Formation of angiotensin III by angiotensin-converting enzyme.

Angiotensin III is formed from des-Asp1 -angiotensin I by angiotensin-converting enzyme. The Km (11 muM) of the reaction is one-third of that for the conversion of angiotensin I into angiotensin II. As suggested by the Km values, bradykinin, peptide BPP9a and angiotensins II and III are better inhibitors of the formation of angiotensin II than of the formation of angiotensin III.     

Role of converting enzyme in the cardiovascular and adrenal cortical responses to (des-Asp1)-angiotensin I.

(Des-Asp1)-angiotensin I, angiotensin II and III were evaluated for pressor activities in conscious nephrectomized rats and for steroidogenic actions in rat adrenal zona glomerulosa. The pressor effect of this angiotensin nonapeptide was similar to that found with mole-equivalent doses of angiotensin III (one-third as active as angiotensin II) and was significantly attenuated by pretreatment with the 0. jararaca nonapeptide converting enzyme inhibitor. Hence, (des-Asp1)-angiotensin I is a substrate for converting enzyme in vivo, and the rapid conversion indicates that an alternate pathway for the formation of angiotensin III could exist. (Des-Asp1)-angiotensin I possessed only 0.1% of the activity of angiotensin III as a steroidogenic agent in cell suspensions of rat adrenal zona glomerulosa. Angiotensin I was a weak steroidogenic agent in vitro (1%) and was not blocked by an inhibitor of converting enzyme. Adrenal cells dispersed from the outer zone of the cortex would appear to be devoid of significant converting enzyme activity.     

Study of peptides inhibiting the angiotensin-converting enzyme of human seminal plasma.

Several peptides were investigated for their inhibitory capacity against dipeptidyl carboxypeptidase (angiotensin-converting enzyme) from human seminal fluid. The strongest inhibitor was the nonapeptide SQ 20881. A marked inhibition was also shown by the compounds Phe-Ala-Pro and Boc-Phe-Ala-Pro, which behaved as competitive inhibitors. Among the peptides related to angiotensin, angiotensin III was the strongest inhibitor, followed by angiotensin II and the C-terminal hexapeptide of angiotensin II. The results indicate the dipeptidyl carboxypeptidase of human semen is very similar to pulmonary dipeptidyl carboxypeptidase in its susceptibility to peptide inhibitors. In view of these and other previously reported similarities, it is possible that both enzymes are identical.     

Affinity chromatography and some properties of the angiotensin-converting enzyme from human heart

Human heart angiotensin-converting enzyme has been purified by affinity chromatography on immobilized N-[1(S)-carboxy-5-aminopentyl]-Gly-Gly. The isolation procedure permitted a 1650-fold-purified enzyme to be obtained. The specific activity of angiotensin-converting enzyme was 38 units per mg protein. The molecular weight of enzyme determined by polyacrylamide gel electrophoresis in denaturing conditions was 150,000. The isoelectric point (5.3) of the enzyme was determined by chromatofocusing. The Km values of the enzyme for Bz-Gly-His-Leu and angiotensin I are 1.2 mM and 10 microM, respectively. Substrate inhibition of heart angiotensin-converting enzyme with a K's of 14 mM has been shown. The human heart enzyme is inhibited by SQ 20881 (IC50 = 40 nM). It was shown that NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- are activators of the heart angiotensin-converting enzyme, whereas CH3COONa and NaNO3 have no effect on a catalytic activity of the enzyme.     

Isolation and molecular kinetic properties of the angiotensin-converting enzyme from the human heart

An angiotensin-converting enzyme was isolated from human heart using N[-1(S)-carboxy-5-aminopentyl]glycyl-glycine as an affinity adsorbent. The isolation procedure resulted in an enzyme purified 1650-fold. The enzyme specific activity was 38.0 u./mg protein, Mr = 150 kD. The pH optimum for the angiotensin-converting enzyme towards Hip-His-Leu lies at 7.8, Km = 1.2 mM. The enzyme was inhibited by the substrate (Ks' = 14 mM). The enzyme effectively catalyzed the hydrolysis of angiotensin I (Km = 10 microM; kcat = 250 s-1). NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- activated the enzyme, whereas CH3COONa and NaNO3 did not influence the enzyme activity. It was found that the bradykinin-potentiating factor inhibited the cardiac angiotensin-converting enzyme with IC50 = 4.0 X 10(-8) M.     

Evolution of angiotensin-converting enzyme inhibitors

The renin-angiotensin system is perhaps the most important hormonal system in the regulation of blood pressure. Its influence on blood pressure is mediated by the potent vasoconstrictor angiotensin II. Since angiotensin-converting enzyme performs the last step in the biosynthesis of angiotensin II, inhibition of this enzyme has attracted the attention of many researchers as a novel approach in the control of high blood pressure. The evolution of inhibitors of this enzyme will be traced from the early snake venom peptide inhibitors to the drugs currently available for the treatment of high blood pressure and congestive heart failure.     

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.     

Pulmonary angiotensin-converting enzyme. Interspecies homology and inhibition by heterologous antibody in vivo.

Goat antibody against pure rabbit pulmonary angiotensin-converting enzyme was used to probe homology of converting enzymes from other species. Immunologically cross-reactive material was found in detergent-solubilized extracts of lung particles from rat, guinea pig, and dog by double immunodiffusion, radioimmunoassay, and inhibition of enzyme activity. No homology was demonstrable with bovine, frog, or chicken lung extracts. Antibodies from different individual goats yielded comparable estimates of homology by immunodiffusion and radioimmunoassay. In contrast, they varied greatly in extent and specificity of their inhibitory action on heterologous enzyme activity. The vasopressor effect of angiotensin I and the vasodepressor effect of bradykinin were diminished and potentiated, respectively, in rats treated with anti-rabbit enzyme antibody. A smaller but significant immune-dependent inhibition of the vasopressor response to angiotensin II was also observed.     

Angiotensin-converting enzyme activity in isolated brain microvessels.

The localization of angiotensin-converting enzyme (kininase II; ACE) in bovine cerebral cortex was studied by mechanically isolating microvessels from surrounding brain parenchyma. ACE specific activity, as assayed by generation of L-histidyl-L-leucine from the synthetic substrate hippuryl-L-histidyl-L-leucine, was enriched approximately 30 times in microvessels compared to homogenates of intact cerebral cortical gray matter. The nonapeptide 9a, SQ20,881), the orally active anti-hypertensive drug, 2-D-methyl-3-mercaptopropanoyl-L-proline (SQ14,225), and the vasoactive peptides bradykinin and angiotensin II inhibited this activity in a dose-dependent fashion. Brain microvessel ACE required chloride for optimal activity, was potentiated by cobalt nitrate, and was inhibited by the chelating agents EDTA and o-phenanthroline. Enzymatic generation of histidyl-leucine also was observed with the naturally occurring decapeptide substrate angiotensin I. In addition, microvessels obtained from bovine cerebellar cortex, hippocampus and corpus striatum, as well as from the cerebral cortex of Sprague-Dawley rats, were enriched in ACE activity. The presence of angiotensin-converting enzyme in brain microvessels suggests that cellular components of the blood-brain barrier may participate in the metabolism of peptide hormones such as angiotensin I and bradykinin within the central nervous system.     

Mechanism of increased angiotensin-converting enzyme activity stimulated by platelet-activating factor

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 x 10(-10) to 1 x 10(-6) M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 x 10(-6) M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca2+ influx and inositol 1,4,5-trisphosphate (IP3) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP3. To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca2+ influx and IP3 release stimulated by 10(-6) M of PAF were suppressed by about 60-70%. ACE activity was also inhibited up to 70% in the presence of PAF (10(-10) - 10(-6) M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activity in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP3-mediated Ca2+ release from intracellular stores.     

CI-906 and CI-907: new orally active nonsulfhydryl angiotensin-converting enzyme inhibitors

CI-906, [3S-[2[R*(R*)]], 3R*]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]-amino]-1-oxopropyl] 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid, monohydrochloride, and CI-907, [2S-[1[R*(R*)]], 2 alpha, 3a beta, 7a 7a beta]-1-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino] 1-oxopropyl]octahydro-1H-indole-2-carboxylic acid, monohydrochloride, are two new nonsulfhydryl-type angiotensin-converting enzyme (ACE) inhibitors. Monoester (prodrug) and diacid forms produced concentration-related ACE inhibition in guinea pig serum (IC50 for CI-906 = 8.3 X 10(-9) M, diacid = 2.8 X 10(-9) M; CI-907 = 1.0 X 10(-7) M, diacid = 2.6 X 10(-9) M). In isolated rabbit aortic rings and in in vivo rat and dog autonomic studies, both compounds were highly specific in suppressing the contractile or pressor responses to angiotensin I. In two-kidney, one-clip Goldblatt (renin-dependent) hypertensive rats there was a good correlation between the inhibition of vascular converting enzyme and blood pressure lowering and a poor correlation between blood pressure lowering and plasma and brain converting enzyme inhibition. Cardiovascular, pulmonary, and central nervous system performance evaluations showed no side effects or gross toxicity. The preclinical profile shows CI-906 and CI-907 to be specific, potent, orally active ACE inhibitors. They are expected to have therapeutic utility in hypertension and in any other condition where converting enzyme inhibition would be useful.     

Isolation and properties of the angiotensin-converting enzyme from human lungs

Using chromatofocusing, an angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human lung. The procedure allows for 24 300-fold purification of the enzyme. The enzyme specific activity is 36.3 u. per mg protein; Mr as determined by polyacrylamide gel electrophoresis is 150 000. The lung enzyme after solubilization by trypsin treatment was found to be heterogeneous. Four isoforms of the enzyme with pI 5.3, 4.9, 4.8 and 4.6 were identified. The pH-optimum for the enzyme with respect to hippuryl-L-histidyl-L-leucine hydrolysis lies at 8.3; Km = 2.8 mM. The effect of Cl- on the enzyme activity was studied. It was found that the bradykinin-potentiating factor (SQ 20 881) inhibits the human lung angiotensin-converting enzyme (I50 = 1.6 X 10(-8) M).     

血管紧张素转换酶2-血管紧张素1-7-Mas轴对血管作用的研究进展

血管紧张素转换酶2（ACE2）和Mas受体的发现使人们对肾素-血管紧张素（RAS）有了更全面的认识。ACE2可水解血管紧张素Ⅰ和血管紧张素Ⅱ直接或间接生成血管紧张素1-7（Ang 1-7）,并与高血压的形成密切相关。Ang 1-7主要通过Mas受体引起血管舒张、抑制细胞增殖。ACE2-Ang1-7-Mas轴的发现为RAS的研究、高血压等心血管疾病的防治和新药开发提供了新的思路和方向。     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Isolation of human liver angiotensin-converting enzyme by chromatofocusing

Angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human liver by chromatofocusing. The isolation procedure permitted us to obtain a 9000-fold purified enzyme with a 22% yield. Specific activity of the angiotensin-converting enzyme was 10 units/mg of protein. The molecular mass of enzyme determined by polyacrylamide gel electrophoresis under denaturing conditions was 150,000. The isoelectric point (4.2-4.3) was also determined by chromatofocusing. The Km values of the enzyme for hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine are 5000 and 125 microM, respectively. The human liver angiotensin-converting enzyme is inhibited by bradykinin-potentiating factor SQ 20881 (IC50 = 18 nM).     

Angiotensin I converting enzyme from human plasma.

The angiotensin I converting enzyme was purified 101 000-fold to homogeneity from human plasma by a combination of chromatographic and electrophoretic techniques. The enzyme is similar to other angiotensin I converting enzymes. It is an acidic glycoprotein consisting of a single polypeptide chain of molecular weight 140 000 with an isoelectric point of 4.6. The enzyme requires chloride ion for activity and is inhibited by ethylenediaminetetraacetic acid, angiotensin II, bradykinin, bradykinin potentiating factor nonapeptide, and 3-mercapto-2-D-methylpropanoyl-L-proline (SQ-14,225). The purified preparation cleaves bradykinin as well as angiotensin II and hippuryl-L-histidyl-L-leucine. Its specific activity with angiotensin I is 2.4 units per mg and with hippuryl-L-histidyl-L-leucine is 31.4 units per mg.     

Rapid affinity chromatographic purification of human lung and kidney angiotensin-converting enzyme with the novel N-carboxyalkyl dipeptide inhibitor N-[1(S)-carboxy-5-aminopentyl]glycylglycine

Human angiotensin-converting enzyme has been purified, in a single chromatographic step, using a novel N-carboxyalkyl dipeptide CA-GlyGly (N-[1(S)-carboxy-5-aminopentyl]glycylglycine) synthesised in our laboratory. CA-GlyGly is a weak competitive inhibitor, Ki = 0.18 mM, and its inhibitory profile is markedly pH-dependent. Human lung and kidney angiotensin-converting enzyme were solubilised with Triton X-100 and after ammonium sulphate fractionation the crude extract was applied to a column containing CA-GlyGly coupled to agarose via a 2.8 nm spacer group. Electrophoretically pure human angiotensin-converting enzyme could be eluted by raising the pH of the chromatography buffer from 7.50 to 9.50. The specific activity of human angiotensin-converting enzyme purified from lung was 104 units/mg, while that from kidney was 88 units/mg. Molecular weight for both enzymes was estimated to be 160,000. The Km with respect to hippuryl-L-histidyl-L-leucine was 1.9 mM in the case of lung angiotensin-converting enzyme and 1.7 mM in that of kidney angiotensin-converting enzyme, while for the substrate angiotensin I Km values were 62 microM and 76 microM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

Kinetic-controlled hydrolysis of Leu-Val-Val-hemorphin-7 catalyzed by angiotensin-converting enzyme from rat brain

Leu-Val-Val-hemorphin-7 (LVV-H7, LVVYPWTQRY), an opioid peptide, was found to be hydrolyzed sequentially by rat brain angiotensin-converting enzyme (ACE) in three steps through dipeptidyl carboxypeptidase activity. The kinetic constants evaluated were in order of: k(1) (0.19 min(-1))>k(2) (0.0008 min(-1)) approximately k(3) (0.0006 min(-1)) in 10 mM NaCl at pH 7.5 giving rise to LVV-H5 almost quantitatively. The decapeptide was noted to be hydrolyzed 164- and 346-fold more efficiently than angiotensin I (Ang I) in k(cat) and kcat/Km values, respectively, at their optimal conditions. The kinetic-controlled preferential action of the brain enzyme on LVV-H7 is suggestive of its multiple roles in vivo.     

The functional role of tyrosine-200 in human testis angiotensin-converting enzyme.

The active site of angiotensin-converting enzyme (ACE) has been shown by chemical modification to contain a critical tyrosine residue, identified as Tyr-200 in human testis ACE (hTACE). We have expressed a mutant hTACE containing a Tyr-200 to Phe mutation. The mutant exhibits a marked decrease in kcat: 15-fold and 7-fold for the hydrolysis of furanacryloyl-Phe-Gly-Gly and angiotensin I, respectively, whereas its Km increases by only 1.6- and 2.2-fold, respectively. We conclude that Tyr-200 is not required for substrate binding. Instead, the effect on kcat together with a 100-fold decrease in affinity for the ACE inhibitor lisinopril indicates that Tyr-200 may participate in catalysis by stabilizing the transition state complex. Thus, Tyr-200 in hTACE has a role analogous to that of Tyr-198 in carboxypeptidase A.