Purification and characterization of angiotensin-converting enzyme (ACE) from sheep lung.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the renin-angiotensin system regulates blood pressure by catalyzing angiotensin I to the vasoconstrictor angiotensin II. In this study, the ACE was purified and characterized from sheep lung. The kinetic properties of the ACE were designated. The inhibition effect of captopril, a specific ACE inhibitor, was determined. ACE was purified from sheep lung using the affinity chromatography method in one step. NHS-activated Sepharose 4 Fast Flow as column filler and lisinopril as a ligand in this method used. The molecular weight and purity of ACE were designated using the SDS-PAGE method. Optimum temperature and optimum pH were found for purified ACE. KM and Vmax values from Lineweaver-Burk charts determined. The inhibition type, IC50, and Ki values of captopril on purified ACE were identified. ACE was 6405-fold purified from sheep lung by affinity chromatography in one step and specific activity was 16871 EU/mg protein. The purity and molecular weight of ACE were found with SDS-PAGE and observed two bands at around 60 kDa and 70 kDa on the gel. Optimum temperature and optimum pH were designated for purified ACE. Optimum temperature and pH were found as 40 °C and pH 7.4, respectively. Vmax and KM values were calculated to be 35.59 (µmol/min).mL-1 and 0.18 mM, respectively. IC50 value of captopril was found as 0.51 nM. The inhibition type of captopril was determined as non-competitive from the Lineweaver-Burk graph and the Ki value was 0.39 nM. As a result, it was observed in this study that the ACE enzyme can be successfully purified from sheep lungs in one step. Also, it was determined that captopril, which is a specific ACE inhibitor, has a significant inhibitory effect with a very low IC50 value of 0.51 nM.

Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation.

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that potently degrades angiotensin II to angiotensin 1-7. Previous studies showed that injection of the enzymatic ectodomain of recombinant ACE2 (rACE2) markedly increases circulatory levels of ACE2 activity, and effectively lowered blood pressure in angiotensin II-induced hypertension. However, due to the short plasma half-life of rACE2, its therapeutic potential for chronic use is limited. To circumvent this, we generated a chimeric fusion of rACE2 and the immunoglobulin fragment Fc segment to increase its plasma stability. This rACE2-Fc fusion protein retained full peptidase activity and exhibited greatly extended plasma half-life in mice, from less than two hours of the original rACE2, to over a week. A single 2.5 mg/kg injection of rACE2-Fc increased the overall angiotensin II-conversion activities in blood by up to 100-fold and enhanced blood pressure recovery from acute angiotensin II induced hypertension seven days after administration. To assess rACE2-Fc given weekly on cardiac protection, we performed studies in mice continuously infused with angiotensin II for 28 days and in a Renin transgenic mouse model of hypertension. The angiotensin II infused mice achieved sustained blood pressure control and reduced cardiac hypertrophy and fibrosis. In chronic hypertensive transgenic mice, weekly injections of rACE2-Fc effectively lowered plasma angiotensin II and blood pressure. Additionally, rACE2-Fc ameliorated albuminuria, and reduced kidney and cardiac fibrosis. Thus, our chimeric fusion strategy for rACE2-Fc is suitable for future development of new renin angiotensin system-based inhibition therapies.

Purification of angiotensin-converting enzyme from human plasma and investigation of the effect of some active ingredients isolated from Nigella sativa L. extract on the enzyme activity.

In the present study, one-step purification of angiotensin-converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860-fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35-40°C and pH 7.4-7.5, respectively. The purity of ACE was determined by SDS-PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC-MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1-6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half-maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver-Burk graph.

Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization.

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.

Purification of Angiotensin-Converting Enzyme (ACE) from Sheep Kidney and Inhibition Effect of Reduced Nicotinamide Adenine Dinucleotide (NADH) on Purified ACE Activity.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a significant enzyme that regulates blood pressure. ACE inhibitors are often used in the treatment of hypertension. In this work, ACE was purified and characterized in one step with affinity chromatography from sheep kidneys. ACE was 10305-fold purified and specific activity was 19,075 EU/mg protein. The molecular weight and purity of ACE were found with SDS-PAGE and observed two bands at about 60 kDa and 70 kDa on the gel. The effects of reduced nicotinamide adenine dinucleotide (NADH), an antioxidant compound, on purified ACE activity were also researched. NADH on ACE activity showed an inhibition effect. The inhibition type of NADH was determined to be non-competitive inhibition by the Lineweaver-Burk chart and IC50 and Ki values for NADH were 244.33 and 175.08 µM, respectively. These results suggest that antioxidant substances might be efficient in preventing hypertension.

Inhibition effect of nicotinamide (vitamin B3) and reduced glutathione (GSH) peptide on angiotensin-converting enzyme activity purified from sheep kidney.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) plays a significant role in blood pressure regulation and inhibition of this enzyme is one of the significant drug targets for the treatment of hypertension. In this work, ACE was purified from sheep kidneys with the affinity chromatography method in one step. The purity and molecular weight of ACE were designated using the SDS-PAGE method and observed two bands at around 60 kDa and 70 kDa on the gel. The effects of nicotinamide (vitamin B3) and reduced glutathione (GSH) peptide on purified ACE were researched. Nicotinamide and GSH peptide on purified ACE showed an inhibition effect. IC50 values for nicotinamide and GSH were calculated as 14.3 µM and 7.3 µM, respectively. Type of inhibition and Ki values for nicotinamide and GSH from the Lineweaver-Burk graph were determined. The type of inhibition for nicotinamide and GSH was determined as non-competitive inhibition. Ki value was calculated as 15.4 µM for nicotinamide and 6.7 µM for GSH. Also, GSH peptide showed higher inhibitory activity on ACE activity than nicotinamide. In this study, it was concluded that nicotinamide and GSH peptide compounds, which show an inhibition effect on ACE activity, may have both protective and therapeutic effects against hypertension.

Enhanced purification protocol for the angiotensin-converting enzyme from bovine systems and investigation of the in vitro effect of some active substances.

Angiotensin-converting enzyme (ACE, EC 3.4.15.1) synthesized by endothelial cells and responsible for the regulation of blood pressure was purified from the bovine lung with affinity chromatography method. The purification rate of the ACE of the bovine lung was calculated as 1748- fold. Optimum pH and optimum temperature for the purified ACE were found to be 7.6 and 35-40 °C, respectively. The purity and molecular weight of the ACE were designated with SDS-PAGE. The ACE was found to have three subunits with molecular weights of 57 kDa, 66 kDa, and 190 kDa. Then, the total molecular weight of the ACE was designated as 303 kDa with gel filtration chromatography. The effects of ACE inhibitors captopril, fosinopril, lisinopril, and beta-blockers propranolol, atenolol, and diuretic triamterene on ACE activity were studied. ACE inhibitors lisinopril, captopril, fosinopril, and diuretic triamterene demonstrated an inhibition effect on ACE activity. Beta-blockers indicated no effect on ACE. IC50 values of captopril, fosinopril, lisinopril, and triamterene from the graphical equation were calculated as 0.835 nM, 1.159 µM, 4.085 nM, and 227 µM, respectively. The inhibition type and Ki values of these compounds were determined from Lineweaver-Burk plots. Captopril, fosinopril, lisinopril, and triamterene demonstrated a non-competitive inhibition effect on ACE activity. Ki constants were found as 1.057 nM, 1.675 µM, 6.449 nM, and 419.5 µM, respectively. Captopril indicated the highest inhibitor effect with an IC50 value of 0.835 nM.

Impact of ultrafiltration and nanofiltration of an industrial fish protein hydrolysate on its bioactive properties.

BACKGROUND: Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. RESULTS: The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation. CONCLUSION: Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature.

Polyphenols composition from leaves of Cuphea spp. and inhibitor potential, in vitro, of angiotensin I-converting enzyme (ACE).

ETHNOPHARMACOLOGICAL RELEVANCE: Cuphea is the largest genus of the Lythraceae family. It is popularly known as "sete-sangrias" in Brazil used in folk medicine as a diuretic, antipyretic, anti-inflammatory, laxative and antihypertensive agent. The raw material of Cuphea has shown promising results in the production of fitotherapics, which are chemically characterized by quercetin core flavonoids. AIMS OF THE STUDY: Present work aims to investigate the chemical composition of Cuphea calophylla, Cuphea carthagenensis, Cuphea glutinosa and Cuphea racemosa by UHPLC-MS using ESI-Q-TOF, and also to investigate the inhibition of angiotensin-converting enzyme (ACE) in vitro. MATERIALS AND METHODS: Leaves extraction was conducted by an ultrasound-assisted system under the following conditions: 40% ethanol, particle size ≤180 µm, plant:solvent ratio 1:20 (w/v) for 30 min. The leaf extracts were analyzed by UHPLC-MS positive mode ionization. For the inhibition of ACE, the leaf extracts used were obtained from different Cuphea species collected from several regions of Rio Grande do Sul (Brazil). RESULTS: In total 26 polyphenolic compounds were proposed, which were mostly derived from quercetin, myricetin, and kaempferol. Of these compounds, ten are described in the genus for the first time. The ACE-inhibiting activities are presented in descending order: miquelianin (32.41%), C. glutinosa 1 (31.66%), C. glutinosa 5 (26.32%) and C. carthagenensis 1 (26.12%). CONCLUSION: The obtained results suggest that the ACE-inhibiting potential may be increased by the interactions among the different phytoconstituents present in the crude extract. These results corroborate with the popular usage of Cuphea genus as diuretic and antihypertensive agents in folk medicine.

In vitro effect of oxidized and reduced glutathione peptides on angiotensin converting enzyme purified from human plasma.

Angiotensin converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1) plays an important role in the regulation of blood pressure. In this study, ACE was purified from human plasma by affinity chromatography in single step. The enzyme purified in 5367-fold from human plasma and specific activity was found to be 1208 EU/mg protein. The purity and molecular weight of ACE were determined by SDS-PAGE, which indicated two bands at around 60 kDa and 70 kDa on the gel. Effect of oxidized glutathione (GSSG) peptide and reduced glutathione (GSH) peptide on purified ACE activity were also investigated in which lisinopril was used as reference inhibitor. GSSG showed activation effect on ACE activity whereas GSH provided inhibition effect. In the lights of activity (%) versus activator graph for GSSG and activity (%) versus inhibitor graphs for GSH and lisinopril; IC50 values for GSH and lisinopril were determined to be 16.2 µM and 0.781 nM, respectively. Type of inhibition for GSH and lisinopril from graph Lineweaver-Burk was found to be reversible non-competitive inhibition and Ki constants for GSH and lisinopril were calculated as 11.7 µM and 0.662 nM, respectively.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Expression of angiotensin I-converting enzymes and bradykinin B2 receptors in mouse inner medullary-collecting duct cells.

We described in mouse inner medullary-collecting duct cells (mIMCD-3) the somatic and the N-domain ACE synthesis and its interaction with the kallikrein-kinin system co-localized in the same cells. We purified two ACE forms from culture medium, M1 (130 kDa) and M2 (N-domain, 60 kDa), and cellular lysate, C1 (130 kDa) and C2 (N-domain, 60 kDa). Captopril and enalaprilat inhibited the purified enzymes. The immunofluorescence studies indicated that ACE is present in the membrane, cytoplasm and in the cell nucleus. Kinin B1 and B2 receptors were detected by immunofluorescence and showed to be activated by BK and DesR9 BK, increasing the acidification rate which was enhanced in the presence of enalaprilat. The presence of secreted and intracellular ACE in mIMCD-3 confirmed the hypothesis previously proposed by our group for a new site of ACE secretion in the collecting duct.

Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone.

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.

Human lung angiotensin converting enzyme. Purification and antibody preparation.

To enable its immunohistologic localization, angiotensin converting enzyme (EC 3.4.15.1) from human lung was solubilized by trypsinization and purified approximately 2,660-fold to apparent homogeneity from a washed lung particulate fraction. The specific activity of pure enzyme was estimated to be 117 mumol/min per mg protein with the substrate hippuryl-l-histidyl-l-leucine. Consistent with previously described lung enzyme studies, catalytic activity was strongly inhibited by EDTA, O-phenanthroline, SQ 20,881, and SQ 14,225 and increased by CoCl(2). SQ 20,881 was a somewhat more potent inhibitor than SQ 14,225, unlike rabbit lung enzyme. The Michaelis constant (K(m)) with hippuryl-l-histidyl-l-leucine was 1.6 mM. The molecular weight was estimated at 150,000 from sucrose density gradient centrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide chain estimated at 130,000 daltons. Rabbit antibody to human lung enzyme was prepared by parenteral administration of pure angiotensin-converting enzyme in Freund's adjuvant. Rabbit antibody to human lung angiotensin-converting enzyme appeared to crossreact weakly with the rabbit enzyme and strongly inhibited the catalytic activity of the enzymes from human serum, lung, and lymph node. The specificity of the rabbit antibody and purity of the final human lung enzyme preparation was suggested by the single precipitin lines obtained by radial double immunodiffusion, and by the coincidence of enzyme catalytic activity and immunoreactivity on polyacrylamide gel electrophoresis, with both relatively pure and highly impure enzymes. Generally applicable sensitive analysis of acrylamide gels for immunoreactivity (and subsequently for any other activity) by use of intact gel slices in radial double immunodiffusion was devised. Human lung enzyme was very tightly bound to and catalytically active on anti-human enzyme antibody covalently bound to Sepharose 4B, and could not be readily dissociated without inactivation. Antibody to human lung angiotensin converting enzyme has permitted tissue localization of the enzyme, which appears to be clinically useful in diseases associated with abnormal abundance of angiotensin-converting enzyme in tissues, such as sarcoidosis.

Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats.

The long-term administration of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, produces coronary vascular remodeling and myocardial hypertrophy in animals. This study used a rat model to investigate the role of angiotensin I converting enzyme (ACE) in the pathogenesis of such changes. We studied the following groups, all of which received drug treatment in their drinking water: untreated controls, and those administered L-NAME, L-NAME, and an ACE inhibitor (ACEI), and L-NAME and hydralazine. Cardiovascular structural changes and tissue ACE activities were evaluated after the first, fourth, and eighth week of treatment. In rats treated with L-NAME alone, vascular remodeling was evident at the fourth and eighth week, and myocardial hypertrophy was present at the eighth week of treatment. The vascular and myocardial remodeling were characterized by increased tissue ACE activities and immunodetectable ACE in those tissues. These changes were markedly reduced by ACEI, but not by hydralazine treatment. Increased local ACE expression may thus be important in the pathogenesis of cardiovascular remodeling in this model.

Presence of angiotensin converting enzyme isoforms in larval lepidoptera (Spodoptera littoralis).

In this research the presence of angiotensin converting enzyme (ACE) in larvae of the lepidopteran Spodoptera littoralis was evaluated. Making use of the substrate Abz-FRK-(Dnp)P-OH and the specific inhibitor captopril at 10 microM, ACE activity was determined in a fluorescence assay for intact larvae, hemolymph, head, midgut and dorsal tissue. In dorsal tissue and hemolymph, ACE activity was highest. These data are consistent with a possible role for ACE in contractions of the dorsal vessel and metabolism of circulating peptide hormones in the hemolymph. After the presence of ACE was confirmed, a sequential procedure of anion exchange and size exclusion chromatography was applied to purify ACE from whole wandering larvae (last stage). With this procedure, three different ACE pools were collected that cleaved the fluorogenic substrate Abz-FRK-(Dnp)P-OH. Activity could be inhibited by a final concentration of 2.5 microM captopril. In addition, two out of three samples eluted at different salt concentration and thus ACE 1, 2 and 3 represent at least two different ACE isoforms. These data reveal that ACE is present in S. littoralis and that at least two out of three isolated ACE forms are truly isoforms.

Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene.

Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptidyl carboxypeptidase that catalyzes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II. By analyzing cDNA and genomic DNA, we have constructed a consensus sequence encoding the testis isozyme of mouse ACE. Testis ACE cDNA contains 2,435 base pairs and encodes a protein of 732 amino acids. The N-terminal 66 amino acids are unique to the testis isozyme, while the remaining 666 are identical to the carboxyl half of mouse somatic ACE. The overall conservation of amino acid sequence between the testis isozymes of the mouse, rabbit, and human is 78 to 84%. The conservation of amino acids for the N-terminal domain uniquely expressed within the testis is 63 to 67% between these species. Primer extension and RNase protection experiments show that RNA transcription of the testis ACE isozyme begins 16 or 17 bases upstream from the translation start site. A sequence element resembling a TATA box is found 25 bases 5' of the transcription start site. To create its unique isozyme of ACE, the testis begins mRNA transcription in the middle of the exonic-intronic structure of somatic ACE, within a sequence treated as an intron by somatic tissues. Testis ACE is not the result of alternative RNA splicing but seems due to the start of transcription at a unique site within the ACE gene.

Porcine germinal angiotensin I-converting enzyme: isolation, characterization and molecular cloning.

Germinal angiotensin I-converting enzyme (gACE) was purified to homogeneity from porcine seminal plasma. The molecular weight of the purified enzyme was calculated to be 182,000 on non-denaturing PAGE and 94,000 and 93,000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of two identical subunits in seminal plasma. The K(m), V(max), K(cat) and K(cat)/K(m) values of gACE at optimal pH (pH 7.2) were 680 microM, 1.0 micromol/mg/min, 33.1 s(-1) and 4.87 x 10(4) s(-1) M(-1) for Z-Val-Lys-Met-MCA, respectively. gACE was potently inhibited by EDTA, 1,10-phenanthroline, captopril and lisinopril, and it promptly released the dipeptides His-Leu and Phe-Arg from angiotensin I and bradykinin. Met- and Leu-enkephalins, neuromedine B and beta-neo-endorphin were also good natural substrates for gACE. We determined the structure of gACE cDNA from the porcine testis, and deduced the amino acid sequence of gACE. The cDNA is composed of 2508 bp of nucleotides in length and encodes 745 amino acids in the coding region. The overall homology of amino acid sequences between porcine, human, sheep and rat gACEs is 72.6 to 84.7%. Zinc-binding motif, chloride-binding site and positions of cysteine residues were well conserved.

Angiotensin converting enzyme from sheep mammary, lingual and other tissues.

Occurrence of angiotensin converting enzyme (ACE) in mammary gland and tongue taste epithelium was demonstrated for the first time. Six times higher ACE activity in lactating mammary gland, than non-lactating mammary gland, suggested pregnancy and lactation hormonal dependent expression of ACE in female mammals. ACE activity was highest in choroid plexus, less in spinal cord and moderate in cerebrum, medulla, cerebellum and pons. Distribution of ACE in different regions of skin, kidney and among other tissues was different. Presence of ACE in adrenal glands, pancreas, bone marrow and thyroid gland indicated functions other than blood pressure homeostasis for this enzyme.