Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical "brush border" membrane of the kidney proximal tubules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydrophobic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Characterization of the secreted recombinant protein indicated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the enzyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopeptidase.
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PMID:Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells. 267 Sep 43

The messenger RNA (mRNA) encoding enkephalinase (EC. 3.4.24.11; neutral endopeptidase) has been localized in rat brain by in situ hybridization using 35S- or 32P-labelled cRNA probes. Hybridization was observed only in few brain areas, and was particularly strong in the striatum, olfactory bulb and pontine nuclei. The enkephalinase protein was also localized in brain sections using a radiolabelled monoclonal antibody. While some brain regions contained both the mRNA and its translation product, others, including in particular the substantia nigra, were rich in enkephalinase but did not contain any detectable amount of enkephalinase mRNA. Enkephalinase mRNA-containing cells could be identified in regions containing neurons known to project to the substantia nigra. The discrepancy between the mRNA and the protein labelling is likely to reflect the fact that the mRNA is exclusively located within the soma of the cells while the translated protein may be found anywhere along the axonal processes.
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PMID:Localization of enkephalinase mRNA in rat brain by in situ hybridization: comparison with immunohistochemical localization of the protein. 281 91

The simple determination of the Neutral Metalloendopeptidase (NEP, Enkephalinase A) with the known fluorogenic substrate Dansyl-D-Ala-Gly-(pNO2)Phe-Gly is disturbed by high concentrations of the Angiotensin-Converting-Enzyme (ACE). ACE hydrolyzes this substrate too but to a smaller degree. In some tissues and body fluids a further substrate hydrolysis takes place by any indefinite proteases. Finally the enzymatic hydrolysis of the NEP-substrate is inhibited by phosphate ions. A method is proposed for the elimination of this disturbances in the NEP-determination with a phosphate-free buffer using two comparison tests with Lisinopril and o-Phenanthroline. The resulting NEP-activity is calculated very simple thereafter.
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PMID:[The determination of neutral metalloendopeptidase (enkephalinase A) in biological material]. 285 71

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.
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PMID:Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site. 289 75

The localization of neutral endopeptidase-24.11 (E.C. 3.4.24.11; enkephalinase) in rat spinal cord was investigated by a novel fluorescent histochemical method. Enkephalinase was localized by using a coupled enzyme assay based upon the sequential cleavage of the synthetic peptide substrate glutaryl-ala-ala-phe-4-methoxy-2-naphthylamide by enkephalinase and exogenous aminopeptidase M. Enzyme distribution was examined in segments from cervical, thoracic, lumbar, and sacral cord. At all spinal cord levels, enkephalinase was localized to discrete regions of the gray matter. The substantia gelatinosa displayed rich enkephalinase staining which overlapped the inner and outer zones of lamina II. A staining pattern similar to that observed in lamina II was observed in the spinal trigeminal nucleus in the medulla. In lamina III the enzyme was associated with small and medium-sized cells. Lamina IV showed staining associated with medium-sized and large cell bodies. The medial boundary of the dorsal gray of laminae IV and V had medium-sized fusiform cells which stained for enkephalinase. In the lateral reticulated areas of lamina V, enkephalinase reaction product was localized to scattered medium-sized and large cells compressed against the white matter of axon bundles. Staining in lamina VI was similar in appearance to lamina V. Enkephalinase reaction product was widely distributed in the ventral horn. Numerous ventral horn motor neurons of varied size and morphology in laminae VIII and XI stained richly for the enzyme. The enzyme was also localized to medium-sized and large cells in lamina X and to cells of the central cervical nucleus. The size and morphology of the cell types associated with the enzyme supported a neuronal association for enkephalinase. The regional distribution of the enzyme overlapped that of enkephalin- and substance-P rich regions of the spinal cord. These findings support a role for enkephalinase in the metabolic regulation of centrally acting neuropeptides.
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PMID:Fluorescent histochemical localization of neutral endopeptidase-24.11 (enkephalinase) in the rat spinal cord. 291 2

The tripeptide Tyr-Gly-Gly (YGG) was established as an endogenous constituent in rat brain. Its origin from enkephalin neurons is suggested by its regional distribution paralleling that of (Met5)-enkephalin (YGGFM), its decrease following kainate-induced ablation of the striato-pallidal neurons and its enhanced formation following depolarization of pallidal slices. Enkephalinase (EC 3.4.24.11) is selectively responsible for endogenous YGG formation in vitro and in vivo.
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PMID:The endogenous tripeptide Tyr-Gly-Gly as an extracellular metabolite of enkephalins in rat brain: origin and metabolism. 312 44

[3H]Thiorphan, a potent inhibitor of enkephalinase (EC 3.4.24.11), was used to label the enzyme in membranes from rat kidney cortex an to explore its specificity at the active site. [3H]Thiorphan binding occurred reversibly, with low non-specific binding and to a single class of sites. The dissociation constant, determined by either kinetics or saturation studies was approximately 0.4 nM. The ratio of the maximal velocity of enkephalinase with enkephalins as substrates to the maximal binding of [3H]thiorphan was consistent with the catalytic constant of the enzyme. Enkephalinase inhibitors competed with [3H]thiorphan and had inhibitory constants in agreement with the corresponding values derived from measurement of the enzyme catalytic activity, whereas inhibitors of other metallopeptidases were ineffective. The inhibitory potencies of a series of systematically varied oligopeptides regarding [3H]thiorphan binding and enkephalinase activity were also highly correlated. Structure-activity relationships among competitors indicated that the main subsites of enkephalinase are: (1) the hydrophobic pocket in P'1, the requirements of which are best satisfied by aromatic amino acid side chain residues (2) the P'2 subsite, the requirements of which are best satisfied by amino acids with a short, uncharged side chain and a free terminal carboxyl group. This novel binding assay should facilitate the exploration of the active site of enkephalinase and the development of new inhibitors.
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PMID:Labelling and exploration of the active site of enkephalinase (EC 3.4.24.11) in kidney membranes with [3H]thiorphan as ligand. 316 67

Neutral endopeptidase (NEP) (enkephalinase, EC 3.4.24.11) and angiotensin converting enzyme (ACE) are two peptidases that can cleave the C-terminal dipeptide bradykinin(8-9) from bradykinin. To determine whether these peptidases play roles in modulating kinin-induced contractions in the airways, we studied the effects of captopril, an ACE inhibitor, and of leucine-thiorphan and phosphoramidon, two NEP inhibitors, on the contractile responses to bradykinin and lysyl-bradykinin in isolated segments of ferret trachea. Bradykinin and lysyl-bradykinin-induced contractions in a concentration-dependent fashion (P less than .001), with a threshold of 10(-7) M and 5 x 10(-7) M, respectively. In contrast, the bradykinin(8-9) and the N-terminal heptapeptide bradykinin(1-7), the major fragments of hydrolysis of bradykinin by NEP and ACE, had a very weak or no effect on tracheal contraction in concentrations as great as 10(-5) M. Captopril, leucine-thiorphan and phosphoramidon (each inhibitor at 10(-5) M, 15 min) shifted the concentration-response curves to lower concentrations by approximately 1 to 1.5 log U (P less than .05). Both NEP inhibitors and the ACE inhibitor potentiated the response to bradykinin in a concentration-dependent fashion (P = .0001), and the combination of phosphoramidon and captopril resulted in an additive potentiation of bradykinin-induced contraction (P less than .02). [D-Pro2-D-Trp7,9]-substance P, a substance P antagonist, did not modify the potentiation of bradykinin-induced contraction by NEP inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Neutral endopeptidase and angiotensin converting enzyme inhibitors potentiate kinin-induced contraction of ferret trachea. 327 79

cDNA clones encoding rat enkephalinase (neutral endopeptidase, EC 3.4.24.11) have been isolated in lambda gt10 libraries from both brain and kidney mRNAs and the complete 742 amino acid sequence of rat enkephalinase is presented. The enzyme possesses a single transmembrane spanning domain near the N-terminal of the molecule but lacks a signal sequence. Because enkephalinase has it active site located extracellularly and is thus an ectopeptidase, we suggest that the N-terminal transmembrane region of the enzyme anchors the protein in membranes and that the majority of the protein, including the carboxy terminus, is extracellular. Enkephalinase, a zinc-containing metallo enzyme, displays homology with other zinc metallo enzymes such as carboxypeptidase A, B and E, suggesting enzymatic similarities in these enzymes.
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PMID:Molecular cloning and amino acid sequence of rat enkephalinase. 355 89

There are at least two types of enzymes in brain, endopeptidases and aminopeptidases, which metabolize enkephalins. Evidence is presented to suggest that enkephalinase, an endopeptidase cleaving at the Gly-Phe bond, is specific for the endogenous enkephalinergic system. Selective inhibitors are described for each enzyme. These are parachloromercuriphenylsulfonic acid and puromycin in the case of aminopeptidases and various enkephalin fragments in the case of enkephalinase. Some characteristics of the two types of enzymes are described. Enkephalinase has many properties in common with the well-characterized brain angiotensin-converting enzyme. These two enzymes, however, behaved differently when tested for chloride dependence, for activity in several buffers and for susceptibility to specific inhibitors.
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PMID:Enkephalinase: selective inhibitors and partial characterization. 626 6


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