Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

Abstract

Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine‐ and leucine‐enkephalins. For this reason this enzyme is often called ‘enkephalinase’. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N‐terminal cytoplasmic domain (27 amino acids), a single membrane‐spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn‐metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase.

Solid‐phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1–93 by tetanus toxin L chain

“…The inhibition by EDTA and ophenanthroline has already been reported by Schiavo et al (1992b) using an in vitro test and Sanders and Habermann (1992) in an in vivo test by preincubation of the toxin with different inhibitors and subsequent injection into permeabilized bovine adrenomedullary cells. The zinc contained in the protein has been shown to be necessary for activity (Schiavo et al, 1992a) and the L chain contains the consensus sequence HEXXH found in zinc endopeptidases (Devault et al, 1987;Jongeneel et al, 1989). The histidines are implicated in zinc binding whereas the glutamate has been shown to be essential for polarization of the water molecule, a precondition for hydrolysis of the peptide bond (Matthews, 1988).…”

“…The amino-acid sequence of the TeTx L chain presents the H-E-X-X-H sequence correspondi.ng to the signature of the zinc binding site of metallopeptidases (Devault et al, 1987;Matthews, 1988;Jongeneel et al, 1989;Vallee and Auld, 1990) and was recently found to contain a zinc atom (Schiavo et al, 1992b;Fraser-Wright et al, 1992). The deduced enzymic activity of TeTx L chain was formerly established by the demonstration that the toxin, but not its apoform, is able to cleave specifically rat synaptobrevin I1 at a single peptide bond (Gln76-Phe77) (Schiavo et al, 1992a).…”

Rat kidney endopeptidase 24.16

“…8B). Thus, endopeptidase 24.11, which was shown to display an N-terminal transmembrane spanning domain (Devault et al, 1987), was nearly equally solubilized by the seven detergents while alkaline phosphatase, which is covalently anchored by a glycosyl-phosphatidylinositol tail, was only released by octyl glucoside, Chaps and sodium deoxycholate (Fig. 8 B).…”

“…Primary structures of several membrane-bound peptidases have revealed that their attachment to the membrane could occur through a NH,-terminal signal sequence (dipeptidyl peptidase IV; Ogata et al, 1989) or by a transmembrane hydrophobic sequence located at the C terminus (angiotensinconverting enzyme; Soubrier et al, 1988) or N-terminally near after a short cytoplasmic domain (endopeptidase 24.1 1 ; Devault et al, 1987;Malfroy et al, 1987). Molecular cloning of endopeptidase 24.16 should establish the type of anchor responsible for attachment of the peptidase to the membrane.…”

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…NEP has a single transmembrane domain of 22 residues, an intracellular N-terminal tail of 27 residues and a large extracellular C-terminus that contains the active site [22,23]. In contrast with the high specificity of acetylcholinesterase, NEP degrades several peptides, albeit with graded affinity.…”