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)

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.
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PMID:Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane. 838 41

Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.
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PMID:Characterization of neutral endopeptidase 24.11 in dog glomeruli. 848 5

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP) dilates resistance arterioles in the in situ systemic circulation and whether inhibitors of neutral endopeptidase (NEP) and angiotensin I-converting enzyme (ACE), two membrane-bound metalloenzymes that are widely distributed in the microcirculation and cleave and inactive VIP, potentiate this response. Using intravital microscopy, we found that VIP (0.05 and 0.1 nmol) induced significant vasodilation in the hamster cheek pouch (13 +/- 1 and 20 +/- 2% increase from baseline, respectively; mean +/- SE; P < 0.05). These responses were significantly potentiated by topical application of phosphoramidon and thiorphan, two relatively selective NEP inhibitors, but not by captopril, a relatively selective ACE inhibitor. Furthermore, suffusion of a mixture of proteinase inhibitors consisting of leupeptin, Bestatin, and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid to inhibit serine proteinases, including mast cell tryptase, aminopeptidases, and carboxypeptidase N, respectively, had no significant effects on VIP-induced responses. These data indicate that VIP elicits vasodilation in the in situ systemic microcirculation and that NEP modulates this response.
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PMID:Neutral endopeptidase modulates VIP-induced vasodilation in hamster cheek pouch vessels in situ. 877 Jan 40

Sertoli cells play a key role in spermatogenesis. To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated. Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis. Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11. The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis. This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II. Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes. A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities. This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors. The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.
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PMID:Enzymatic digestion of bradykinin by rat Sertoli cell cultures. 888

Cells capable of de novo angiotensin (Ang)II generation in the heart remain unidentified. High-density angiotensin converting enzyme (ACE) binding has been localized to sites of high collagen turnover, such as heart valve leaflets and their valvular interstitial cells (VIC). VIC express ACE mRNA and their membrane-bound ACE utilizes AngI as substrate. Whether VIC also express angiotensinogen (Ao) and an aspartyl protease, and whether they generate AngI and II de novo, is presently unknown. We sought to address these questions in serum-deprived cultured VIC. Ao, renin and cathepsin D (Cat-D) mRNA expression was addressed by RT-PCR. Production of Ao, AngI and AngII peptides were measured in VIC-culture media by radioimmunoassay (RIA). Immunoreactive Cat-D was detected by immunofluorescein labeling and Western blotting. Cat-D and renin activities were determined by spectrofluorometric and autoradiographic methods and AngI generation by RIA. Results showed (a) expression of Ao and Cat-D both at mRNA and protein levels; (b) AngI and AngII peptides in culture media; (c) acceleration of AngII production by exogenous AngI (1 nmol/l), which was blocked by lisinopril (0.1 mumol/l); (d) that dexamethasone (0.1 mumol/l) increased AngII production; (e) a 46 kDa immunoreactive Cat-D protein by Western blotting; (f) aspartyl protease activity, using chromogenic and 125I-labeled Ao as substrates, inhibited by pepstatin-A; and (g) the absence of renin mRNA and activity. It is concluded that at both the mRNA and protein levels, cultured VIC express Ao and Cat-D, and can generate AngI and AngII peptides by the action of a non-renin protease Cat-D and ACE, respectively. VIC therefore appear to represent a constitutive nonendothelial cell found in adult rat heart valve leaflets, which are capable of de novo Ang peptide generation.
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PMID:Valvular interstitial cells express angiotensinogen and cathepsin D, and generate angiotensin peptides. 892 11

Vascular tolerance develops rapidly in isolated vascular strips exposed to millimolar concentrations of nitroglycerin. Several mechanisms, including depletion of sulfhydryl groups, reduced biotransformation of nitrates to NO or nitrosothiols, oxygen free radical injury, and downregulation of a membrane-bound enzyme or a nitrate receptor, have been proposed, but the exact mechanism responsible for in-vitro tolerance remains unknown. In-vivo tolerance of the beneficial effects of nitrates on hemodynamics, myocardial ischemia, and exercise performance develops rapidly. It has been suggested, but remains to be proven, that development of venous tolerance and not arterial tolerance is responsible for the attenuation of nitrate effects during long-term nitrate therapy. Several mechanisms, including neurohormonal activation, depletion of sulfhdryl groups, and the shift of fluid from the extravascular to intravascular compartment have been implicated. However, the use of agents to counteract these mechanisms (ACE inhibitors, sulfhydryl donors, diuretics) has produced conflicting results. Thus, at present the mechanism responsible for in vivo tolerance to nitrates remains unknown. Both in vitro and in vivo vascular tolerance to nitrates can be prevented or minimized by providing nitrate-free or low-nitrate intervals. However, during nitrate-free periods, rebound phenomena (rest angina in patients with ischemic heart disease or a deterioration in exercise performance prior to the renewal of the morning dose in patients with stable angina) remain a clinical concern. When treating patients with stable angina pectoris, it must be recognized that none of the nitrate preparations or formulations can provide round-the-clock antianginal or antiischemic prophylaxis. In these patients, beneficial antianginal and antiischemic effects of nitrates for 10-14 hours during the daytime can be maintained by using formulations and dosing regimens that avoid or minimize the development of tolerance (standard formulation of isosorbide-5-mononitrate, 20 mg in the morning and 7 hours later; slow-release formulation of isosorbide-5-mononitrate, 120-240 mg once a day; or nitroglycerin patch delivering 0.6 nitroglycerin per hour for 10-12 hours each day). Only the patch on and off treatment is associated with nitrate rebound. Although intermittent nitrate therapy is not associated with the development of tolerance, this strategy cannot be recommended for treating unstable angina because rebound angina during nitrate-free periods complicates clinical decision making. In the acute phase of unstable angina, continuous treatment with intravenous nitroglycerin is recommended because it permits rapid up- or down-titration. Tolerance towards antianginal and antiischemic effects does develop in a substantial number of patients with 24 hours, but this can be overridden by dose escalation and restoration of the therapeutic effectiveness of nitroglycerin. Tolerance towards the beneficial effects of nitrates on hemodynamics and on exercise performance also develops rapidly during continuous or long-term nitrate therapy, and for these reasons nitrates are not used as first-line therapy to treat chronic heart failure. In combination with hydralazine, high-dose isosorbide dinitrate (30-40 mg four times a day) improves survival, but this combination therapy is inferior to ACE inhibitors.
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PMID:Nitrate tolerance, rebound, and their clinical relevance in stable angina pectoris, unstable angina, and heart failure. 911 Jan 17

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.
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PMID:Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats. 917 84

We enzymatically deglycosylated pig lung angiotensin I-converting enzyme (ACE) to study the involvement of its glycanic chains in its physicochemical and catalytic properties. The effects of endoglycosidases F2 and H, and of N-glycanase were assessed by ACE mobility in SDS-PAGE. N-Glycanase only was completely effective with or without previous denaturation, leading to a shift in ACE M(r) from 172 to 135 kDa; endoglycosidase F2 produced the same shift but only without previous denaturation. Deglycosylated ACE had the same kcat as native ACE for the substrate hippuryl-histidyl-leucine, and an identical Stokes radius as measured by size-exclusion high performance liquid chromatography. Neuraminidase had no effect on ACE Stokes radius but slightly decreased its kcat which could be related to variations in ionization of the active site. The isoelectric point of ACE, as, determined by isoelectric focusing, increased from 4.5-4.8 to 5.0-5.3 after either endoglycosidase F2 or neuraminidase digestion, but still with microheterogeneities which thus did not seem to be related to ACE glycans. Deglycosylated ACE did not bind onto agaroselectins in contrast to native ACE which bound strongly to concanavalin A showing interactions involving oligomannosidic or biantennary and sialylated N-acetyl-lactosaminic isoglycans. Finally, tunicamycin, an inhibitor of N-glycosylation, did not modify ACE secretion by endothelial cells. Thus, ACE glycans have no drastic effects on structural and biological properties of the protein, but they may have a functional role on intracellular targeting of both secreted and membrane-bound ACE isoforms, also for the protection of the soluble plasma form against hepatic lectins and the maintenance of its hydrosolubility.
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PMID:Structural and biological roles of glycosylations in pulmonary angiotensin I-converting enzyme. 918 38

Scar tissue found at the site of myocardial infarction (MI) contains phenotypically transformed fibroblast-like cells termed myofibroblasts (myoFb). In injured cardiac tissue, autoradiography and immunolabeling have localized high density angiotensin (Ang) converting enzyme (ACE) and Ang II receptor binding to these cells, suggesting that they may regulate local concentrations of Ang II and transduce signals at this site. Ang II is known to modulate type I collagen gene expression of fibroblasts and myoFb, and to promote fibrous tissue contraction, each of which may contribute to tissue repair. It is unknown whether myoFb themselves generate Ang peptides de novo via expression of angiotensinogen (Ao), an aspartyl protease needed to convert Ao to Ang I, and ACE. We therefore isolated and cultured myoFb from 4-week-old scar tissue of the adult rat left ventricle with transmural MI. In cultured myoFb we found: (a) immunoreactive membrane-bound ACE, cytosolic cathepsin D (Cat-D), and AT, receptors by immunofluorescence and confocal microscopy, (b) mRNA expression for Ao, ACE, and Cat-D, but not renin, by reverse transcriptase-polymerase chain reaction, (c) production of Ang I and II in serum-free culture media; (d) absence of renin activity; (e) a time-dependent conversion of Ao to Ang I by myoFb cytosol, which was inhibited by pepstatin A, but not by renin inhibitor; and (f) significant increase in Ang II production (P < 0.05) by exogenous Ao and Ang I (10 nM), which was significantly blocked by lisinopril (0.1 microM: P < 0.05). Thus, cultured myoFb express requisite components and are able to generate Ang I and II de novo. In an autocrine and/or paracrine manner, Ang II may regulate myoFb collagen turnover and fibrous tissue contraction.
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PMID:Cultured myofibroblasts generate angiotensin peptides de novo. 920 23

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.
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PMID:Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer. 935 32


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