT3 and KAT1, it was difficult to assay the specific activity of each KAT and to study the enzyme localization in animals.

Methods: KAT3 transcript and protein levels as well as enzyme activity in the liver and kidney were analyzed by regular reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, biochemical activity assays combined with a specific inhibition assay, and western blotting using a purified and a highly specific antibody, respectively.

Results: This study concerns the comparative biochemical characterization and localization of KAT 3 in the mouse. The results showed that KAT3 was present in both liver and kidney of the mouse, but was much more abundant in the kidney than in the liver. The mouse KAT3 is more efficient in transamination of glutamine with indo-3-pyruvate or oxaloacetate as amino group acceptor than the mouse KAT1.

Conclusions: Mouse KAT3 is a major glutamine transaminase in the kidney although it was named a liver type transaminase.

General significance: Our data highlights KAT3 as a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney. This suggests tissue localizations of KAT3/GTL/CCBL2 in other animals may be carefully checked.

Keywords: Aminotransferase; CCBL, cysteine conjugate beta-lyase; Cysteine conjugate beta-lyase 2; GTK, glutamine transaminase K; GTL, glutamine transaminase L; Glutamine transaminase; KAT, kynurenine aminotransferase; KYNA, kynurenic acid; Kynurenic acid; Kynurenine; Kynurenine aminotransferase; PLP, pyridoxal-5′-phosphate..

Fig. 1

Transamination activity of two KATs towards different α-ketoacids. Purified recombinant mouse KAT1 and KAT3 were incubated with each of 16 α-ketoacids at 2 mM in the presence of 5 mM kynurenine in a reaction mixture including 40 μM PLP and 100 mM phosphate, pH 7.5. The activity was quantified by the amount of KYNA produced in the reaction mixture. (A) mouse KAT1; (B) mouse KAT3.

Fig. 2

HPLC UV–vis detection of glutamine transaminase activity. The reaction mixtures, standards and buffers were mixed with an equal volume of 0.8 M formic acid before being injected into an HPLC reverse-phase column (150×4.6 mm, Varian, Palo Alto, CA) for analysis. The mobile phase consists of 10 mM potassium phosphate (monobasic) buffer containing 10% (v/v) acetonitrile for the analysis of phenylalanine in the reaction mixtures. The formation of transamination product, phenylalanine was monitored by an in-line UV detector at a wavelength of 257 nm. A, B, C, and D illustrate chromatograms of boric acid buffer, 1 mM phenylpyruvate standard, 1 mM phenylalanine standard, and the reaction mixture without incubation, respectively. Chromatogram E illustrates the product, phenylalanine (arrowed) formed in 100 μl reaction mixture including 5 mM glutamine, 2 mM phenylpyruvate and 2 μg recombinant mouse KAT3, 100 mM boric acid buffer, pH 9.0, in 15 min at 38 °C. The reaction was stopped by adding an equal volume of 0.8 M formic acid.

Fig. 3

Inhibition of glutamine transaminase activity of mouse recombinant KAT1 and KAT3 by methionine. The reaction mixture consisted of 5 mM glutamine, 2 mM phenylpyruvate, 40 μM PLP, 2 μg recombinant protein, KAT1 or KAT3 in 100 mL 100 mM boric acid buffer, pH 9.0. The mixture was incubated at 38 °C for 15 min and the reaction was stopped by adding an equal volume of 0.8 M formic acid. Measurement of phenylalanine product was performed by HPLC with UV detection at a wavelength of 257 nm. Panel B shows methionine significantly inhibited KAT3 activity.

Fig. 4

Inhibition of glutamine transaminase activity of mouse liver and kidney crude protein extracts by methionine. The reaction mixture consisted of 5 mM glutamine, 2 mM phenylpyruvate, 40 μM PLP, 20 μl crude liver protein extract or kidney protein extract in 100 mL of 100 mM boric acid buffer, pH 9.0. The mixture was incubated at 38 °C for 2 h and the reaction was stopped by adding an equal volume of 0.8 M formic acid. Measurement of phenylalanine was performed by HPLC with UV detection at a wavelength of 257 nm. Panel B shows methionine significantly inhibited KAT activity in the kidney.

Fig. 5

mRNA and protein levels of KATs in the mouse liver and kidney. The mRNA transcripts of mouse kat1 and kat3 in the liver and kidney were analyzed using both RT-PCR and qRT-PCR. All final products in RT-PCR experiments were analyzed by 1% agarose gel electrophoresis (A). Relative expression levels of kat1 and kat3 genes were shown as percentages of gapdh gene expression in qRT-PCR tests (B). The specificity of the purified mouse KAT3 antibody was tested by Western blotting. Both recombinant mouse KAT1 and KAT3 proteins were run in a SDS-PAGE, transferred to a PVDF membrane and immuno-stained with the purified mouse KAT3 antibody. The antibody recognized the recombinant mouse KAT3 protein very well, without cross-reaction with recombinant mouse KAT1 (C). Using the purified mouse KAT3 antibody, KAT3 protein was detected both in liver and kidney, but a more intensive band was seen in the kidney (D).