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)

To investigate the role of neuropeptides in allergic inflammation, we examined the effect of peptides on eosinophil chemotaxis. Eosinophils were purified from the blood of allergic and normal subjects using a discontinuous Percoll density gradients. Chemotaxis was induced by platelet-activating factor (PAF) and leukotriene B4, and was assayed by a modified Boyden's chamber technique. Four neuropeptides were examined in this study: substance P (SP), neurokinin A, calcitonin gene-related peptide (CGRP), and cholecystokinin octapeptide. Peptides alone (10 nM to 10 microM) were not chemotactic for eosinophils. However, when eosinophils were pre-treated with peptides (100 nM) at 37 degrees C for 30 min, chemotactic response to PAF (10 nM) was significantly enhanced (p < 0.01) in allergic subjects; % control by SP, neurokinin A, CGRP and cholecystokinin octapeptide was 269 +/- 42, 243 +/- 32, 227 +/- 21, and 251 +/- 42, respectively (n = 8). Similar results were obtained in leukotriene B4-induced eosinophil chemotaxis. In contrast, no enhancement was observed in normal subjects. Potentiating effect of SP and CGRP on PAF-induced eosinophil chemotaxis in allergic subjects was significantly attenuated by SP antagonist [D-Pro2,D-Trp7,9]-SP and human CGRP (8-37) receptor antagonist, respectively. Neutral endopeptidase inhibitors (phosphoramidon, leupeptin, and bestatin) failed to significantly augment the PAF-induced eosinophil chemotaxis when the cells were pretreated with various peptides and neutral endopeptidase inhibitors. The C-terminal fragment of SP (SP6-11) had an effect similar to that of the intact SP molecule, whereas no potentiating effect by the N-terminal of SP (SP1-9) was observed. These results suggest that neuropeptides may play a significant role in eosinophil infiltration by priming cells in allergic inflammation.
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PMID:Neuropeptides modulate human eosinophil chemotaxis. 138 21

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.
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PMID:Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells. 226 8

The participation of a serine endopeptidase, previously shown to be involved in endogenous cholecystokinin inactivation [Rose, Camus and Schwartz (1989) Neuroscience 29, 583-594], in the hydrolysis of various exogenous cholecystokinin peptides was studied with slices from rat cerebral cortex. In order to protect intermediate fragments from further degradation and mimick experimental conditions in this previous study, most experiments were performed in the presence of Thiorphan, an enkephalinase inhibitor, and bestatin, an aminopeptidase inhibitor, which did not significantly affect the rate of cholecystokinin-8 hydrolysis. All peptide fragments formed after incubation of cholecystokinin-8, non-sulphated cholecystokinin-8, cholecystokinin-6, cholecystokinin-5, cholecystokinin-4 or Asp-Tyr-Met-Gly-Trp were identified by isocratic high-performance liquid chromatography in several systems, fluorescence spectra and/or amino acid analysis. When identified, the appearing fragments were quantified by u.v. spectrophotometry and found to fully account for the substrate disappearance. The hydrolysis rate was higher for short cholecystokinin peptides than for the octapeptide and was, in all cases, diminished by 30-50% in the presence of diisopropyl fluorophosphate, a serine peptidase inhibitor. One of the main hydrolysis products of cholecystokinin-8, or its non-sulphated analogue, was cholecystokinin-5, whose formation was impaired in the presence of diisopropyl fluorophosphate. Cholecystokinin-5 itself was apparently a substrate for a serine peptidase leading to the formation of the tripeptide Gly-Trp-Met, later cleaved into Trp-Met and Trp. Hence a serine endopeptidase(s) appears to be responsible for cleavage of the two peptides bonds of the cholecystokinin-8 molecule where the carboxyl group is donated by a methionine residue.2+n addition,
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PMID:Role of a serine endopeptidase in the hydrolysis of exogenous cholecystokinin by brain slices. 266 53

The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.
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PMID:Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides. 273 61

Endogenous cholecystokinin immunoreactivity released by depolarization of slices of rat cerebral cortex undergoes extensive degradation (85% of released immunoreactivity) before reaching the incubation medium. In order to identify the responsible peptidases, a large number of inhibitors of the four catalytic classes were tested for their protective effects. Inhibitors of metallopeptidases (bestatin, amastatin, puromycin, Thiorphan, captopril, o-phenantroline), thiol-peptidases, (leupeptin, antipain, p-hydroxymercuribenzoate) or carboxyl-peptidases (pepstatin) had generally low if any protective effect. By contrast, several serine peptidase inhibitors, i.e. diisopropyl-fluorophosphate, phenylmethylsulphonylfluoride or the chloromethylketone Ala-Ala-Pro-Val-CH2Cl, doubled the recovery of cholecystokinin immunoreactivity and the effect was amplified in the co-presence of bestatin, an aminopeptidase inhibitor and/or Thiorphan, an enkephalinase inhibitor. High-performance liquid chromatographic analysis of the cholecystokinin immunoreactivity recovered in medium in the absence of any inhibitor showed cholecystokinin-8 to be the major peak, representing 8% of the released immunoreactive material. Non-sulphated cholecystokinin-8 represented less than 1%, indicating that desulphation does not constitute a major inactivation pathway for the endogenous octapeptide. Cholecystokinin-5 was the major clearly identifiable immunoreactive fragment, representing 9% of released immunoreactivity in the absence of inhibitors. Its formation was decreased by about 50% in the presence of either diisopropyl-fluorophosphate or bestatin and Thiorphan and abolished when they were associated, suggesting that it resulted from the actions of a serine peptidase(s) and an aminopeptidase(s). Cholecystokinin-6 (or cholecystokinin-7) was less abundant, representing 4% of the released immunoreactivity, and its level was augmented in the presence of diisopropyl-fluorophosphate. Hence a serine endopeptidase cleaving the Met3-Gly4 bond of cholecystokinin-8 may represent a major inactivating peptidase for the endogenous neuropeptide. Additional metabolic pathways not blocked by serine peptidase inhibitors and resulting in the formation of cholecystokinin-6 (or cholecystokinin-7) and, possibly, cholecystokinin-4, are also suggested by the present approach.
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PMID:Protection by serine peptidase inhibitors of endogenous cholecystokinin released from brain slices. 273 3

1. Cholecystokinin-octapeptide (CCK-OP, 10(-10)-3 x 10(-6) M) produced a concentration-dependent contractile response in guinea-pig trachea which was enhanced by both the mechanical removal of the epithelium and by indomethacin (10(-5) M), with an EC50 of 6.18 +/- 0.10 x 10(-8) M. 2. Sub-threshold concentrations of CCK-OP, which did not alter the resting tone of the smooth muscle, did not alter responses produced to electrical field stimulation (EFS) or to vagal nerve stimulation in an intact tracheal tube preparation. Atropine (2 x 10(-6) M) did not alter the concentration-response curve to CCK-OP, indicating that CCK-OP contraction is not mediated by cholinergic mechanisms. 3. The inhibition of neutral endopeptidase (endopeptidase-24.11) by phosphoramidon (10(-5) M) gave a leftward shift in the CCK-OP concentration-response curve in tissues with intact epithelium obtained from normal animals, but had no effect in tissues denuded of epithelium or in tissues obtained from animals which had been actively sensitized and challenged with ovalbumin (OA). 4. CCK-OP-induced contractile responses were antagonized by the CCK-receptor antagonists dibutyryl cyclic guanosine monophosphate (pA2 = 4.3) and L-364,718 (pA2 = 9.6). 5. CCK-OP induced bronchoconstriction in large, but not small, human airways and was antagonized by the CCK-receptor antagonist L-364,718. CCK-OP had no effect on cholinergic neural responses elicited by EFS in human airways.
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PMID:Cholecystokinin-octapeptide constricts guinea-pig and human airways. 275 37

Peptidases present in central nervous system (CNS) synaptic membranes, hydrolyze the neuroactive peptide cholecystokinin-octapeptide (CCK-8; Asp-Tyr-SO3H-Met-Gly-Trp-Met-Asp-Phe-NH2). In order to determine the pathway of degradation, synthetic CCK-8 was incubated at 37 degrees C with purified synaptic membranes; at various intervals reaction samples were removed from the reaction mixture and analysed by high-performance liquid chromatography to identify and quantify the peptide fragments. The results indicate an initial endopeptidase cleavage at the Met-Gly bond producing CCK-5 (Gly-Trp-Met-Asp-Phe-NH2). The carboxyl-terminal pentapeptide is further proteolysed to CCK-4 (Trp-Met-Asp-Phe-NH2) by a puromycin-sensitive aminopeptidase and to CCK-3 (Met-Asp-Phe-NH2) and Gly-Trp by an endopeptidase action. CCK-3 and CCK-2 appear to be relatively stable end-products. Moreover, these proteolytic fragments are shown to bind to the CCK receptor in brain with varying potencies.
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PMID:Products of cholecystokinin (CCK)-octapeptide proteolysis interact with central CCK receptors. 298 58

A serine endopeptidase was characterized as a major inactivating enzyme for endogenous cholecystokinin (CCK) in brain. CCK-8 released by depolarization of slices of rat cerebral cortex, as measured by its immunoreactivity (CCK-ir), undergoes extensive degradation (approximately 85% of the amount released) before reaching the incubation medium. However, recovery of CCK-ir is enhanced up to 3-fold in the presence of serine-alkylating reagents (i.e., phenylmethylsulfonyl fluoride) as well as selected active site-directed inactivators (i.e., peptide chloromethyl ketones) or transition-state inhibitors (i.e., peptide boronic acids) of serine peptidases. Among these compounds, elastase inhibitors were the most potent protecting agents, whereas trypsin or chymotrypsin inhibitors were ineffective. HPLC analysis of endogenous CCK-ir recovered in media of depolarized slices indicated that endogenous CCK-5 [CCK-(29-33)-pentapeptide] was the most abundant fragment and that its formation was strongly decreased in the presence of an elastase inhibitor. HPLC analysis of fragments formed upon incubation of exogenous CCK-8 [CCK-(26-33)-octapeptide] with brain slices showed CCK-5, Gly-Trp-Met, and Trp-Met to be major metabolites of CCK-8 whose formation was prevented or at least diminished in the presence of the elastase inhibitor. It is concluded that there is an elastase-like serine endopeptidase in brain that cleaves the two peptide bonds of CCK-8 where the carboxyl group is donated by a methionine residue and constitutes a major inactivation ectoenzyme for the neuropeptide.
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PMID:A serine peptidase responsible for the inactivation of endogenous cholecystokinin in brain. 318 27

The purpose of this investigation is to examine the metabolism and inactivation of human and porcine gastrin 17 (nonsulfated) (G-17) and cholecystokinin octapeptide (sulfated) (CCK-8) by gastric endopeptidase 24.11. Endopeptidase 24.11 was isolated by immunoaffinity chromatography using a monoclonal antibody to the kidney enzyme. Peptides were incubated with endopeptidase 24.11. The digests were either fractionated by reverse-phase high-pressure liquid chromatography and the products identified by amino acid analysis or they were used for bioassays. Digests of human gastrin were assayed for stimulation of acid secretion in the anesthetized rat, and cholecystokinin digests were assayed for the stimulation of amylase secretion from isolated rat pancreatic acini. Human G-17 was degraded by cleavage of the Trp4-Leu5,Ala11-Tyr12,Gly13-Trp14,Trp14 -Met15, and Asp16-Phe17-NH2 bonds, and the fragments (1-16), (1-13), (1-11), (1-4), (5-11), (5-13), (12-13), (12-14), (14-16), and (17-NH2) were identified. Porcine G-17 was degraded by hydrolysis of the Ala11-Tyr12,Gly13-Trp14, and Asp16-Phe17-NH2 bonds producing (1-16), (1-13), (1-11), (12-13), (14-16), and (17-NH2) fragments. CCK-8 was degraded by hydrolysis of the Gly4-Trp5 and Asp7-Phe8-NH2 bonds, and the fragments (1-7), (1-4), (5-7), (5-8), and (8-NH2) were identified. There was a progressive decline in the biological activity with incubation time.
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PMID:Metabolism of gastrin and cholecystokinin by endopeptidase 24.11 from the pig stomach. 318 56

Cholecystokinin octapeptide and the C-terminal tetrapeptide are hydrolysed by a highly purified preparation of "enkephalinase" (EC 3.4.24.11). In both cases the Asp-PheNH2 bond is hydrolysed and the Gly4-Trp5 bond of the octapeptide is also cleaved, though more slowly. Evaluated from the appearance of Phe-NH2, the Km for the hydrolysis of the octapeptide by the purified peptidase is 57 microM and that for the tetrapeptide 65 microM. The apparent affinities of these peptides for the enzyme in striatal membranes are similar. The importance of this hydrolysis in the inactivation of endogenous cholecystokinin was assessed by studying the fate of cholecystokinin immunoreactivity released from slices of rat cerebral cortex and striatum by depolarization with potassium. In the absence of any peptidase inhibitor only 16% of the peptide released from the tissue was recovered in immunoreactive form in the medium, indicating that endogenous cholecystokinin octapeptide is, like other neuropeptides, rapidly and extensively hydrolysed following release. Selective inhibition of "enkephalinase" by Thiorphan (DL-3-mercapto-2-benzylpropanoyl glycine) did not significantly alter the recovery from slices of cerebral cortex and had only a very slight effect in the case of striatal slices. This suggests that, while cholecystokinin octapeptide is a substrate for "enkephalinase", this enzyme plays a less important (if any) role in the inactivation of endogenous cholecystokinin than for the opioid peptides.
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PMID:Assessment of the role of "enkephalinase" in cholecystokinin inactivation. 389 59


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