Isolation of endopeptidase-24.11 (EC 3.4.24.11, "enkephalinase") from the pig stomach. Hydrolysis of substance P, gastrin-releasing peptide 10, [Leu⁵] enkephalin, and [Met⁵] enkephalin

The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immuno-adsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 μg/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu⁵] enkephalin, and [Met⁵] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10^-6 M) and strongly by 1,10-phenanthroline (10^-3 M), but was unaffected by captopril (10^-5 M).