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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
Author(s) -
Indig Fred E.,
Ben-Meir Daniella,
Spungin Anya,
Blumberg Shmaryahu
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81098-1
Subject(s) - chemistry , enzyme , neutral red , neutral protease , biochemistry , chromatography , cytotoxicity , in vitro , protease
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 × 10 2 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 × 10 3 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.