Arthur J. L. Cooper
A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases
Cooper, Arthur J. L.; Conway, Myra; Hutson, Susan M.
Myra Conway Myra.Conway@uwe.ac.uk
Occasional Associate Lecturer - HAS DAS
Susan M. Hutson
A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with α-ketoglutarate results in formation of α-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between α-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (α-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure. © 2002 Elsevier Science (USA). All rights reserved.
Cooper, A. J. L., Conway, M., & Hutson, S. M. (2002). A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases. Analytical Biochemistry, 308(1), 100-105. https://doi.org/10.1016/S0003-2697%2802%2900243-9
|Journal Article Type||Article|
|Publication Date||Sep 1, 2002|
|Peer Reviewed||Not Peer Reviewed|
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