Proteolytic inactivation of substance P and neurokinin A in the longitudinal muscle layer of guinea pig small intestine.
Membrane vesicles, showing a 21 +/- 2-fold enrichment in the activity of 5'-nucleotidase and a 11 +/- 4-fold enrichment in the activity of angiotensin-converting enzyme relative to homogenate, were prepared from the myenteric plexus-containing longitudinal muscle layer of guinea pig ileum. Incubation of the vesicles with substance P and neurokinin A led to degradation of the peptides, and metabolites were isolated by reverse-phase HPLC and identified by amino acid composition. Cleavages of substance P between Glu6-Phe7, Phe7-Phe8, and Gly9-Leu10 and of neurokinin A between Gly8-Leu9 were observed and could be inhibited in a dose-dependent manner by phosphoramidon, an inhibitor of neutral endopeptidase 24.11. Formation of these metabolites was not completely inhibited by this agent, indicating that a phosphoramidon-insensitive form of endopeptidase 24.11 was present in the gut. Substance P was resistant to degradation by aminopeptidases, but neurokinin A was a substrate for bestatin-sensitive aminopeptidase(s), so that the neurokinin A (3-10) fragment represented the predominant metabolite in the chromatograms. The rate of formation of all the metabolites was not inhibited by enalapril and not enhanced by an increased Cl- concentration, indicating that angiotensin-converting enzyme was unimportant in the degradation process. Degradation of neurokinin A by the vesicles (Km 30 microM; Vmax 7.2 +/- 0.8 nmol min-1 mg of protein-1) was more rapid than degradation of substance P (Km 25 microM; Vmax 4.4 +/- 0.4 nmol min-1 mg of protein-1).
D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism; D004848:Epithelium / Q000201:enzymology; D006168:Guinea Pigs; D007421:Intestine, Small / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000201:enzymology*; D015260:Neprilysin; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D015288:Neurokinin A; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D013373:Substance P; D015288:Neurokinin A; D010450:Endopeptidases; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D015260:Neprilysin