Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.15.1 (ACE)
18,300 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1-5 as the primary stable product, which was then degraded to GnRH1-3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCl2 and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH1-9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro9-Gly10-NH2 bond, inhibitable by TPCK, ZnCl2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1-5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.
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PMID:Characterization of membrane-associated peptidase activities expressed by endothelial cells of the ovine median eminence. 804 22

[D-Ala2,Leu5]Enkephalin was readily metabolized by membranes (40,000 g pellet) prepared from heads of the housefly, Musca domestica, with Gly3-Phe4 being the major site of cleavage. This hydrolysis was only partially inhibited (40%) by 10 microM phosphoramidon, an inhibitor of endopeptidase-24.11, but was almost totally abolished in the presence of a mixture of 10 microM phosphoramidon and 10 microM captopril, a potent inhibitor of mammalian angiotensin-converting enzyme (ACE). An assay for ACE employing Bz-Gly-His-Leu as the substrate was used to confirm the presence of an ACE-like peptidyl dipeptidase activity in fly head membranes. The peptidase had a Km of 1.91 mM for Bz-Gly-His-Leu and a pH optimum of 8.2. The activity was inhibited by 100 microM EDTA and was greatly activated by ZnCl2 but not other bivalent metal ions. Captopril, lisinopril, fosinoprilat and enalaprilat, all selective inhibitors of mammalian ACE, were also good inhibitors of the insect enzyme with IC50 values of 400 nM, 130 nM, 16 nM and 290 nM respectively. An M(r) value of around 87,000 was obtained for this enzyme from gel-filtration chromatography, indicating that the insect enzyme is similar in size to mammalian testicular ACE (M(r) = 90,000-110,000) and not the larger form of the enzyme (M(r) = 150,000-180,000) found in mammalian somatic tissues. The fly peptidyl dipeptidase was released from membranes into a soluble fraction by incubating the head membranes at 37 degrees C but not at 0 degree C, suggesting that the insect ACE-like enzyme can be solubilized from cell surfaces through the activity of a membrane-bound enzyme activity. In conclusion, we have shown the existence of a peptidyl dipeptidase in membranes from the heads of M. domestica, which has similar properties to those of mammalian ACE.
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PMID:Identification and properties of a peptidyl dipeptidase in the housefly, Musca domestica, that resembles mammalian angiotensin-converting enzyme. 819 53

Peptides function as chemical signals between cells of multicellular organisms, or different organisms, via specific receptors on target cells. Many hormones, neuromodulators, and growth factors are peptides. Because there is no known reuptake system for peptides at the nerve terminal, the biological activity of peptides in the extracellular space is regulated by enzymatic degradation and extracellular metabolism. For example, angiotensin I is processed extracellularly in the lung by angiotensin-converting enzyme (ACE; E.C. 3.4.15.1), a peptidyl dipeptidase, to form the potent vasoconstrictor hormone angiotensin II. When neuropeptides are released from neurons into the extracellular space, specific peptidases also can modulate the peptidergic signal by generating smaller, biologically active fragments via products with similar or dissimilar characteristics of the parent peptide. Therefore, receptor-binding selectivity of a released peptide hormone can be regulated by peptidases. Because peptidases may play a key role in the extracellular regulation of peptidergic signaling, alterations in peptidase activities by drugs or disease states may lead to disruptions in biological homeostasis. The subject of this article is the role of peptidases in the central nervous system in the formation of biologically active, receptor-specific peptides from peptide E, beta-endorphin, neurotensin, and cholecystokinin.
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PMID:Peptidases in the CNS: formation of biologically active, receptor-specific peptide fragments. 822 10

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate transport and hydrolysis of two enkephalin peptides, Met-enkephalin (TGGPM) and [D-Ala2]Met-enkephalinamide (TAGPM), in pulmonary epithelium. Isolated alveolar type II cells formed continuous monolayers when grown on microporous tissue culture-treated polycarbonate filters in serum-free, hormonally defined medium. Transport and hydrolysis studies of enkephalins in the monolayer system obtained after 6 days in culture, using fluorescence reversed-phase HPLC, indicate a reduced but significant degradation of enkephalins in the alveolar epithelium compared to most other epithelia previously reported. Aminopeptidases and dipeptidyl carboxypeptidase represent two major hydrolytic enzymes for TGGPM, as indicated by the formation of the degradative products Tyr and Tyr-Gly-Gly, while dipeptidyl peptidase, which is responsible for the formation of Tyr-Gly, contributes much less. The enkephalinase inhibitor thiorphan failed to prevent the hydrolysis of TGGPM whereas the enkephalin analog TAGPM was relatively resistant to enzymatic cleavage. The rate of enkephalin transport across the alveolar epithelium was directly proportional to drug concentration and occurred irrespective of transport direction, suggesting passive diffusion as the major mechanism for transepithelial transport. Agents that affect paracellular transport pathways, e.g., EGTA and the calcium ionophore A-23187, greatly promoted the transport rate. The ionophore at high doses, in addition to promoting tight junction permeability, also caused cellular damage associated with a sustained rise in intracellular calcium levels, as indicated by nuclear propidium iodide fluorescence. The cultured monolayer of alveolar epithelium may be used to study pulmonary drug absorption, degradation, and toxicity.
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PMID:Transport and hydrolysis of enkephalins in cultured alveolar epithelial monolayers. 829 Apr 82

We investigated the release of carboxypeptidase M (CPM), neutral endopeptidase 24.11 (enkephalinase, NEP), and angiotensin I converting enzyme (kininase II, ACE) and their contribution to bradykinin metabolism in the rat lung. The P3, membrane-enriched fraction of the homogenized lung was rich in all three peptidases. The activities of CPM and NEP were high in bronchoalveolar lavage fluid but lower in alveolar macrophages indicating that they originate from other cells present on the alveolar surface. In situ perfusion of rat lung with buffer that contained either deoxycholate or melittin or compound 48/80, produced lung edema. CPM, NEP, and ACE activities were recovered both in edema and perfusate fluid. The level of CPM and NEP was higher in edema fluid whereas, in contrast, more ACE activity was released into the perfusate. To evaluate the effect of peptidase inhibitors on changes in vascular permeability induced by bradykinin in the in situ perfused rat lung we measured the increase in lung weight as an index of increased vascular permeability or edema. Combined inhibition of either ACE plus NEP or ACE plus CPM augmented the effect of a subthreshold dose of bradykinin. Inhibitors of ACE, NEP, or CPM given alone and a combination of NEP plus CPM inhibitors did not enhance the bradykinin effect. Our results indicate that CPM, NEP, and ACE although present on different lung cells, synergistically modulate bradykinin effects. The different ratios of distribution of these enzymes in the perfusate and in edema fluid may not be due only to their presence on different pulmonary cells but also to their different anchoring mechanisms to plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Metabolism of bradykinin by peptidases in the lung. 838 9

The inhibitory effects of opioid peptides such as [Met5]-enkephalin and [Met5]-enkephalin-Arg6 on the electrically-evoked contractions of guinea-pig ileum, mouse vas deferens and rat vas deferens were enhanced by aminopeptidase inhibitors such as amastatin and bestatin, a peptidyl dipeptidase A inhibitor like captopril, and endopeptidase-24.11 inhibitors such as phosphoramidon and thiorphan. The magnitude of the enhancement by each peptidase inhibitor depended on both the preparation and opioid peptide employed. Additionally, enkephalin had been shown to be almost exclusively hydrolyzed at least in the ileal and striatal guinea pig membrane preparation by 3 kinds of enzymes, amastatin-sensitive aminopeptidase(s), captopril-sensitive peptidyl dipeptidase A and phosphoramidon-sensitive endopeptidase-24.11, which were indicated to be located very close to opioid receptors.
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PMID:[Enkephalin-inactivating enzymes]. 839 Mar 90

The presence of neutral endopeptidase 24.11 was demonstrated in human umbilical vein endothelial cells by immunostaining. Enzymatic activity of neutral endopeptidase was determined as 0.167 +/- 0.02 mU/mg protein in the membrane fraction of human umbilical vein endothelial cells, using the fluorogenic peptide substrate, dansyl-D-Ala-Gly-Phe(pNO2)-Gly. No activity was found in the cytosolic fraction of endothelial cells. The role of this peptidase in the degradation of the endogenous vasodilator bradykinin was investigated by incubating human umbilical vein endothelial cell monolayers with bradykinin (10(-8) mol/l). The inhibitor of neutral endopeptidase, phosphoramidon (10(-8) mol/l), decreased the degradation of bradykinin in the supernatant of endothelial cells; the half-life of bradykinin was then increased from 29 +/- 1 to 46 +/- 2 minutes. The angiotensin-converting enzyme inhibitor, lisinopril (10(-8) mol/l), increased the half-life of bradykinin to 244 +/- 20 minutes; the combination of both inhibitors increased the half-life of bradykinin to 381 +/- 51 minutes. Inhibitors of aminopeptidase (amastatin) and carboxypeptidase (2-mercaptomethyl-3-guanidinoethyl-thiopropionic acid) caused no significant effect. The effect of phosphoramidon was small in comparison with that of lisinopril, but was pronounced in combination with lisinopril. Neutral endopeptidase activity is localized in the membranes of human endothelial cells and seems to be involved in the degradation of bradykinin by the vascular endothelium, particularly during angiotensin converting enzyme inhibition.
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PMID:Degradation of bradykinin by neutral endopeptidase (EC 3.4.24.11) in cultured human endothelial cells. 839 30

To evaluate the feasibility of transmucosal delivery of methionine enkephalin (Tyr-Gly-Gly-Phe-Met; Met-Enk), it is important to first investigate its physicochemical and enzymatic stability. The kinetics of degradation of Met-Enk in aqueous solution was determined at pH 2.01-9.84 and 37-45 degrees C by high-performance liquid chromatography. The first-order rate constant (k) was calculated, and the log k-pH profile showed that Met-Enk is most stable at pH approximately 5.0. Various mucosae excised from rabbit were mounted on Valia-Chien permeation cells and exposed to isotonic phosphate buffer at physiologic pHs. Mucosal and serosal extracts were collected from the donor and receptor solutions, respectively. The degradation of Met-Enk in the extracts followed first-order kinetics, but no significant difference in the degradation rates was observed between mucosal and serosal extracts, regardless of the type of mucosa used. Degradation was most rapid in the extracts of rectal mucosa, followed by vaginal and nasal mucosae. The major metabolites were Des-Tyr-Met-Enk and Tyrosine (Tyr), indicating the enzymatic hydrolysis by aminopeptidases. However, the data also suggested that dipeptidyl peptidase and dipeptidyl carboxypeptidase could play some roles in the degradation of Met-Enk. The degradation pathways of Met-Enk were further explored by concomitantly determining the formation of smaller metabolites of primary hydrolytic fragments of Met-Enk in the mucosal extracts.
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PMID:Transmucosal delivery of methionine enkephalin. I: Solution stability and kinetics of degradation in various rabbit mucosa extracts. 846 81

To determine whether lysylbradykinin (LBK, kallidin) causes bronchoconstriction in animals and if peptidase inhibitors modulate the response, we studied the effects of LBK administered by aerosol in rats and assessed whether pretreatment with aerosolized solutions of enalaprilat, an inhibitor of angiotensin converting enzyme (ACE), or phosphoramidon, an inhibitor of endopeptidase 24.11 (EP 24.11, neutral endopeptidase), altered the response. Accordingly, LBK-induced bronchoconstriction was measured in anesthetized, mechanically ventilated, specific pathogen-free, Sprague-Dawley rats by body plethysmography and followed by continuous determination of lung resistance (RL) and maximal expiratory flow (MEF). Incremental doses of aerosolized LBK were administered by nebulization to obtain a concentration that caused a 5-15% increase in RL, which was designated the BC10 dose. We found that pretreatment with aerosolized enalaprilat (1 mM) 3 min prior to a BC10 dose of LBK significantly increased RL as compared to the BC10 dose alone (129 +/- 4.1% vs. 105 +/- 2.4%, P < 0.002, n = 4) and significantly decreased MEF (83 +/- 1.5% vs. 97 +/- 1.4%, P < 0.008, n = 4). Following pretreatment with aerosolized phosphoramidon (1 mM), significant increases in RL (113 +/- 1.4% vs. 106 +/- 1.6%, P < 0.019, n = 7) and decreases in MEF (92 +/- 0.9% vs. 95 +/- 0.9%, P < 0.035, n = 7) were observed (paired Student's t-test). The above findings demonstrate the effects of LBK on airway caliber for the first time in an animal model, and suggest that ACE and EP 24.11 contribute to degradation of the peptide in the airway.
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PMID:Peptidase inhibitors potentiate lysylbradykinin-induced bronchoconstriction in the rat. 873 81


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