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)

With the aim of confirming our previous spectrophotometric binding studies ((1978) Eur. J. Biochem. 85, 345-350 and (1980) Eur. J. Biochem. 104, 249-254) and of ascertaining the full physiological significance of ion binding, we investigated the effects of ions and thiol reagents on the proteolysis of yeast phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). The single non-essential thiol of the enzyme was modified with 5,5'-dithiobis(2-nitrobenzoic acid) or 2-chloromercuri-4-nitrophenol. Both modifications greatly increased the susceptibility of the kinase to inactivation by trypsin or yeast proteinase A, when compared with that of the native kinase. Electrophoresis in sodium dodecyl sulfate (SDS) revealed that limited proteolysis had occurred. The time courses for the proteolysis and loss of catalytic activity were followed and the active and inactive fragments identified. The molecular masses of the major proteolytic fragments differed with the two endopeptidases. Substrate and non-substrate anions in a concentration-dependent fashion, protected the native and mercurial-labelled kinase from inactivation by trypsin or yeast proteinase A. However, Zn2+, in a concentration-dependent fashion, increased the susceptibility of the native kinase to inactivation by each endopeptidase. The time courses for the inactivation and for the proteolysis allowed the active and inactive fragments to be identified. Zn2+ decreased the rate of inactivation of the mercurial-labelled kinase by proteinase A. The effects of these ions were detected at concentrations compatible with occupancy of an anion binding site and a low affinity Zn2+ binding site, both of which have been indicated from our previous binding studies.
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PMID:The effects of anions, substrates, metal ions and sulfhydryl reagents on the proteolytic susceptibility of yeast phosphoglycerate kinase. 703

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2' residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2' residue by formation of a unique hydrogen bond.
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PMID:Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2' residue of substrates and inhibitors. 748 5

Pancreatic acinar cells, a model cell type for the study of exocytosis in non-excitable cells, were used here to test the hypothesis that molecular mechanisms regulating exocytosis from neuronal and neuroendocrine cells may also be utilized in exocrine cells. Using specific antisera raised against vesicle-associated membrane protein (VAMP) isoforms 1 and 2, we have identified VAMP-2, but not VAMP-1, immunoreactive proteins on rat pancreatic acinar cell secretory (zymogen) granules. This 18-kDa protein co-migrated with rat brain synaptic vesicle VAMP-2. Tetanus toxin (TeTx) light chain caused complete cleavage of this protein, which was prevented by the addition of EDTA. This toxin also inhibited in a dose-dependent manner Ca(2+)-stimulated enzyme secretion by up to approximately 30% in streptolysin O-permeabilized acini. This effect of TeTx on secretion was prevented by the zinc endopeptidase inhibitor captopril or by boiling the toxin. Incomplete inhibition of secretion by TeTx may suggest that TeTx-insensitive or VAMP-2-independent mechanisms also regulate exocytosis. In support, TeTx light chain was without effect on Rab3AL (effector domain peptide of Rab3)-mediated potentiation of Ca(2+)-stimulated secretion. These results indicate that a TeTx-sensitive VAMP-2-like protein on zymogen granules participates in Ca(2+)-regulated pancreatic enzyme secretion and that undefined Rab3AL-activated mechanisms may act independently to regulate exocytosis.
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PMID:Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion. 751 31

Botulinum neurotoxin C1 inhibited Ca(2+)-evoked norepinephrine secretion from digitonin-permeabilized PC12 cells. The inhibition by the neurotoxin was dependent on the presence of Zn2+ added exogenously. This zinc-dependent inhibition was neutralized by monoclonal antibodies that recognize the sites close to the putative zinc-binding motif in the light chain. The neurotoxin was found to have an endopeptidase activity toward small peptide, substance P. The presence of exogenous Zn2+ was also indispensable to the full expression of this endopeptidase activity. Thus both the inhibition of neurotransmitter release by the C1 neurotoxin and its endopeptidase activity are dependent on exogenous Zn2+, which suggests a strong link between the two activities.
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PMID:Exogenous zinc ion is required for inhibitory activity of botulinum neurotoxin C1 against norepinephrine release and its endopeptidase activity toward substance P. 751 76

Infective larvae of a pathogenic nematode of humans, Strongyloides stercoralis, release a potent zinc endopeptidase activity which has a broad substrate specificity for constituents of extracellular dermal matrix, including elastin. Specific inhibitors of zinc endopeptidases prevent the penetration of mammalian skin by S. stercoralis larvae. We now report the molecular size and isoelectric point of the S. stercoralis zinc endopeptidase at 40 kDa, pI 5 by zymogram analysis. The activity was not influenced by incubation with beta-mercaptoethanol at 22 degrees C, but was inactivated by incubation at 100 degrees C for 2 min. The enzyme, which we term Ss40, is immunogenic and stimulates humoral IgG antibodies during infection of humans; the activity was immunoprecipitable from ES with pooled infection sera. In addition, a HPLC-enriched Ss40 preparation stimulated the release of histamine from peripheral blood leukocytes of S. stercoralis-infected persons, suggesting that Ss40 is allergenic in humans.
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PMID:Ss40: the zinc endopeptidase secreted by infective larvae of Strongyloides stercoralis. 752 15

A secreted dibasic cleaving peptidase capable of converting dynorphins into Leu-enkephalin-Arg6 was purified from the medium of EL-4 mouse thymoma cells. The enzyme is a novel metalloendopeptidase with a neutral pH optimum (6.9) and a molecular weight of approximately 130 000. The dibasic cleaving enzyme was completely inhibited in the presence of 20-50 mM amine buffers, 0.1 mM EDTA, 0.5 mM 1,10-phenanthroline, 0.5 mM N-ethylmaleimide, and 1mM DTNB. Unlike the Kex2 family of proteases, Ca2+ did not activate the endopeptidase, but high concentrations (1 mM) of metal ions such as Cu2+, Ni2+, Zn2+, and Co2+ completely inhibited the enzyme. Inhibition was not seen with 0.2 mM TLCK, 1 mM DTT, and 1 mM PMSF. The enzyme will cleave Arg-Arg and Arg-Lys bonds, but not Lys-Arg or Lys-Lys bonds in identical environments, and no aminopeptidase or carboxypeptidase activity was seen. The size of the substrate does not seem to be a determining factor, since dynorphin A(1-12) is cleaved at a rate similar to prodynorphin B(228-256) containing 29 amino acids. The identity of the residues on either side of the cleavage site influences the rate of processing, as noted by different rates of cleavage for the same size peptides dynorphin A(1-13) vs dynorphin A(1-9) vs beta-neoendorphin. The presence of proline in the P3' (alpha-neoendorphin), P4' (dynorphin A(1-11)), or P5' (bovine adrenal medulla dodecapeptide) position does not prevent cleavage, but neurotensin and its (1-11) fragment containing both P2 and P2' proline residues are not cleaved.
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PMID:Purification and characterization of a secreted arginine-specific dibasic cleaving enzyme from EL-4 cells. 754 86

Tetanus and botulinum neurotoxins are produced by several Clostridia and cause the paralytic syndromes of tetanus and botulism by blocking neurotransmitter release at central and peripheral synapses, respectively. They consist of two disulfide-linked polypeptides: H (100 kDa) is responsible for neurospecific binding and cell penetration of L (50 kDa), a zinc-endopeptidase specific for three protein subunits of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxin serotypes B, D, F and G cleave at single sites, which differ for each neurotoxin, VAMP/synaptobrevin, a membrane protein of the synaptic vesicles. Botulinum A and E neurotoxins cleave SNAP-25, a protein of the presynaptic membrane, at two different carboxyl-terminal peptide bonds. Serotype C cleaves specifically syntaxin, another protein of the nerve plasmalemma. The target specificity of these metallo-proteinases relies on a double recognition of their substrates based on interactions with the cleavage site and with a non-contiguous segment that contains a structural motif common to VAMP, SNAP-25 and syntaxin.
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PMID:The metallo-proteinase activity of tetanus and botulism neurotoxins. 758 Dec 98

1. We have examined several phosphorus-containing peptides as potential mixed inhibitors of two neurotensin-degrading zinc metallopeptidases, endopeptidase 3.4.24.15 and endopeptidase 3.4.24.16. 2. Among a series of 13 phosphonamide peptides, N-(2-(2-naphtyl)ethylphosphonyl-glycyl-prolyl-norleucine (phosphodiepryl 08) was found to inhibit potently the hydrolysis of neurotensin by purified endopeptidase 3.4.24.15 and 3.4.24.16 with an identical Ki value of 0.4 nM. 3. Phosphodiepryl 08 displayed a strong selectivity towards the two peptidases since it failed to inhibit several other zinc-containing peptidases such as endopeptidase 3.4.24.11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase and carboxypeptidases A and B. 4. The protective effect of phosphodiepryl 08 on neurotensin degradation was examined in vitro and in vivo in central and peripheral bioassays. 5. Phosphodiepryl 08 virtually abolished neurotensin degradation by 4-day-old plated pure cultured neurones from mouse embryos and greatly potentiated neurotensin-induced antinociception in the mouse hot plate test. 6. In the periphery, phosphodiepryl 08 inhibited neurotensin degradation by membranes prepared from isolated longitudinal smooth muscle of guinea-pig ileum and greatly potentiated the neurotensin-induced contraction of the same longitudinal smooth muscle preparation. 7. Our study indicates that phosphodiepryl 08 behaves as a potent and selective mixed inhibitor of endopeptidase 3.4.24.15 and 3.4.24.16 and can be used as a powerful agent to prevent neurotensin degradation, in vitro and in vivo, in central and peripheral assays.
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PMID:Phosphorus-containing peptides as mixed inhibitors of endopeptidase 3.4.24.15 and 3.4.24.16: effect on neurotensin degradation in vitro and in vivo. 758 3

We have isolated by immunological screening of a lambda ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase 3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis. We have transiently transfected COS-7 cells with pcDNA3 containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical, and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.
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PMID:Molecular cloning and expression of rat brain endopeptidase 3.4.24.16. 759 86

Endopeptidase-24.11, which is identical with the common acute lymphoblastic leukaemia antigen CD10 (CALLA), is a cell surface Zn2+ metalloprotease that regulates peptide-induced responses in different tissues, including the nervous and immune systems. In the peripheral nervous system, high levels of the enzyme are present in all neonatal and early postnatal Schwann cells, while as myelination proceeds it is gradually suppressed in the majority of cells that form myelin but retained in non-myelin-forming cells in the adult animal. In the present study we have investigated the effects of transection, crush and regeneration of the adult rat sciatic nerve on the expression of the endopeptidase by Schwann cells in situ. Endopeptidase-24.11 was monitored by immunocytochemistry using the monoclonal anti-endopeptidase antibody 23B11. For comparison, a parallel study was carried out with a monoclonal antibody directed against the rat nerve growth factor receptor. We found that (i) all Schwann cells of the distal segment re-expressed endopeptidase-24.11 as early as 4 days after axotomy, the level of immunostaining reaching a maximum after 2 weeks, (ii) axonal regeneration repressed Schwann cell expression of endopeptidase-24.11, and (iii) the induction of the nerve growth factor receptor followed a similar pattern to that of endopeptidase-24.11 in the transected and crushed nerve. Enzymatic amplification of endopeptidase-24.11 cDNA from normal and axotomized adult rat sciatic nerve confirmed the expression of endopeptidase-24.11 in these tissues. Our results show that the expression of endopeptidase-24.11 in Schwann cells, as is the case with the nerve growth factor receptor, is induced by the loss of the normal axon-Schwann cell contact. The significant increase in the expression of endopeptidase-24.11 by Schwann cells after axonal damage suggests that the enzyme could play a role in axonal regeneration.
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PMID:Expression of endopeptidase-24.11 (common acute lymphoblastic leukaemia antigen CD10) in the sciatic nerve of the adult rat after lesion and during regeneration. 761 30


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