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 immunoadsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 micrograms/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).
Bunnett
N W
NW
University of Washington, Department of Surgery, Seattle.
Turner
A J
AJ
Hryszko
J
J
Kobayashi
R
R
Walsh
J H
JH
D000818:Animals; D001839:Bombesin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004743:Enkephalin, Leucine / Q000378:metabolism*; D004744:Enkephalin, Methionine / Q000378:metabolism*; D005753:Gastric Mucosa / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000502:physiology; D015260:Neprilysin; D010446:Peptide Fragments / Q000378:metabolism*; D010449:Peptide Mapping; D013373:Substance P / Q000378:metabolism*
D010446:Peptide Fragments; D013373:Substance P; D004744:Enkephalin, Methionine; D004743:Enkephalin, Leucine; C040794:neuromedin C; D008666:Metalloendopeptidases; D015260:Neprilysin; D001839:Bombesin