Section 69
Chapter 68,634

Membrane-bound kidney neutral metalloendopeptidase: interaction with synthetic substrates, natural peptides, and inhibitors

Almenoff, J.; Orlowski, M.

Biochemistry 22(3): 590-599


ISSN/ISBN: 0006-2960
PMID: 6340719
DOI: 10.1021/bi00272a011
Accession: 068633480

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A neutral metalloendopeptidase with a thermolysin-like specificity was purified to apparent homogeneity, from the particulate fraction of rabbit kidney homogenates. After preparation of a deoxycholate extract, the enzyme was released from membranes by papain treatment and separated from other membrane-bound enzymes including dipeptidyl aminopeptidase IV, aminopeptidase M, and .gamma.-glutamyl transpeptidase by chromatography on Sephadex G-200, phenyl-Sepharose, and carboxymethylcellulose columns. The isolated enzyme has a MW of about 95,000 and is inhibited by thiols, metal chelators, phosphoramidon, and thiorphan. It is apparently identical with the kidney neutral metalloendopeptidase [Kerr and Kenny, (1974)] and similar to the bovine pituitary metalloendopeptidase and to an enzyme designated as enkephalinase. Studies with a series of synthetic substrates showed that the enzyme preferentially cleaves bonds in which the amino group is provided by a hydrophobic amino acid residue. Several biologically active peptides such as methionine and leucine enkephalin, dynorphin, bradykinin, and angiotensin I are degraded by cleavage of the same type of bond. The endopeptidase acts as a dipeptidyl carboxypeptidase on peptides having a hydrophobic residue in the penultimate position. N-[1-(R,S)-Carboxy-2-phenylethyl] derivatives of phenylalanyl- and alanyl-p-aminobenzoate were synthesized and tested as potential inhibitors. The 2 diastereomers of N-[1(R,S)-carboxy-2-phenylethyl]phenylalanyl-p-aminobenzoate were separated by high-pressure liquid chromatography; the more potent isomer had a KI of 2.9 .times. 10-8 M. The inhibitory potency of the alanyl derivatives was lower by almost 2 orders of magnitude. As with thermolysin, a hydrophobic residue in the P1' position and the carboxylate group complexing with the active site Zn atom may account for the inhibitory action of these derivatives.

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