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

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10-Tyr11 bond, generating the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70-75-kDa monomer as determined by SDS-PAGE analysis in reducing or non-reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol-blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10-phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin-converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl-peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 microM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9-13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C-terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C-terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro-Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa-Pro dipeptides tested were totally ineffective. (e) The neurotensin-related peptides: neuromedin N, xenopsin and [Lys8-Asn9]neurotensin(8-13), as well as angiotensins I and II and dynorphins(1-8) and (1-13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.
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PMID:Peripheral inactivation of neurotensin. Isolation and characterization of a metallopeptidase from rat ileum. 340 80

A neutral endopeptidase (EC 3.4.24.5) previously thought to be unique to the eye lens has been found to be closely similar if not identical in native molecular size, component polypeptides and antigenic structure to a neutral proteinase from pituitary. Here we investigated some subtle differences in properties of the two enzymes, such as the effects of temperature, divalent cations and SDS on their activities with respect to different substrates. We conclude that the pituitary enzyme may have a relatively more compact structure requiring relaxation by low SDS concentration or higher temperature for maximum activity.
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PMID:Comparative studies on lens neutral endopeptidase and pituitary neutral proteinase: two closely similar enzymes. 351 Jan 28

Endopeptidase-24.11 (EC 3.4.24.11), a widely distributed cell-surface endopeptidase in pig tissues, was purified by immunoaffinity chromatography from its second most abundant source, lymph nodes. The detergent-solubilized enzyme is a glycoprotein with an apparent subunit Mr of 91,000, by electrophoresis in the presence of SDS. This value is intermediate between those observed in preparations from kidney and intestine. The specific activity (125I-labelled insulin B-chain as substrate) was similar to that prepared from other sources. Immuno-peroxidase and immunofluorescent cytochemical methods with either a monoclonal antibody, GK7C2, or an affinity-purified polyclonal antiserum, RP109, were used to establish the distribution and localization of the antigen in lymph nodes. Examination of many nodes confirmed the variability of endopeptidase-24.11 content from node to node. Pig lymph nodes are composed of functionally discrete nodelets and are anatomically inverted, with medulla being located peripheral to the cortex. Endopeptidase-24.11 was present in medulla, paracortex and cortex. The medulla, containing relatively few lymphocytes, stained more intensely than other zones. Lymphocyte-rich areas stained only weakly, but antigen was detectable in the centres of follicles and more strongly in a band surrounding them. The pattern of staining was reticular in appearance in all zones. In primary cell cultures, set up after enzymic disruption of nodes, the immuno-positive cells were found to be adherent to glass or plastic and to exhibit a fibroblastic morphology. Diffuse surface immunofluorescence and brighter intracellular immunofluorescence in granules were observed in these cells in the first few days of culture, but by the fourth day no immuno-positive cells remained and the fibroblasts that grew to confluence were somewhat different in morphology. The cells expressing the endopeptidase-24.11 antigen did not express Ia antigen and were clearly distinct from antigen-presenting dendritic cells. In appearance and properties they belong to the group described as reticular cells.
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PMID:Endopeptidase-24.11 in pig lymph nodes. Purification and immunocytochemical localization in reticular cells. 353 5

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.
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PMID:A high-molecular-mass neutral endopeptidase-24.5 from human lung. 355 24

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 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

Considerable attention has been focused recently on alpha 2-macroglobulin (alpha 2M), a major endopeptidase inhibitor in blood plasma, as a possible source of the primary defect in cystic fibrosis (CF). We report here studies designed to compare the structure of CF alpha 2M with normal alpha 2M to determine if there is a difference. The physicochemical properties of purified alpha 2M as revealed by various electrophoretic techniques, covalent proteinase binding properties, and primary structural studies on a variety of partial hydrolyzates of CF alpha 2M and normal alpha 2M are compared. These studies were carried out on eight different individual isolates of CF alpha 2M and three age-matched normal alpha 2M preparations and alpha 2M isolated from fetal cord blood. Three properties of CF alpha 2M were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): (1) the existence of four identically-sized subunits in the native molecule (10), (2) the cleavage of this subunit into fragments of approximately 100,000 daltons upon interaction with proteinases (10), and (3) the cleavage of an alkaline/heat sensitive bond to produce 120,000 and 60,000 dalton fragments (11). Both CF and normal alpha 2M were cleaved to the extent of 79-87%, CF alpha 2M behaves identically with normal alpha 2M with regard to all these properties. Salvesen and Barrett (24) have demonstrated that varying proportions of several [125I]-labeled proteinases form SDS-stable, non-reducible links to normal alpha 2M. Two of the CF alpha 2M preparations were studied to determine if similar covalent binding of proteinases occurred. The positions of the labeled and % of proteinase bound bands in SDS/reduced PAGE system were identical for normal alpha 2M and CF alpha 2M. These results indicate that CF alpha 2M behaves normally with regard to covalent binding of proteinases. Qualitative comparison of the peptide fragments separated by SDS-PAGE or isoelectric focusing of CF and normal alpha 2M produced by partial proteolysis with trypsin, chymotrypsin or Staphylococcus aureus V-8 proteinase did not reveal any difference unique to CF alpha 2M. The cyanogen bromide fragmentation studies and the cysteine cleavage studies also indicated that no major change in the positions of methionyl residues or cysteinyl/cystinyl residues has occurred in CF alpha 2M. The failure of all these different studies and those reported by others to demonstrate any differences between CF and normal alpha 2M makes it highly unlikely that there is a primary defect in alpha 2M in CF.
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PMID:Comparison of the structure and aspects of the proteinase-binding properties of cystic fibrotic alpha-macroglobulin with normal alpha 2-macroglobulin. 617 35

Fifteen human melanoma cells lines were tested by an antibody-binding radioimmunoassay using a monoclonal antibody (A12) directed against the common acute lymphoblastic leukemia antigen (CALLA). Cells from six melanoma lines were found to react with this antibody. The level of antigen and the percentage of positive cells in these six melanoma lines showed wide variation, as demonstrated by analysis in the fluorescence-activated cell sorter (FACS). Immunoprecipitation of solubilized 125I-labeled membrane proteins from CALLA positive melanoma cells with A12 monoclonal antibody revealed a major polypeptide chain with an apparent m.w. of 100,000 daltons, characteristic for CALLA as determined on SDS-polyacrylamide gel electrophoresis. The expression of CALLA on MP-6 melanoma cells was modulated when the cells were cultured in the presence of A12 antibody. Reexpression of CALLA on these cells occurred within 5 days after transfer of the modulated cells into medium devoid of monoclonal antibody.
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PMID:Expression of common acute lymphoblastic leukemia antigen (CALLA) on human malignant melanoma cell lines. 633 29

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.
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PMID:Purification and characterization of cathepsin B from monkey skeletal muscle. 672 39

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.
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PMID:Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme. 677 72


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