Recommendation for the measurement of "alanine aminopeptidase" in urine

A method is recommended for the measurement of the catalytic concentration of alanine aminopeptidase in the urine of man, rat, and dog, using L-alanine-4-nitroanilide as substrate. In currently used methods, substrate concentrations between 1.7 and 2.0 mmol/l are used. Kinetic experiments show, however, that the reaction is inhibited by substrate concentrations exceeding 0.8 (man), 0.3 (rat) and 0.5 mmol/l (dog); these concentrations lie in the range of the Km values. Assay conditions were therefore chosen to give the lowest possible Km. The Km value depends on the type of buffer, and it increases with pH and temperature. The recommended assay conditions are: triethanolamine buffer 70 mmol/l, pH 7.6; L-alanine-4-nitroanilide concentrations depending on the species; incubation temperature 25 degrees C.