Blood ACE Phenotyping for Personalized Medicine: Revelation of Patients with Conformationally Altered ACE.

Background: The angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated blood ACE is a marker for granulomatous diseases and elevated ACE expression in tissues is associated with increased risk of cardiovascular diseases. Objective and Methodology: We applied a novel approach -ACE phenotyping-to find a reason for conformationally impaired ACE in the blood of one particular donor. Similar conformationally altered ACEs were detected previously in 2-4% of the healthy population and in up to 20% of patients with uremia, and were characterized by significant increase in the rate of angiotensin I hydrolysis. Principal findings: This donor has (1) significantly increased level of endogenous ACE inhibitor in plasma with MW less than 1000; (2) increased activity toward angiotensin I; (3) M71V mutation in ABCG2 (membrane transporter for more than 200 compounds, including bilirubin). We hypothesize that this patient may also have the decreased level of free bilirubin in plasma, which normally binds to the N domain of ACE. Analysis of the local conformation of ACE in plasma of patients with Gilbert and Crigler-Najjar syndromes allowed us to speculate that binding of mAbs 1G12 and 6A12 to plasma ACE could be a natural sensor for estimation of free bilirubin level in plasma. Totally, 235 human plasma/sera samples were screened for conformational changes in soluble ACE. Conclusions/Significance: ACE phenotyping of plasma samples allows us to identify individuals with conformationally altered ACE. This type of screening has clinical significance because this conformationally altered ACE could not only result in the enhancement of the level of angiotensin II but could also serve as an indicator of free bilirubin levels.

Chitosan-covered calcium phosphate particles as a drug vehicle for delivery to the eye.

Formulations on the base of an inhibitor of angiotensin-converting enzyme, enalaprilat, were prepared by the inclusion of the drug into calcium phosphate (CaP)-particles in situ, followed by the covering of the particles with 5 kDa chitosan or 72 kDa glycol chitosan and cross-linking with sodium tripolyphosphate. Physicochemical characterization of the resulted hybrid particles was conducted using dynamic light scattering, as well as scanning and transmission electron microscopy. Enalaprilat-containing particles had a mean hydrodynamic diameter 180 nm and 260 nm and ζ-potential +7 mV and +16 mV for 5 kDa and 72 kDa chitosans, respectively. In vivo studies showed that enalaprilat within particles stayed longer in the tear fluid after single instillation and caused a significantly pronounced and prolonged decrease of intraocular pressure in rabbits, especially in the case of CaP-particles, covered by glycol chitosan. Thus, such formulations demonstrate potential as prospective therapeutic agents for the treatment of eye diseases.

Superoxide Dismutase 1 Nanoparticles (Nano-SOD1) as a Potential Drug for the Treatment of Inflammatory Eye Diseases.

Inflammatory eye diseases remain the most common clinical problem in ophthalmology. The secondary processes associated with inflammation, such as overproduction of reactive oxygen species (ROS) and exhaustion of the endogenous antioxidant system, frequently lead to tissue degeneration, vision blurring, and even blindness. Antioxidant enzymes, such as copper-zinc superoxide dismutase (SOD1), could serve as potent scavengers of ROS. However, their delivery into the eye compartments represents a major challenge due to the limited ocular penetration. This work presents a new therapeutic modality specifically formulated for the eye on the basis of multilayer polyion complex nanoparticles of SOD1 (Nano-SOD1), which is characterized by appropriate storage stability and pronounced therapeutic effect without side reactions such as eye irritation; acute, chronic, and reproductive toxicity; allergenicity; immunogenicity; mutagenicity even at high doses. The ability of Nano-SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with a model immunogenic uveitis-the inflammation of the inner vascular tract of the eye. It was shown during preclinical studies that topical instillations of Nano-SOD1 were much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as corneal and conjunctival edema, iris hyperemia, and fibrin clots. Moreover, Nano-SOD1 penetrates into interior eye structures more effectively than SOD itself and retains enzyme activity in the eye for a much longer period of time, decreasing inflammation and restoring antioxidant activity in the eye. Thus, the presented Nano-SOD1 can be considered as a potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

Tissue ACE phenotyping in prostate cancer.

Epithelial cells of prostate express significant level of ACE and, as a result, seminal fluid has 50-fold more ACE than plasma. The substitution of highly specialized prostate epithelial cells by tumor cells results in dramatic decrease in ACE production in prostate tissues. We performed detailed characterization of ACE status in prostate tissues from patients with benign prostate hyperplasia (BPH) and prostate cancer (PC) using new approach- ACE phenotyping, that includes evaluation of: 1) ACE activity with two substrates (HHL and ZPHL); 2) the ratio of the rates of their hydrolysis (ZPHL/HHL ratio); 3) the ratio of immunoreactive ACE protein to ACE activity; 4) the pattern of mAbs binding to different epitopes on ACE - ACE conformational fingerprint - to reveal conformational changes in prostate ACE due to prostate pathology. ACE activity dramatically decreased and the ratio of immunoreactive ACE protein to ACE activity increased in PC tissues. The catalytic parameter, ZPHL/HHL ratio, increased in prostate tissues from all patients with PC, but was did not change for most |BPH patients. Nevertheless, prostate tissues of several patients diagnosed with BPH based on histology, also demonstrated decreased ACE activity and increased immunoreactive ACE protein/ACE activity and ZPHL/HHL ratios, that could be considered as more early indicators of prostate cancer development than routine histology. Thus, ACE phenotyping of prostate biopsies has a potential to be an effective approach for early diagnostics of prostate cancer or at least for differential diagnostics of BPH and PC.

Conformational fingerprint of blood and tissue ACEs: Personalized approach.

BACKGROUND: The pattern of binding of monoclonal antibodies (mAbs) to 18 epitopes on human angiotensin I-converting enzyme (ACE)-"conformational fingerprint of ACE"-is a sensitive marker of subtle conformational changes of ACE due to mutations, different glycosylation in various cells, the presence of ACE inhibitors and specific effectors, etc. METHODOLOGY/PRINCIPAL FINDINGS: We described in detail the methodology of the conformational fingerprinting of human blood and tissue ACEs that allows detecting differences in surface topography of ACE from different tissues, as well detecting inter-individual differences. Besides, we compared the sensitivity of the detection of ACE inhibitors in the patient's plasma using conformational fingerprinting of ACE (with only 2 mAbs to ACE, 1G12 and 9B9) and already accepted kinetic assay and demonstrated that the mAbs-based assay is an order of magnitude more sensitive. This approach is also very effective in detection of known (like bilirubin and lysozyme) and still unknown ACE effectors/inhibitors which nature and set could vary in different tissues or different patients. CONCLUSIONS/SIGNIFICANCE: Phenotyping of ACE (and conformational fingerprinting of ACE as a part of this novel approach for characterization of ACE) in individuals really became informative and clinically relevant. Appreciation (and counting on) of inter-individual differences in ACE conformation and accompanying effectors make the application of this approach for future personalized medicine with ACE inhibitors more accurate. This (or similar) methodology can be applied to any enzyme/protein for which there is a number of mAbs to its different epitopes.

ACE phenotyping in Gaucher disease.

BACKGROUND: Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. METHODS: ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. RESULTS: We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen. CONCLUSIONS: The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease.

ACE phenotyping in human heart.

AIMS: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, is expressed as a type-1 membrane glycoprotein on the surface of different cells, including endothelial cells of the heart. We hypothesized that the local conformation and, therefore, the properties of heart ACE could differ from lung ACE due to different microenvironment in these organs. METHODS AND RESULTS: We performed ACE phenotyping (ACE levels, conformation and kinetic characteristics) in the human heart and compared it with that in the lung. ACE activity in heart tissues was 10-15 lower than that in lung. Various ACE effectors, LMW endogenous ACE inhibitors and HMW ACE-binding partners, were shown to be present in both heart and lung tissues. "Conformational fingerprint" of heart ACE (i.e., the pattern of 17 mAbs binding to different epitopes on the ACE surface) significantly differed from that of lung ACE, which reflects differences in the local conformations of these ACEs, likely controlled by different ACE glycosylation in these organs. Substrate specificity and pH-optima of the heart and lung ACEs also differed. Moreover, even within heart the apparent ACE activities, the local ACE conformations, and the content of ACE inhibitors differ in atria and ventricles. CONCLUSIONS: Significant differences in the local conformations and kinetic properties of heart and lung ACEs demonstrate tissue specificity of ACE and provide a structural base for the development of mAbs able to distinguish heart and lung ACEs as a potential blood test for predicting atrial fibrillation risk.

Lysozyme and bilirubin bind to ACE and regulate its conformation and shedding.

Angiotensin I-converting enzyme (ACE) hydrolyzes numerous peptides and is a critical participant in blood pressure regulation and vascular remodeling. Elevated tissue ACE levels are associated with increased risk for cardiovascular and respiratory disorders. Blood ACE concentrations are determined by proteolytic cleavage of ACE from the endothelial cell surface, a process that remains incompletely understood. In this study, we identified a novel ACE gene mutation (Arg532Trp substitution in the N domain of somatic ACE) that increases blood ACE activity 7-fold and interrogated the mechanism by which this mutation significantly increases blood ACE levels. We hypothesized that this ACE mutation disrupts the binding site for blood components which may stabilize ACE conformation and diminish ACE shedding. We identified the ACE-binding protein in the blood as lysozyme and also a Low Molecular Weight (LMW) ACE effector, bilirubin, which act in concert to regulate ACE conformation and thereby influence ACE shedding. These results provide mechanistic insight into the elevated blood level of ACE observed in patients on ACE inhibitor therapy and elevated blood lysozyme and ACE levels in sarcoidosis patients.

Superoxide Dismutase 1 Nanozyme for Treatment of Eye Inflammation.

Use of antioxidants to mitigate oxidative stress during ocular inflammatory diseases has shown therapeutic potential. This work examines a nanoscale therapeutic modality for the eye on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), termed "nanozyme." The nanozyme is produced by electrostatic coupling of the SOD1 with a cationic block copolymer, poly(L-lysine)-poly(ethyleneglycol), followed by covalent cross-linking of the complexes with 3,3'-dithiobis(sulfosuccinimidylpropionate) sodium salt. The ability of SOD1 nanozyme as well as the native SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with immunogenic uveitis. Results suggested that topical instillations of both enzyme forms demonstrated anti-inflammatory activity; however, the nanozyme was much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as the intensities of corneal and iris edema, hyperemia of conjunctiva, lens opacity, fibrin clots, and the protein content in aqueous humor. Clinical findings were confirmed by histological data. Thus, SOD1-containing nanozyme is potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

BACKGROUND: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. METHODS/PRINCIPAL FINDINGS: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. CONCLUSIONS: Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.

Conformational changes of blood ACE in chronic uremia.

BACKGROUND: The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. HYPOTHESIS: Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. METHODOLOGY/PRINCIPAL FINDINGS: The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The "short" ACE inhibitor enalaprilat (tripeptide analog) and "long" inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. CONCLUSIONS/SIGNIFICANCE: The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy.

Conformational fingerprinting of the angiotensin I-converting enzyme (ACE). 1. Application in sarcoidosis.

Fine epitope mapping of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) revealed that the epitopes of all mAbs contained putative glycosylation sites. ACE glycosylation is both cell- and tissue-specific and, therefore, the local conformation of ACE produced by different cells could be also unique. The pattern of ACE binding by a set of mAbs to 16 epitopes of human ACE - "conformational fingerprint of ACE" - is the most sensitive marker of ACE conformation and could be cell- and tissue-specific. The recognition of ACEs by mAbs to ACE was estimated using an immune-capture enzymatic plate precipitation assay. Precipitation patterns of soluble recombinant ACE released from Chinese hamster ovary (CHO)-ACE cells was influenced by conditions that alter ACE glycosylation. This pattern was also strongly cell type specific. Patients with sarcoidosis exhibited conformational fingerprints of tissue ACE (lungs and lymph nodes), as well as blood ACE, which were distinct from controls. Conformational fingerprinting of ACE may detect ACE originated from the cells other than endothelial cells in the blood and when combined with elevated blood ACE levels in patients with sarcoidosis may potentially reflect extrapulmonary sarcoidosis involvement (bone marrow, spleen, liver). If proven true, this would serve as a biomarker of enormous potential clinical significance.

Simultaneous determination of ACE activity with 2 substrates provides information on the status of somatic ACE and allows detection of inhibitors in human blood.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of 2 homologous domains, each bearing a Zn-dependent active site. The ratio of the rates of hydrolysis of 2 synthetic substrates, Z-Phe-His-Leu (ZPHL) and Hip-His-Leu (HHL), is characteristic for each type of ACE: somatic 2-domain 1, N-domain 4.5, and C-domain 0.7 (Williams et al, 1996). We hypothesized that inactivation or selective inhibition of the C-domain within the somatic ACE should increase the ratio from 1 toward higher values, whereas inactivation or inhibition of the N-domain should decrease the ratio to lower values. Temperatures in the 40-60 degrees C range cause preferential inactivation of the C-domain in somatic ACE and an increase in the ZPHL/HHL ratio. Determination of the ZPHL/HHL ratio in freshly 100-fold concentrated urine ruled out the existence of the N-domain fragment in human urine, which appears only as a result of long storage. Inhibition of ACE by common inhibitors also increases the ZPHL/HHL ratio for 2-domain ACE, thus providing a sensitive method for the detection of ACE inhibitors in biological fluids. Therefore, simultaneous measurements of ACE activity with 2 substrates (ZPHL and HHL) and calculation of their ratio allow us to monitor the status of the ACE molecule and detect ACE inhibitors (endogenous and exogenous) in human blood and other biological fluids. This method should find wide application in monitoring clinical trials with ACE inhibitors and in the development of the approach for personalized medicine by these effective drugs.

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.

Monoclonal Antibodies 1G12 and 6A12 to the N-domain of human angiotensin-converting enzyme: fine epitope mapping and antibody-based detection of ACE inhibitors in human blood.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N- and C-), each bearing a Zn-dependent active site. ACE inhibitors are among the most prescribed drugs in the treatment of hypertension and cardiac failure. Fine epitope mapping of two monoclonal antibodies (mAb), 1G12 and 6A12, against the N-domain of human ACE, was developed using the N-domain 3D-structure and 21 single and double N-domain mutants. The binding of both mAbs to their epitopes on the N-domain of ACE is significantly diminished by the presence of the C-domain in the two-domain somatic tissue ACE and further diminished by the presence of sialic acid residues on the surface of blood ACE. The binding of these mAbs to blood ACE, however, increased dramatically (5-10-fold) in the presence of ACE inhibitors or EDTA, whereas the effect of these compounds on the binding of the mAbs to somatic tissue ACE was less pronounced and even less for truncated N-domain. This implies that the binding of ACE inhibitors or removal of Zn2+ from ACE active centers causes conformational adjustments in the mutual arrangement of N- and C-domains in the two-domain ACE molecule. As a result, the regions of the epitopes for mAb 1G12 and 6A12 on the N-domain, shielded in somatic ACE by the C-domain globule and additionally shielded in blood ACE by sialic acid residues in the oligosaccharide chains localized on Asn289 and Asn416, become unmasked. Therefore, we demonstrated a possibility to employ these mAbs (1G12 or 6A12) for detection and quantification of the presence of ACE inhibitors in human blood. This method should find wide application in monitoring clinical trials with ACE inhibitors as well as in the development of the approach for personalized medicine by these effective drugs.

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action.

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

Kinetic probes for inter-domain co-operation in human somatic angiotensin-converting enzyme.

s-ACE (the somatic form of angiotensin-converting enzyme) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. Negative co-operativity between the two domains has been demonstrated for cow and pig ACEs. However, for the human enzyme there are conflicting reports in the literature: some suggest possible negative co-operativity between the domains, whereas others indicate independent functions of the domains within s-ACE. We demonstrate here that a 1:1 stoichiometry for the binding of the common ACE inhibitors, captopril and lisinopril, to human s-ACE is enough to abolish enzymatic activity towards FA {N-[3-(2-furyl)acryloyl]}-Phe-GlyGly, Cbz (benzyloxycarbonyl)-Phe-His-Leu or Hip (N-benzoylglycyl)-His-Leu. The kinetic parameters for the hydrolysis of seven tripeptide substrates by human s-ACE appeared to represent average values for parameters obtained for the individual N- and C-domains. Kinetic analysis of the simultaneous hydrolysis of two substrates, Hip-His-Leu (S1) and Cbz-Phe-His-Leu (S2), with a common product (His-Leu) by s-ACE at different values for the ratio of the initial concentrations of these substrates (i.e. sigma=[S2]0/[S1]0) demonstrated competition of these substrates for binding to the s-ACE molecule, i.e. binding of a substrate at one active site makes the other site unavailable for either the same or a different substrate. Thus the two domains within human s-ACE exhibit strong negative co-operativity upon binding of common inhibitors and in the hydrolysis reactions of tripeptide substrates.

Evidence for the negative cooperativity of the two active sites within bovine somatic angiotensin-converting enzyme.

The somatic isoform of angiotensin-converting enzyme (ACE) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. We have used the two-domain ACE form and its individual domains to compare characteristics of different domains and to probe mutual functioning of the two active sites within a bovine ACE molecule. The substrate Cbz-Phe-His-Leu (N-carbobenzoxy-L-phenylalanyl-L-histidyl-L-leucine; from the panel of seven) was hydrolyzed faster by the N-domain, the substrates FA-Phe-Gly-Gly (N-(3-[2-furyl]acryloyl)-L-phenylalanyl-glycyl-glycine) and Hip-His-Leu (N-benzoyl-glycyl-L-histidyl-L-leucine) were hydrolyzed by both domains with equal rates, while other substrates were preferentially hydrolyzed by the C-domain. The inhibitor captopril ((2S)-1-(3-mercapto-2-methylpropionyl)-L-proline) bound to the N-domain more effectively than to the C-domain, whereas lisinopril ((S)-N(alpha)-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) bound to equal extent with all ACE forms. However, active site titration with lisinopril assayed by hydrolysis of FA-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either two-domain or single-domain ACE forms, indicating that a single active site functions in bovine somatic ACE. Neither of the k(cat) values obtained for somatic enzyme was the sum of k(cat) values for individual domains, but in every case the value of the catalytic constant of the hydrolysis of the substrate by the two-domain ACE represented the mean quantity of the values of the corresponding catalytic constants obtained for single-domain forms. The results indicate that the two active sites within bovine somatic ACE exhibit strong negative cooperativity.

Epitope-dependent blocking of the angiotensin-converting enzyme dimerization by monoclonal antibodies to the N-terminal domain of ACE: possible link of ACE dimerization and shedding from the cell surface.

In a biomembrane modeling system, reverse micelles, somatic ACE forms dimers via carbohydrate-mediated interaction, providing evidence for the existence of a carbohydrate-recognizing domain on the ACE molecule. We localized this putative region on the N-domain of ACE using monoclonal antibodies (mAbs) to seven different epitopes of ACE. Two mAbs, 9B9 and 3G8, directed to distinct, but overlapping, epitopes of the N-domain of ACE shielded the CRD. Only "simple" ACE-antibody complexes were found in the system. Five mAbs allowed the formation of "double" antibody-ACE-ACE-antibody complexes via carbohydrate-mediated interactions. The results were confirmed using the ACE N- and C-domains. Testicular ACE was unable to form carbohydrate-mediated ACE dimers in the reverse micelles, while the N-domain of ACE, obtained by limited proteolysis of the parent full-length ACE, retained the ability to form dimers. Furthermore, mAb 3G8, which blocked ACE dimerization in micelles, significantly inhibited ACE shedding from the surface of ACE-expressing cells. Galactose prevented ACE dimerization in reverse micelles and also affected antibody-induced ACE shedding in an epitope-dependent manner. Restricted glycosylation of somatic ACE, obtained by the treatment of CHO-ACE cells with the glucosidase inhibitor N-butyldeoxynojirimycin, significantly increased the rate of basal ACE shedding and altered antibody-induced ACE shedding. A chemical cross-linking approach was used to show that ACE is present (at least in part) as noncovalently linked dimers on the surface of CHO-ACE cells. These results suggest a possible link between putative ACE dimerization on the cell surface and the proteolytic cleavage (shedding) of ACE.