Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two‐stage enzymatic reaction

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Abstract
A sensitive two‐stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3‐carboxypropanoyl‐alanyl‐alanyl‐leucine‐4‐nitroanilide supplemented with Streptomyces griseus amino‐peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25°C) with a catalytic efficiency (k cat=1.2 × 102 s−1, K m=0. 15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (k cat=1.2 × 103 s−1) but the overall efficiency is diminished by a higher K m (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidase and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.