RESUMO
A convenient continuous spectrophotometric assay for estimation of branched-chain L-amino acid aminotransferase activity was established: Branched-chain 2-oxo acid-dependent transamination of L-glutamate was coupled-via 2-oxoglutarate-to L-aspartate aminotransferase plus L-malate dehydrogenase or to L-alanine aminotransferase plus L-lactate dehydrogenase as indicator systems. The rate of transamination can be monitored specifically by measuring the decrease in NADH absorbance at 334 nm over time. The method was applied, e.g., for evaluation of some kinetic properties of the rat heart (iso)enzyme.
Assuntos
Cetoácidos/química , Mitocôndrias Cardíacas/enzimologia , Transaminases/análise , Animais , Bovinos , Sistema Livre de Células/enzimologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Hemiterpenos , Isoenzimas/análise , Modelos Lineares , Ratos , Especificidade por Substrato , TemperaturaRESUMO
A spectrophotometric assay for the determination of branched-chain L-amino acid aminotransferase activity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4-methyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum as coupling enzyme by measuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evaluated for the (iso)enzyme from rat heart.
