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)

Endopeptidase-24.11 (EC 3.4.24.11), purified to homogeneity from pig kidney, was shown to hydrolyse a wide range of neuropeptides, including enkephalins, tachykinins, bradykinin, neurotensin, luliberin and cholecystokinin. The sites of hydrolysis of peptides were identified, indicating that the primary specificity is consistent with hydrolysis occurring at bonds involving the amino group of hydrophobic amino acid residues. Of the substrates tested, the amidated peptide substance P is hydrolysed the most efficiently (Km = 31.9 microM; kcat. = 5062 min-1). A free alpha-carboxy group at the C-terminus of a peptide substrate is therefore not essential for efficient hydrolysis by the endopeptidase. A large variation in kcat./Km values was observed among the peptide substrates studied, a finding that reflects a significant influence of amino acid residues, remote from the scissile bond, on the efficiency of hydrolysis. These subsite interactions between peptide substrate and enzyme thus confer some degree of functional specificity on the endopeptidase. The inhibition of endopeptidase-24.11 by several compounds was compared with that of pig kidney peptidyldipeptidase A (EC 3.4.15.1). Of the inhibitors examined, only N-[1(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate inhibited endopeptidase-24.11 but not peptidyldipeptidase. Captopril (D-3-mercapto-2-methylpropanoyl-L-proline), Teprotide (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and MK422 [N-[(S)-1-carboxy-3-phenylpropyl]-L-Ala-L-Pro] were highly selective as inhibitors of peptidyldipeptidase. Although not wholly specific, phosphoramidon was a more potent inhibitor of endopeptidase-24.11 than were any of the synthetic compounds tested.
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PMID:The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. 614 47

Neuroleptic drugs have been shown to affect the level and messenger ribonucleic acid of specific neuropeptides. The effect of subchronically administered neuroleptics on neuropeptide metabolism, however, has not been systematically characterized. In the present study, the effect of neuroleptics and other dopaminergic compounds on substance P (SP), cholecystokinin and met-enkephalin degradation was determined on intact, regional, rat brain slices. After 7-day administration of haloperidol (1 mg/kg) or chlorpromazine (20 mg/kg), SP degradation was decreased in caudate-putamen and nucleus accumbens. After administration of the dopaminergic agonist apomorphine (5 mg/kg, b.i.d.), SP degradation was increased in the nucleus accumbens. The dopamine D2-receptor antagonist sulpiride (100 mg/kg, b.i.d.) produced no effect on SP degradation. Met-enkephalin degradation was decreased after haloperidol administration in both frontal cortex and caudate-putamen and unaffected by apomorphine administration. The metabolism of cholecystokinin was not affected by neuroleptic treatment. Studies performed with specific peptidase inhibitors suggested that neutral endopeptidase 24.11, metalloendopeptidase 24.15 and aminopeptidases degrade SP on caudate-putamen and nucleus accumbens slices. Therefore, alterations in these peptidases may be responsible for the change noted in SP degradation after dopaminergic compound administration. These metabolic changes noted after neuroleptic administration may therefore contribute to neuroleptic-induced alterations in regional peptide levels.
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PMID:Neuropeptide metabolism on intact, regional brain slices: effect of dopaminergic agents on substance P, cholecystokinin and Met-enkephalin degradation. 754 72

Cholecystokinin (CCK) and bombesin stimulate dose-dependent amylase secretion from dispersed pancreatic acini. To establish whether cellular proteases can reduce secretion by degrading these regulatory peptides, the effect of protease inhibition on CCK and bombesin stimulated amylase secretion was investigated. A spectrum of protease inhibitors, including bacitracin, phenylmethylsulfonylfluoride, captopril, bestatin, phosphoramidon, and 1,10-phenanthroline, were investigated. Bacitracin (0.35 mM) increased the acinar amylase secretory response to CCK and bombesin substantially, suggesting that these two peptides are degraded by an endopeptidase from pancreatic acinar cells. In contrast, PMSF (1 mM) inhibited CCK and bombesin stimulated amylase release, suggesting a covalent interaction with this inhibitor and CCK or bombesin receptors. Other protease inhibitors either had minimal or no effects on acinar cell secretion. These results suggest bacitracin is a valuable enzyme inhibitor that can potentiate the effect of CCK and bombesin on acinar cells. In contrast, PMSF should be avoided when using these secretagogs to study pancreatic function.
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PMID:Effect of protease inhibitors on peptide-stimulated amylase secretion from dispersed pancreatic acini. 754 17

The metabolism of Met-enkephalin and cholecystokinin (CCK) 8-(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met-enkephalin (400 microM) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met-enkephalin was high in caudate-putamen (5,160 +/- 120) and lower in nucleus accumbens (3,630 +/- 110) and frontal cortex (3,180 +/- 120). Inhibition of aminopeptidases decreased Met-enkephalin degradation (50-97% vs. control) in frontal cortex but was less effective in caudate-putamen (20-34%). Tyr-Gly-Gly and Phe-Met were recovered in caudate-putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met-enkephalin degradation in caudate-putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met-enkephalin from degradation (recovery > 95%) in caudate-putamen. CCK 8-(sulfated) degradation on slices from caudate-putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13-30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.
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PMID:Regional metabolism of Met-enkephalin and cholecystokinin on intact ratbrain slices: characterization of specific peptidases. 759 77

Neutral endopeptidase (E.C.3.4.24.11) was visualized at the ultrastructural level in the external zone of the rat median eminence by using 125I-labelled IgG of a monoclonal serum. A precise analysis of the localization of the immunolabelling, which appears in the form of individual stray silver grains, was undertaken. Among the 1,045 grains counted, 82% were localized over membrane appositions involving nerve endings only and nerve endings plus tanycytes. The difference between the real and a randomly generated population of grains was statistically significant. Our results provide morphological arguments in support of the view of a paracrine action of neuropeptides present in the median eminence especially enkephalins but possibly, substance P, angiotensin, cholecystokinin and neurotensin. These neuropeptides are known to be inactivated by neutral endopeptidase. The action of these peptides may be exerted on nerve endings (autocrine or paracrine) but an intervention on tanycytes cannot be excluded.
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PMID:Radioimmunocytochemical distribution of neutral endopeptidase (enkephalinase E.C.3.4.24.11) at the ultrastructural level in the rat median eminence. 768 59

Both the sulphated and non-sulphated forms of cholecystokinin (CCK) octapeptide are susceptible to hydrolysis by the cell-surface peptidases endopeptidase-24.11 (NEP), angiotensin converting enzyme and aminopeptidase N (AP-N). Indirect studies have previously implicated an elastase-like serine endopeptidase in CCK metabolism in brain. We have therefore compared the hydrolysis of CCK, in both sulphated and non-sulphated forms by solubilized membrane preparations from the human astrocytoma clone D384 and the neuroblastoma line SH-SY5Y. Selective peptidase inhibitors were used to elucidate the principal activities involved in CCK metabolism. In the glial cell line the hydrolysis of cholecystokinin octapeptide (CCK-8), sulphated or non-sulphated, was inhibited predominantly by the NEP inhibitor, phosphoramidon (PR). In contrast, in the neuroblastoma line, angiotensin converting enzyme (ACE) was seen to play a major role in metabolism of CCK-8 with a lesser effect attributable to NEP but with some differences between sulphated and non-sulphated forms reflecting the preference of ACE for CCK-8ns. In neither cell line was a significant effect of the serine peptidase inhibitor Dip-F seen on CCK metabolism arguing against the presence of a putative CCK-degrading serine peptidase in these cell lines. Both NEP and ACE remain as candidates for inactivation of CCK at the cell surface.
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PMID:Comparison of cholecystokinin metabolism by membrane preparations from the human astrocytoma clone D384 and the neuroblastoma line SH-SY5Y. 791 87

The human cholinergic neuroepithelioma cell line SK-N-MCIXC, which expresses high levels of cholecystokinin (CCK) mRNA and secretes intact CCK into the media, was used to examine CCK processing and metabolism. Our data provide evidence for the existence of specific candidate processing enzymes in SK-N-MCIXC cells which may be involved in processing proCCK in the brain and indicate that SK-N-MCIXC cells provide a model system for studying the regulation of these enzymes. mRNAs for the intracellular processing enzymes, prohormone convertase 1 (PC1), PC2 and furin were present in SK-N-MCIXC cells. PC1 and/or PC2 and/or furin may cleave at the dibasic amino acid pairs Arg-Arg at the C-terminal part of proCCK, and Arg-X-X-Arg at the N-terminal of the CCK-58 sequence in proCCK. The SK-N-MCIXC cell line demonstrated spontaneous and regulated release of CCK and large amounts of CCK-precursors, as measured with region specific radioimmunoassays coupled to high performance liquid chromatography. Storage granules containing glycine-extended CCK were shown in SK-N-MCIXC cells using indirect immunofluorescence. The extracellularly localized CCK-metabolizing enzyme, neutral endopeptidase 24.11 (EC 3.4.24.11), was present in membranes from both SK-N-MCIXC cells and in intact slices of rat cerebral cortex. The rat cerebral cortex is a brain region known to be rich in CCK. The SK-N-MCIXC cell line provides an in vitro model to study the regulation of CCK synthesis and metabolism in neuronal systems since it contains the storage granules, mRNA, intact peptide, and complement of enzymes necessary for biosynthesis and metabolism of CCK.
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PMID:Processing, release and metabolism of cholecystokinin in SK-N-MCIXC cells. 841 49

Cholecystokinin (CCK) may act as an endogenous anti-opioid and blockade of CCK receptors can enhance the potency and efficacy of morphine. This effect is blocked by opioid delta (delta) receptor antagonists, suggesting a tonic inhibitory action of CCK to diminish the release and/or availability of endogenous enkephalins. The present studies have further evaluated this possibility by studying the antiallodynic actions of a CCKB antagonist (L365,260) alone, or in the presence of thiorphan (a neutral endopeptidase inhibitor) in a model of peripheral neuropathy. Animals subjected to nerve injury, but not sham controls, exhibited long lasting, stable mechanical allodynia. Intrathecal (i.t.) administration of L365,260 or thiorphan alone did not alter allodynia. However, co-administration of these compounds produced a significant antiallodynic action which was antagonized by receptor selective doses of naltrindole, an opioid delta receptor antagonist. In addition, antisera to [Leu5]enkephalin, but not to [Met5]enkephalin, also blocked the antiallodynic action of thiorphan plus L365,260. These data suggest that blockade of CCKB receptors may enhance the actions or availability of endogenous [Leu5]enkephalin or a like substance which can elicit a significant antiallodynic action via opioid delta receptors when its degradation is by inhibited by thiorphan. The data suggest that delta opioids are involved in regulation of some aspects of nerve-injury induced pain.
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PMID:Antiallodynic effects of a CCKB antagonist in rats with nerve ligation injury: role of endogenous enkephalins. 889 38

Precursors of the human regulatory peptide cholecystokinin (CCK) have been expressed in Saccharomyces cerevisiae, and the post-translational processing of secreted CCK-related products analyzed. Recombinant plasmids expressing native human prepro-CCK and a hybrid molecule encompassing the prepro leader of the yeast alpha-mating pheromone fused to pro-CCK were examined. The latter construct resulted in considerably higher levels of pro-CCK secretion and was therefore analyzed in more detail. Two of the protein modifications essential for CCK bioactivity, C-terminal alpha-amidation and tyrosyl sulfation, were not detected in S. cerevisiae. Proteolytic cleavage of pro-CCK occurred C-terminally of three basic sites; (i) Arg105-Arg106 which, upon exposure to carboxypeptidase activity, leads to the production of glycine-extended CCK; (ii) Arg95 to produce CCK-8 related processing intermediates; and (iii) Lys81 resulting in CCK-22 related products. To elucidate which protease(s) are involved in these endoproteolytic cleavage events, pro-CCK was expressed in yeast mutants lacking various combinations of the Mkc7, Yap3, and Kex2 proteases. Only in S. cerevisiae strains deficient in Kex2 function was any of the above mentioned pro-CCK cleavages abolished, namely processing at the Arg105-Arg106 and Arg95 sites. This suggests that mammalian Kex2-like serine proteases may process pro-CCK at single arginine residues. Our data suggests that an as yet uncharacterized endopeptidase(s) in the S. cerevisiae secretory pathway is responsible for the lysine-specific cleavage of pro-CCK.
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PMID:Heterologous expression of human cholecystokinin in Saccharomyces cerevisiae. Evidence for a lysine-specific endopeptidase in the yeast secretory pathway. 909 3

Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.
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PMID:Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin. 1196 70


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