Simple radiometric assay for aminoacid:2-oxoacid aminotransferases and 2-hydroxyacid:2-oxoacid oxidoreductases

Abstract

The 1-carboxyl of oxocarboxylic acids is subject to oxidative decarboxylation by H2O2 and acid. The 1-carboxyl of most 2-amino- or 2-hydroxyacids is resistant to the treatment. This differential stability was exploited as an assay for the enzymatic interconversion of labile [1-14C]2-oxocarboxylic acids to stable [1-14C]2-hydroxyacids and 2-aminoacids. The approach appears generally applicable for the assay of small amounts of aminoacid:2-oxoacid aminotransferases and 2-hydroxyacid:2-oxoacid of oxidoreductases and some other enzymes involved in 2-oxocarboxylic acid metabolism in subcellular fractions from small numbers of cultured cells [human fibroblasts].