Elsevier

Peptides

Volume 5, Issue 4, July–August 1984, Pages 769-776
Peptides

Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase

https://doi.org/10.1016/0196-9781(84)90020-2Get rights and content

Abstract

Angiotensin I converting enzyme (ACE) and neutral endopeptidase (“enkephalinase”; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7, Phe7-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (μmol/min/mg): SP1–11=7.8, SP4–11=11.7, SP5–11=15.4, SP6–11=15.6, SP8–11=6.7, NT1–13=2.9, and NT8–13=4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio=4:1). The hydrolysis was Cl dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80–93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (μmol/min/mg): SP1–11=1.2, SP methyl ester=0.7, SP free acid=8.5, SP4–11=2.4, SP5–11=0.9, SP6–11=1.4, SP8–11=0, NT1–13=0.2, and NT8–13=1.3. Peptide substrates were used as inhibitors of ACE (substrate=FA-Phe-Gly-Gly) and NEP (substrate=Leu5-enkephalin). Ki values (μM) were: for ACE, bradykinin=0.4, angiotensin I=4, SP=25, SP free acid=2, NT=14, and Met5-enkephalin=450, and for NEP, bradykinin=162, angiotensin I=36, SP=190, NT=39, and Met5-enkephalin=22. These studies indicate that ACE and NEP, two enzymes widely distributed in the body, may be involved in the metabolism of SP and NT.

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  • Cited by (0)

    Presented in part at the 56th annual meeting of the American Heart Association, November 14–17, 1983, Anaheim, CA.

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    Current address: Department of Biology and Chemistry, University of Osnabrück, Postfach 44 69, D-4500, Osnabrück, Germany.

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