One-step and two-step fluorometric assay methods for general aminotransferases using glutamate dehydrogenase

One-step and two-step assay methods were developed for general aminotransferases (ATs) utilizing Glu and alpha-ketoglutarate (alpha-KG) as the donor and acceptor of the amino group, by coupling a glutamate dehydrogenase (GDH) reaction with the AT reactions. For instance, alpha-KG formed from Glu by AspAT is reduced and aminated back to Glu by GDH, which oxidizes NADPH corresponding to the amount of alpha-KG formed. In the reverse reaction, Glu formed from alpha-KG is oxidized and deaminated back to alpha-KG by GDH, which reduces NADP+ corresponding to the amount of Glu formed. In the one-step assay, both AT and GDH reactions are simultaneously carried out, and the decrease or increase in NADPH fluorescence is directly monitored in 1.0 ml of the reaction mixture for both forward and reverse reactions. In the two-step assay, an AT reaction is carried out and stopped once at the first step. Next, the alpha-KG or Glu formed is determined fluorometrically in a GDH reaction. In order to analyze partially purified or crude samples, the one-step assay is convenient for surveying the relative activities. The two-step assay is useful for analyzing the properties of enzymes and measuring activities under conditions approaching the optimum. AspAT can be replaced by other general ATs using enzyme-specific substrates in place of oxalacetate and Asp in the assay mixture. The present methods were successfully applied to four enzymes (Asp, alanine, gamma-aminobutyrate, and ornithine ATs) in tissue homogenates and a mitochondrial extract.