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)

A novel in vivo binding test was developed in order to evaluate the degree of occupancy of enkephalinase (EC 3.4.24.11), a membrane-bound metallopeptidase, in cerebral and peripheral tissues of mice treated with enkephalinase inhibitors. The probe selected for this purpose was the prodrug [3H]acetorphan, a lipophilic diesterified derivative of the potent enkephalinase inhibitor thiorphan readily releasing the latter by tissue hydrolysis. In order to validate the in vivo binding assay, [3H]thiorphan binding to membranes was first studied in vitro. [3H]Thiorphan binding to cerebral and peripheral tissues (lung and kidney) was saturable over a low nonspecific binding, occurring with a KD of 0.6 nM consistent with the Ki of the compound as enkephalinase inhibitor. [3H]Thiorphan binding varied largely among various tissues and was highly correlated with the catalytic activity of enkephalinase, thus indicating a selective labeling of the peptidase. After the i.v. administration of [3H]acetorphan a large fraction of the radioactivity remained bound to membranes isolated by a rapid filtration assay. Bound radioactivity mainly corresponded to [3H] thiorphan as identified by high-performance liquid chromatography analysis of kidney membranes, whereas unchanged [3H]acetorphan was not detectable. In vivo binding generated by [3H]acetorphan was saturable, with maximum binding sites values which were in rather good agreement with corresponding maximum binding sites values of [3H]thiorphan binding in vitro, particularly in brain. Specific in vivo binding was calculated as the difference between total and a generally low, nonspecific binding evaluated in mice receiving a large dose of nonlabeled acetorphan. Specific in vivo binding varied largely among tissues and generally reflected the abundance of enkephalinase molecules in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evaluation of enkephalinase inhibition in the living mouse, using [3H]acetorphan as a probe. 318 61

Thiorphan and its prodrug, acetorphan, are two inhibitors of enkephalinase (EC 3.4.24.11), a membrane-bound peptidase which plays an important role in the metabolic degradation of enkephalins. Since exogenous opioids have been reported to both stimulate and inhibit gastric secretion, the effects of thiorphan and acetorphan were studied in conscious rats equipped with chronic gastric fistulas. While i.v. thiorphan had no effect, both i.c.v. thiorphan and i.v. acetorphan potently inhibited the basal gastric acid output and the acid output stimulated by pentagastrin. Conversely, neither drug affected the histamine- or methacholine-induced stimulation of acid secretion. Neither thiorphan or acetorphan had any effect on the acid secretion stimulated by a combination of pentagastrin and acetylcholine in vagotomized rats. The results strongly suggest that both drugs inhibit gastric secretion through an effect at the level of the central nervous system, which decreases the vagal drive to the stomach. However, the effects of thiorphan and acetorphan were not prevented by naloxone. This is at variance with most of the effects of these drugs reported to date, including the inhibition of gastric secretion in cats. Furthermore, these effects were observed at doses which could inhibit other enzymes apart from enkephalinase. This suggests that the antisecretory action in rats is related to the protection of some non-opioid peptide(s) from degradation. In conclusion, both peptidase inhibitors inhibit gastric secretion of the rat through a central mechanism involving unknown, non-opioid peptide(s).
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PMID:Thiorphan and acetorphan inhibit gastric secretion by a central, non-opioid mechanism in the rat. 323 79

The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which reaches the anterior pituitary via the hypothalamo-hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site-directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu-His-Trp (LHRH1-3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu-His-Trp-Ser-Tyr (LHRH1-5) and, to a lesser extent, pGlu-His-Trp-Ser-Tyr (LHRH1-6). The degradation of LHRH and the formation of the N-terminal tri- and pentapeptides was blocked by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific, active site directed inhibitor of endopeptidase-24.15. Some inhibition of LHRH degradation and formation of the N-terminal hexapeptide was also obtained in the presence of N-[1-carboxy-2-phenylethyl]-Phe-p-aminobenzoate (cFE-F-pAB), an inhibitor of endopeptidase-24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C-terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D-amino acid are resistant to degradation by both endopeptidase-24.11 and -24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP-AAF-pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane-bound form of endopeptidase-24.15 to yield pGlu-His-Trp-Ser-Tyr and to a lesser extent by endopeptidase-24.11 to yield pGlu-His-Trp-Ser-Tyr-Gly.
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PMID:Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo. 329 5

Vasoactive peptides contain a high proportion of proline residues which make them resistant to hydrolysis by many peptidases. However, post proline cleaving enzyme (PPCE; EC 3.4.21.26), a proline specific endopeptidase which specifically hydrolyzes internal peptide bonds on the carboxyl side of proline residues, has been shown to inactivate numerous vasoactive peptides including angiotensins, kinins, substance P, vasopressin and oxytocin. In order to determine whether PPCE could be involved in vascular metabolism of vasoactive peptides, we carried out localization and characterization studies of PPCE-like activity in hog aorta and mesenteric artery. PPCE was assayed fluorometrically at pH 7.0 using the specific PPCE substrate CBZ-Gly-Pro-4-methyl-coumarinylamide. The subcellular distribution of vascular PPCE was essentially the same as that of the cytosolic marker enzyme lactic dehydrogenase (LDH). PPCE was enriched six-fold in the cytosolic fraction (11.4 +/- 2.7 units/mg) and unlike the plasma membrane-bound proline specific exopeptidase dipeptidyl-(amino)peptidase IV (DAP IV; EC 3.4.14.5), little or no activity could be detected in the microsomal or plasma membrane fractions. Similar to PPCE characterized from other sites, vascular PPCE was stabilized and activated by dithiothreitol and EDTA, and inhibited by DFP, p-chloromercuriphenyl sulfonic acid, L-1-tosylamido-2-phenylethylchloromethyl ketone, Cu++, Ca++, and Zn++. Vascular PPCE was unaffected by inhibitors of trypsin and kallikrein (Aprotinin, ABTI), aminopeptidase M (bestatin, amastatin), neutral endopeptidase (phosphoramidon), angiotensin I converting enzyme (captopril) or carboxypeptidase N (MERGETPA). These data demonstrate that PPCE is present in vascular endothelium and/or smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vascular, post proline cleaving enzyme: metabolism of vasoactive peptides. 354 18

We studied the binding of two radioactive probes, i.e. [3H]thiorphan and a 125I-labeled monoclonal antibody raised against the rabbit kidney enzyme, to enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) from rat cerebral membranes. [3H]Thiorphan binding at equilibrium to striatal membranes was monophasic with a KD (0.7 nM) and a pharmacology consistent with a selective labeling of the enzyme. The ratio of Vmax/Bmax was in the same range as the Kcat of the enzyme purified from peripheral tissues. The monoclonal antibody immunoprecipitated to a similar extent the solubilised enkephalinase activity and [3H]thiorphan binding sites from striatum. The regional distributions of binding sites for the two probes established either on isolated membranes or autoradiographic sections were highly heterogeneous and similar to that of enkephalinase activity. Hence the two probes appear to label membrane-bound enkephalinase in rat brain but, from a technical point of a view, the 125I-monoclonal antibody is a more sensitive and flexible tool.
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PMID:Characterisation of two probes for the localisation of enkephalinase in rat brain: [3H]thiorphan and a 125I-labeled monoclonal antibody. 354 55

A membrane-bound neutral endopeptidase which hydrolyzes Suc-(Ala)3-pNA to succinyl dialanine and Ala-pNA has been purified from human placenta. The enzyme was solubilized from membranes with DOC and papain, and was purified about 5000-fold by successive chromatographies on Sephadex G-200, DEAE-Sephacel, butyl-Toyopearl 650, and Sephacryl S-300. It was found to be homogeneous on SDS-polyacrylamide gel electrophoresis and to have a molecular weight of about 70,000. It was strongly inhibited by phosphoramidon, thiorphan, and metal-chelating agents, but was not affected by most other protease inhibitors. These findings indicate that it can be classified as a phosphoramidon-sensitive neutral endopeptidase. With biologically active peptides as substrates, the enzyme preferentially cleaved the bonds at the amino side of hydrophobic amino acid residues. The physiological significance of this enzyme is discussed with reference to the placental barrier.
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PMID:Purification and characterization of a membrane-bound neutral endopeptidase from human placenta. 355 3

The guinea pig two-domain precursor of MSEL-neurophysin and copeptin has been passed through a trypsin-Sepharose column in order to mimic the enzyme processing by a membrane-bound endopeptidase. Only two cleavages were observed located in the inter-domain sequence (at Arg-94 and Arg-98), in contrast to several additional cleavages found when free neurophysin or copeptin is subjected to soluble trypsin. Because the physiological maturation involves a single cleavage at Arg-94, both local accessibility in the precursor and narrow specificity of the enzyme are implied in the processing.
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PMID:Conformation limited proteolysis in the common neurophysin-copeptin precursor shown by trypsin-sepharose chromatographic proteolysis. 359 48

Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-Ala-Phe-MNA) at a rate of 9.5 nmol X hr-1 per 10(6) cells, and phosphoramidon (1 microM) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA (96 nmol X min-1 X mg-1) and the hydrolysis was inhibited by phosphoramidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol X min-1 mg-1) and 0.1 mM fMet-Leu-Phe (49 nmol X min-1 X mg-1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (Km, 59 X 10(-6) M; kcat, 3654 min-1). Immunocytochemistry at the light microscopic level revealed a high concentration of NEP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.
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PMID:Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. 390 53

The neutral endopeptidase (NEP) is a membrane-bound enzyme, which is solubilized by treatment with the protease, papain. Papain did not affect the apparent catalytic activity or the molecular mass of the purified human enzyme in SDS-PAGE. When NEP was treated with a reducing agent after papain digestion, it dissociated into smaller, lower molecular mass fragments. Amino acid analysis and s-carboxymethylation of the half cystine residues indicated that NEP contains four S-S bridges. We concluded that, although covalent bonds appear to be cleaved in NEP by papain, its activity and structure are sustained by S-S bridges.
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PMID:The importance of disulfide bridges in human endopeptidase (enkephalinase) after proteolytic cleavage. 391 45

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.
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PMID:The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. 442 92


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