A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases Arthur J.L. Cooper, a,b Myra Conway, c and Susan M. Hutson c, * a Departments of Biochemistry and Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10021, USA b Burke Medical Research Institute, White Plains, NY 10605, USA c Department of Biochemistry, Wake Forest University School of Medicine, Winston–Salem, NC 27157, USA Received 5 March 2002 Abstract A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L -leucine with a-ketoglutarate results in formation of a-ketoisocaproate, which is reductively aminated back to L -leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm 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 radio- chemical procedure, which measures transamination between a-ketoiso[1- 14 C]valerate and L -isoleucine. Due to the presence of glutamate dehydrogenase substrates (a-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehy- drogenase) 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 340 nm 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Branched-chain amino acid aminotransferase; Leucine dehydrogenase The whole body metabolism of L -leucine has been well studied (e.g., Refs. [1–3]). For each of the three branched-chain amino acids (BCAAs 1 —leucine, isoleu- cine, and valine), the first step in catabolism involves a transamination with a-ketoglutarate catalyzed by branched-chain amino acid aminotransferase (BCAT). Two isozymic forms of BCAT occur in mammals, namely, a mitochondrial form (BCAT m ) and a cytosolic form (BCAT c ) [4–10]. In the rat and human, BCAT m activity is substantial in most tissues [5,6,8]. In the rat, but not in humans, however, liver has low BCAT m ac- tivity [5,6,8]. BCAT c is confined to nervous tissue, ovary, and placenta [6]. The two human isozymes have been overexpressed in Escherichia coli, purified, and characterized [9]. Skeletal muscle is a major site in the body for the metabolism of the BCAAs [11]. In the muscle, the ni- trogen of the BCAAs is transferred to glutamate by the action of BCAT m and then to alanine in another transamination step. Transamination of glutamate with oxaloacetate yields aspartate. In rat muscle, which lacks glutamate dehydrogenase, aspartate (via the purine nu- Analytical Biochemistry 308 (2002) 100–105 www.academicpress.com ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: 336-716-7671. E-mail address: shutson@wfubmc.edu (S.M. Hutson). 1 Abbreviations used: BCAA, branched-chain amino acid; BCAT, branched-chain amino acid aminotransferase; BCAT c , cytosolic iso- form of BCAT; BCAT m , mitochondrial isoform of BCAT; GDH, glutamate dehydrogenase; EDTA, ethylenediamine tetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)-N ; N ; N 0 ; N 0 -tetraacetic acid; GTP, guanosine 5 0 -triphosphate; h, human; Hepes, N-(2-hydro- xyethyl)piperazine-N 0 -(ethanesulfonic acid); KIC, a-ketoisocaproate; LeuDH, leucine dehydrogenase; mOD, millioptical density. 0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0003-2697(02)00243-9