scholarly journals Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer

1997 ◽  
Vol 327 (1) ◽  
pp. 37-43 ◽  
Author(s):  
S. PARVATHY ◽  
Sylvester Y. OPPONG ◽  
Eric H. KARRAN ◽  
Derek R. BUCKLE ◽  
Anthony J. TURNER ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Angiotensin Converting Enzyme ◽  
Lipid Bilayer ◽  
Proteolytic Processing ◽  
Full Length ◽  
Converting Enzyme ◽  
Pig Kidney ◽  
Zinc Metalloprotease ◽  
Metalloprotease Inhibitors ◽  
Triton X

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 μM, whereas TAPI-2 was slightly less potent (IC50 18 μM). 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 related 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-octylβ-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.

scholarly journals Pig kidney angiotensin converting enzyme. Purification and characterization of amphipathic and hydrophilic forms of the enzyme establishes C-terminal anchorage to the plasma membrane

1987 ◽  
Vol 247 (1) ◽  
pp. 85-93 ◽  
Author(s):  
N M Hooper ◽  
J Keen ◽  
D J C Pappin ◽  
A J Turner
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Converting Enzyme ◽  
Enzyme Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Converting Enzyme ◽  
Pig Kidney ◽  
C Terminus ◽  
Endogenous Activity ◽  
Triton X

Angiotensin converting enzyme from pig kidney was isolated by affinity chromatography after solubilization from the membrane by one of four different procedures. Solubilization with Triton X-100, trypsin or by an endogenous activity in microvillar membranes all generated hydrophilic forms of the enzyme as assessed by phase separation in Triton X-114 and failure to incorporate into liposomes. Only when solubilization and purification was effected by Triton X-100 in the presence of EDTA (10 mM) could an amphipathic form of the enzyme (membrane- or m-form) be generated. The m-form of angiotensin converting enzyme (ACE) appeared slightly larger (Mr approx. 180,000) than the hydrophilic forms (Mr approx. 175,000) after SDS/polyacrylamide-gel electrophoresis, and the m-form incorporated into liposomes, consistent with retention of the membrane anchor. The m-form of ACE showed an N-terminal sequence identical with that of preparations of enzyme isolated after solubilization with detergent alone (d-form), with trypsin (t-form) or by the endogenous mechanism (e-form). These data imply that ACE is anchored to the plasma membrane via its C-terminus, in contrast with the N-terminal anchorage of endopeptidase-24.11. No release of ACE from the membrane could be detected with a variety of phospholipases, including bacterial phosphatidylinositol-specific phospholipases C, although an endogenous EDTA-sensitive membrane-associated hydrolase was capable of releasing a soluble, hydrophilic, form of the enzyme.

scholarly journals Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane

1993 ◽  
Vol 292 (2) ◽  
pp. 597-603 ◽  
Author(s):  
S Y Oppong ◽  
N M Hooper
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Human Kidney ◽  
Soluble Form ◽  
Converting Enzyme ◽  
Ph Optimum ◽  
Secretase Activity ◽  
Sds Page ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Triton X

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.

scholarly journals Human Tissue Angiotensin Converting Enzyme (ACE) Activity Is Regulated by Genetic Polymorphisms, Posttranslational Modifications, Endogenous Inhibitors and Secretion in the Serum, Lungs and Heart

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1708
Author(s):  
Viktor Bánhegyi ◽  
Attila Enyedi ◽  
Gábor Áron Fülöp ◽  
Attila Oláh ◽  
Ivetta Mányiné Siket ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Lung Tissue ◽  
Posttranslational Modifications ◽  
Left Ventricular ◽  
Converting Enzyme ◽  
Tissue Samples ◽  
Treat Heart Failure ◽  
Ace Activity ◽  
Triton X ◽  
Genotype Ii

Objective: Inhibitors of the angiotensin converting enzyme (ACE) are the primarily chosen drugs to treat heart failure and hypertension. Moreover, an imbalance in tissue ACE/ACE2 activity is implicated in COVID-19. In the present study, we tested the relationships between circulating and tissue (lung and heart) ACE levels in men. Methods: Serum, lung (n = 91) and heart (n = 72) tissue samples were collected from Caucasian patients undergoing lung surgery or heart transplantation. ACE I/D genotype, ACE concentration and ACE activity were determined from serum and tissue samples. Clinical parameters were also recorded. Results: A protocol for ACE extraction was developed for tissue ACE measurements. Extraction of tissue-localized ACE was optimal in a 0.3% Triton-X-100 containing buffer, resulting in 260 ± 12% higher ACE activity over detergent-free conditions. SDS or higher Triton-X-100 concentrations inhibited the ACE activity. Serum ACE concentration correlated with ACE I/D genotype (II: 166 ± 143 ng/mL, n = 19, ID: 198 ± 113 ng/mL, n = 44 and DD: 258 ± 109 ng/mL, n = 28, p < 0.05) as expected. In contrast, ACE expression levels in the lung tissue were approximately the same irrespective of the ACE I/D genotype (II: 1423 ± 1276 ng/mg, ID: 1040 ± 712 ng/mg and DD: 930 ± 1273 ng/mg, p > 0.05) in the same patients (values are in median ± IQR). Moreover, no correlations were found between circulating and lung tissue ACE concentrations and activities (Spearman’s p > 0.05). In contrast, a significant correlation was identified between ACE activities in serum and heart tissues (Spearman’s Rho = 0.32, p < 0.01). Finally, ACE activities in lung and the serum were endogenously inhibited to similar degrees (i.e., to 69 ± 1% and 53 ± 2%, respectively). Conclusion: Our data suggest that circulating ACE activity correlates with left ventricular ACE, but not with lung ACE in human. More specifically, ACE activity is tightly coordinated by genotype-dependent expression, endogenous inhibition and secretion mechanisms.

scholarly journals The heterogeneous nature of the Coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia

2020 ◽  
Author(s):  
Vincent J. Manna ◽  
Salvatore J. Caradonna
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Essential Element ◽  
Renin Angiotensin System ◽  
Full Length ◽  
Converting Enzyme ◽  
Ciliated Cells ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Nasal Epithelial Cells ◽  
The Impact

ABSTRACTCoronavirus Disease 2019 (COVID-19) is transmitted through respiratory droplets containing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) particles. Once inhaled, SARS-CoV-2 particles gain entry into respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). It is known that ACE2 functions within the renin-angiotensin system to regulate blood pressure, fluid homeostasis and inflammation. However, it is largely unknown what roles ACE2 has in ciliated cells of the airway. Therefore, understanding the function and nature of ACE2 within airway tissue has become an essential element in combatting the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells isolated from nasal turbinates of donors and the air-liquid interface (ALI) model of differentiation. Using ALI tissue we cloned transcripts for three distinct variants of ACE2, one of which encodes the full-length ACE2 protein, the other two transcripts are truncated isoforms that had only been predicted to exist via sequence analysis software. We demonstrate that all three isoforms have the capacity to be glycosylated, a known modification of full-length ACE2. Immunofluorescence microscopy of individual ACE2 isoform transfected cells reveals distinct localization of variant 1 relative to X1 and X2. Double staining immunohistochemistry of ALI tissue using antibodies to either the N-term or C-term region of ACE2 revealed distinct and overlapping signals in the apical cytosol of ciliated cells. Most notably only the ACE2 C-term antibody displayed plasma-membrane localization in ciliated cells. We also observed a decrease in the total amount of ACE2 in ALI tissue derived from a 33 year-old male donor when compared to a 34 year-old female donor, thus there may be variation in the abundance of ACE2 protein in the airway among the population. Together, our data begins to highlight the dynamic status of the ACE2 protein in airway mucociliary tissue and we propose multiple ACE2 parameters that may impact an individual’s susceptibility to SARS-CoV-2. These parameters include the balance of cytosolic versus membrane bound ACE2, isoform expression levels, maintenance of post-translational modifications and the impact of genetic, environmental and lifestyle factors on these processes.

scholarly journals A comparison of the zinc contents and substrate specificities of the endothelial and testicular forms of porcine angiotensin converting enzyme and the preparation of isoenzyme-specific antisera

1992 ◽  
Vol 288 (3) ◽  
pp. 875-881 ◽  
Author(s):  
T A Williams ◽  
K Barnes ◽  
A J Kenny ◽  
A J Turner ◽  
N M Hooper
Keyword(s):  
Substance P ◽  
Angiotensin Converting Enzyme ◽  
Catalytic Site ◽  
Immunofluorescent Staining ◽  
Seminiferous Tubules ◽  
Converting Enzyme ◽  
Pig Kidney ◽  
Specific Antisera ◽  
Lh Rh ◽  
Hydrolysis Of

Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.

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