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A spectrophotometric rate assay of aminoacylase
H Y Cho1, K Tanizawa, H Tanaka
1Laboratory of Microbial Biochemistry, Kyoto University, Japan.
Abstract:
Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.