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Determination of BCAT2 activity

ML Ming-Zhu Lei
XL Xu-Xu Li
YZ Ye Zhang
JL Jin-Tao Li
FZ Fan Zhang
YW Yi-Ping Wang
MY Miao Yin
JQ Jia Qu
QL Qun-Ying Lei
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BCAT2 activity was assessed as described previously.36 To assay BCAT2 enzymatic activity, reactions were conducted in 96-well plates in 200 μL of 100 mM potassium phosphate buffer (pH 7.4) containing 5 μM PLP, 50 mM ammonium sulfate (100 mM NH4+), 0.05 mM NADH, 5 mM DTT, 5 mM a-ketoglutarate, 10 mM L-leucine, and 25 μg (0.95 U) of LeuDH. The mixture was preincubated at 37 °C, and then, Flag-BCAT2 and Flag-BCAT2 mutants were added separately to the reaction mixture to initiate the reaction. The disappearance of NADH absorbance at 340 nm was monitored continuously in a spectrofluorometer (FL-4600, Hitachi). Triplicate-independent experiments were performed.

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