Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.
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PMID:Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus. 204 32

Recent studies have suggested a role for Zn2+, distinct from that of Ca2+, in the subcellular distribution and activation of protein kinase C (PKC). Here we show that Zn2+ is required for a cellular response mediated by PKC, the rapid loss of expression of a human B cell receptor MER, detected by rosetting with mouse erythrocytes. Zn2+, in the presence of the Zn2+ ionophore pyrithione, caused rapid inhibition of MER rosetting at concentrations which induce the translocation and activation of PKC. This required cellular uptake of Zn2+ and was blocked by 1,10-phenanthroline and TPEN which chelate Zn2+ but not Ca2+. Gold, a metal with similar properties, also induced translocation of PKC and inhibition of MER. By contrast, Ca2+ ionophores A23187 and ionomycin, which induce a different pathway of translocation of PKC, had no effect on MER. Phenanthroline and TPEN also blocked the inhibition of MER induced by the PKC activators phorbol ester and sodium fluoride, suggesting that endogenous cellular Zn2+ is required. We propose that some cellular actions of PKC require a Zn(2+)-dependent event and that these may be a target for gold during chrysotherapy in rheumatoid arthritis.
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PMID:Role for zinc in a cellular response mediated by protein kinase C in human B lymphocytes. 205 69

In this study, plasma levels of magnesium, calcium, zinc and copper were simultaneously determined in pregnancies complicated by either abortion, intrauterine growth retardation (IUGR), diabetes or EPH (edema, proteinuria, hypertension) gestosis. The levels of the four cations in non-pregnant women and in healthy, pregnant women were also determined. Compared with controls, a significant decrease in magnesium, with increase of the Ca/Mg ratio, was found in spontaneous abortions, but not when patients had a successful continuation of pregnancy. In EPH gestosis, total calcium was reduced, with a significant decrease of the plasma Ca/Mg ratio. A slight, but significant, increase in plasma zinc was observed in women affected by either diabetes or IUGR, probably as a result of reduced zinc uptake by the fetus. In addition, higher copper levels were found in the pathologies studied, with the exception of missed abortions. The possible role of an altered Ca/Mg ratio homeostasis in relation to gestational pathologies is discussed.
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PMID:Maternal plasma concentrations of magnesium, calcium, zinc and copper in normal and pathological pregnancies. 227 Apr 73

This report summarizes the recent rapid development of research on neutral endopeptidase 24.11 (enkephalinase; NEP) and on two other metalloenzymes, meprin and endopeptidase 24.15. NEP cleaves a variety of active peptides, including enkephalins, at the amino side of hydrophobic amino acids. The cDNA for human, rat, and rabbit NEP has been cloned and the deduced protein sequences revealed a high degree of homology (93-94%). Site-directed mutagenesis proved that an active site glutamic acid is involved in catalysis and two active site histidines are responsible for binding the zinc cofactor. Although NEP was originally discovered in the kidney, it is widely distributed in the body including specific structures in the central nervous system, lung, male genital tract, and intestine and in neutrophils, fibroblasts, and epithelial cells. In tissues and cells NEP is bound to plasma membrane through a hydrophobic membrane-spanning domain near the NH2 terminus, but it is present in soluble form in urine and blood. In addition to enkephalins, NEP cleaves kinins, chemotactic peptide, atrial natriuretic factor (ANF), and substance P in vivo. NEP in the lung is a major inactivator of substance P, which constricts the airway smooth muscles. Because of the possible involvement of NEP in the metabolism of opioid peptides and the cardiac hormone ANF, orally active inhibitors have been synthesized. Compounds that inhibit both aminopeptidase and NEP were reported to prolong the analgesic effects of enkephalins. Other inhibitors given per os prolonged the renal effects of exogenous ANF. A newly synthesized specific inhibitor of NEP was also active in animal experiments as an analgesic. Studies on the structure and function of NEP should lead to further development of therapeutically applicable inhibitors.
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PMID:Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. 252 10

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein that has been identified recently as the neutral endopeptidase 24.11 [NEP (EC 3.4.24.11)]. Herein, we characterize the organization of the human CALLA/NEP gene and show that it spans more than 80 kilobases (kb) and is composed of 24 exons. Exons 1 and 2 encode 5' untranslated sequences; exon 3 [170 base pairs (bp)] encodes the initiation codon and transmembrane and cytoplasmic domain; 20 short exons (exons 4-23), ranging in size from 36 to 162 bp, encode most of the extracellular portion of the enzyme; and exon 24 (approximately 3400 bp) encodes the COOH-terminal 32 amino acids of the protein and contains the entire 3' untranslated region (UTR). Of note, the pentapeptide sequence (His-Glu-Ile-Thr-His) associated with metalloprotease zinc binding and substrate catalysis is encoded within a single exon (exon 19). Three types of CALLA/NEP cDNAs have been identified: these clones contain 5' UTR sequences differing from one another upstream of exon 3. These human 5' sequences are homologous to those found in rat brain and rabbit kidney NEP cDNAs. The three human CALLA cDNA types result from alternative splicing of exons 1, 2a, or 2b to the common exon 3. Moreover, exons 2a and 2b share the same 5' sequence but differ from each other by the use of two distinct donor splice sites 171 bp apart in the gene. The substantial conservation of 5' untranslated sequences among species and the existence of 5' alternative splicing suggest that CALLA gene expression may be differentially controlled in a tissue-specific and/or developmentally regulated fashion.
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PMID:Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions. 252 30

Peptide retro-inverso modification was applied to the complete hydroxamate inhibitors of the three zinc metallopeptidases (neutral endopeptidase 24-11 (NEP, EC 3.4.24.11), aminopeptidase N (APN, EC 3.4.11.2), and a dipeptidylaminopeptidase (DAP) involved in the in vitro enkephalin degradation by brain tissues. Compounds corresponding to the general formula RN(OH)CO(CH2)nCH(CH2Ph)NHCOCH(R')COOH (n = 0, 1) were synthesized. In the first series of inhibitors (n = 0), the "retro-inverso" modification induced a large decrease in inhibitory potency for NEP as compared to that of the parent compounds. In contrast, the presence of a methylene group between the hydroxamate and CH alpha in the second series (n = 1) led to derivatives with inhibitory potencies in the nanomolar range, similar to their analogues with a natural amide bond. On the other hand, the retro-inverso modification led to a slight improvement in the inhibition of DAP and APN, in the first series of inhibitors, while the inverse result occurred in the second series. Thus, compounds containing an alpha-amino acid moiety in P'1 position behave as weak inhibitors of the three enzymes, with IC50 values in the micromolar range, and compounds bearing a beta-amino acid moiety in the same position are more specific than the parent compounds for NEP inhibition.
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PMID:Retro-inverso concept applied to the complete inhibitors of enkephalin-degrading enzymes. 290 Aug 98

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His-142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.
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PMID:Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. Histidine residues 583 and 587 are essential for catalysis. 316 86

Adipocytic-cytosolic non-receptor protein tyrosine kinase (CytPTK) when activated can substitute for the insulin receptor tyrosine kinase (InsRTK), in manifesting several insulin effects in insulin-receptor independent fashion. Our aims here were to utilize PolyGlu4Tyr, a good experimental exogenous substrate for protein tyrosine kinases (PTKs) in general, for studying qualitative and quantitative parameters of CytPTKs extracted from different tissue cytosols. At the same time, we would search for a unique specific marker specifically characterizing CytPTKs. High speed supernatants of spleen, thymus, smooth muscle, lung and kidney were found to be rich in CytPTK activities. Their specific activities being 6- to 13-fold that of liver or adipose cytosols. Brain, testis and adrenal cytosols were an intermediate source of CytPTK activity, whereas CytPTK activity of heart and skeletal muscle was low. It was also evaluated that the capacity of the cytosol to phosphorylate PolyGlu4Tyr is 15-50% that of the non-stimulated Triton X-100 extractable plasma membrane PTKs. Fractionation of the cytosols on superose 12 column revealed several CytPTKs within the same tissue, their peaks ranging between 30 and 450 kDa. Immunoblotting analysis showed Fyn and Lyn were present in most tissue cytosols. Upon immunoprecipitation, however, with anti-Fyn or anti-Lyn, negligible amounts (< 2%) of the total cellular CytPTK were precipitated. Thus, these general markers of CytPTKs comprise only a minor proportion of the total intracellular PolyGlu4Tyr phosphorylating capacity. To see whether a specific marker for CytPTK could be detected, we also examined the requirement of CytPTKs for divalent ions, their preferred phosphate donor and their sensitivity to inhibition by known PTK inhibitors. We found that the order of reactivity with divalent cations was Co2+ > Mn2+ > Mg2+, while Zn2+ and Ca2+ did not support CytPTK activity. The best phosphate donor was ATP (ED50 = 5 microM), but other nucleoside 3-phosphates could substitute for ATP at high concentrations. With respect to these parameters, no basic difference exists between cytosolic and plasma-membrane PTKs. The PTK inhibitors, genestein and quercetin, inhibited both cytosolic and membranal PTKs at micromolar concentrations. In contrast, staurosporine was a potent inhibitor of CytPTKs (IC50 5-20 nM) and a poor inhibitor of membranal PTKs (IC50 10-40 microM). One of the conclusions we can draw from this study is that tissue cytosols contain PolyGlu4Tyr phosphorylating capacity in quantities greater than previously assumed and that the low level of phosphotyrosine found in cells is not the result of limited intracellular levels of CytPTKs.
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PMID:Non-receptor cytosolic protein tyrosine kinases from various rat tissues. 749 84

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram-positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C-terminus and at selected sites in the N-terminal domain. Four amino acids in the C-terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000-fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10-fold reduction in MHC class II affinity. The combination of these mutations in the N- and C-terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II-dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab-SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A. 754 84

Endopeptidase-24.11 (neutral endopeptidase, neprilysin, 'enkephalinase', EC 3.4.24.11) and endopeptidase-24.18 (endopeptidase-2, meprin, EC 3.4.24.18) are cell-surface zinc-dependent metallo-endopeptidases able to cleave a variety of bioactive peptides including growth factors. We report the first study of the cellular and tissue distribution of both enzymes and of the mRNA for NEP during embryonic development in the rat. Endopeptidase-24.11 protein was first detected at E10 in the lining of the gut and, at E12, the enzyme was present on the notochord, medial and lateral nasal processes, otocyst, mesonephros, heart and neuroepithelium. In contrast, at this time endopeptidase-24.18 was present only on the apical surface of the neuroepithelial cells. By E14 and E16, NEP was also detected in a wide range of craniofacial structures, notably the palatal mesenchyme, the choroid plexus, tongue and perichondrium. The distribution of endopeptidase-24.18 at these stages was restricted to the inner ear, the nasal conchae, and ependymal layer of the brain ventricles and the choroid plexus. Although endopeptidase-24.11 had been detectable in the craniofacial vasculature at E12 and E14, this was no longer apparent at E16. Significantly, the distribution of endopeptidase-24.11 mRNA closely matched the immunolocalization of the protein at all stages investigated. In order to explore the functional role of these enzymes, inhibition studies were carried out using two selective inhibitors of endopeptidase-24.11, phosphoramidon and thiorphan. E9.5 and E10.5 embryos exposed to either inhibitor displayed a characteristic, asymmetric abnormality consisting of a spherical swelling, possibly associated with a haematoma, predominantly on the left side of the prosencephalon, and the severity of this defect appeared to be a dose-dependent phenomenon. This study suggests that these enzymes play previously unrecognized roles during mammalian embryonic development.
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PMID:Distribution of, and a putative role for, the cell-surface neutral metallo-endopeptidases during mammalian craniofacial development. 772 May 64


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