Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase

We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser-Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz-Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase.     

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.     

Enalapril in the treatment of hypertension with renal artery stenosis.

The converting enzyme inhibitor enalapril, in single daily doses of 10-40 mg, was given to 20 hypertensive patients with renal artery stenosis. The blood pressure fall six hours after the first dose of enalapril was significantly related to the pretreatment plasma concentrations of active renin and angiotensin II and to the concurrent fall in angiotensin II. Blood pressure fell further with continued treatment; the long term fall was not significantly related to pretreatment plasma renin or angiotensin II concentrations. At three months, 24 hours after the last dose of enalapril, blood pressure, plasma angiotensin II, and converting enzyme activity remained low and active renin and angiotensin I high; six hours after dosing, angiotensin II had, however, fallen further. The rise in active renin during long term treatment was proportionally greater than the rise in angiotensin I; this probably reflects the fall in renin substrate that occurs with converting enzyme inhibition. Enalapril alone caused reduction in exchangeable sodium, with distinct increases in serum potassium, creatinine, and urea. Enalapril was well tolerated and controlled hypertension effectively long term; only two of the 20 patients required concomitant diuretic treatment.     

The inhibition of angiotensin converting enzyme in chronic renal hypertension by a synthetic peptide.

Renal hypertension was produced in 14 Sprague-Dawley rats by ligating both poles of one kidney followed in one week by unilateral nephrectomy. Biweekly subcutaneous injections of SQ 20858 reduced the blood pressure in the chronic renal hypertensive animals. Upon discontinuing the injections the blood pressure rose to pretreatment levels. No angiotensin II activity was seen in seven of the renal group treated with SQ indicating a complete block in serum converting enzyme activity. Likewise, serum with only angiotensin I activity when added to normal serum containing converting enzyme, continued to show angiotensin I activity. It is concluded that SQ 20858 is effective in lowering blood pressure in chronic renal hypertensive rats presumably by partially inhibiting converting enzyme.     

Angiotensin II-producing proteases from granulomatous tissue reaction in mice infected with Schistosoma mansoni

1. Angiotensin I hydrolases, Mr 140,000 and Mr 70,000 were separated by gel filtration from Tris-HCl buffer extract of hepatic granulomas developed in mice with schistosomiasis. Two enzymes had different substrate specificity. 2. Mr 140,000 hydrolase activity was inhibited by captopril as reported for angiotensin converting enzyme (ACE), while that of Mr 70,000 hydrolase activity was inhibited by potato carboxypeptidase inhibitor. 3. An intermediary, des-Leu10-angiotensin I and then angiotensin II were formed from angiotensin I by Mr 70,000 hydrolase. 4. The findings suggest that Mr 70,000 enzyme is tissue carboxypeptidase A, and it generates angiotensin II in granulomatous inflammation as does ACE.     

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.     

Characteristics of the Angiotensin I Converting Enzyme from Dog Lung

AFTER the demonstration of two forms of angiotensin by Skeggs et al.¹ and their preparation of an enzyme capable of catalysing the conversion of angiotensin I to angiotensin II (converting enzyme) from horse plasma², attention centred round the blood as the physiologically significant site of converting enzyme. But when Ng and Vane³ showed that angiotensin I was converted to angiotensin II in the lungs and that the rate of conversion was sufficient to account for most of the conversion during a single passage through the circulation, attention was directed towards the lung. Bakhle⁴ partially purified the converting enzyme from dog lung but this preparation contained too much angiotensinase activity for extensive analysis of the converting enzyme to be possible. There have been several further studies of the conversion of angiotensin I to angiotensin II by extracts from various animal sources^5–8, including the purification of converting enzyme from hog plasma⁹. We have now obtained a preparation of the enzyme from dog lung with only slight contamination by angiotensinase and have studied its characteristics with particular emphasis on its ionic requirements.     

Peptide inhibitors of converting enzyme.

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the renin-angiotensin system, and by angiotensin II and its analogues. Des-Asp¹ angiotensin II (angiotensin III) 0.1 mM and tridecapeptide renin substrate 0.1 mM are both effective inhibitors of human lung, plasma and porcine plasma converting enzymes. Des-Asp¹-Arg² angiotensin II also was an effective inhibitor of plasma enzymes. Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate. The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system.     

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.     

Differentiation of nephrotensin from the renin angiotensin system.

An investigation of the relationship between nephrotensin and the renin angiotensin system was carred out. Nephrotensin was found in the plasma of rats with renal clip hypertension and with chemically induced kidney damage. There was no demonstrable correlation between presence of nephrotensin and plasma renin activity, and the pressor activity of nephrotensin was not altered by previous immunization of test animals with angiotensin II nor by pretreatment with angiotensin I converting enzyme inhibitor. These results indicate that nephrotensin is different from the components of the renin-angiotensin system.     

In vitro renin inhibition to prevent generation of angiotensins during determination of angiotensin I and II

To prevent in vitro generation of angiotensins, the renin inhibitor CGP 29287 (CGP) was added to blood sampling tubes. Plasma immunoreactive angiotensin (ir-ANG) I and II were simultaneously measured by radioimmunoassay after rapid and quantitative extraction from a single plasma sample on phenylsilylsilica (Bondelut PH). True plasma ANG-(1-8)octapeptide was determined after additional separation of the different angiotensins by high performance liquid chromatography. Ir-ANG II/CGP showed the known linear relationship with ANG-(1-8)octapeptide (r = 0.87, n = 23), but - in contrast to studies without addition of CGP - the y-axis intercept which presumably represents cross-reacting angiotensins other than ANG II was very small. Ir-ANG II/CGP concentrations fell below 1 fmol/ml after converting enzyme inhibition. The results suggest that CGP 29287 prevents in vitro generation of ANG I and ANG II as well as the ANG-metabolites. Ir-ANG I/CGP measured after Bondelut PH extraction of the plasma was strongly correlated with ir-ANG I obtained after blood ethanol extraction (r = 0.97, n = 23). Thus, it is now possible to measure reliably both ANG I and ANG II within the same plasma extract after a simple extraction procedure.     

Effect of captopril (SQ 14225) on blood pressure,plasma renin activity and angiotensin I converting enzyme activity.

Seven patients with essential hypertension and seven patients with hypertension associated with renal artery stenosis received captopril (SQ 14225), an inhibitor of angiotensin I converting enzyme. There was a significant reduction in mean blood pressure, from 176/113 +/- 4/3 mm Hg during the control period to 140/90 +/- 5/3 mm Hg during captopril administration. Five patients received captopril alone and nine patients needed hydrochlorothiazide in addition to control their blood pressure. Captopril produced a significant increase in peripheral plasma renin activity. When measured 12 hours after the administration of captopril the angiotensin I converting enzyme activity was found to be similar to that during the control period even though the blood pressure was at or near normal. These findings indicate that although captopril is an effective antihypertensive agent, its action does not depend only on inhibition of plasma angiotensin I converting enzyme activity.     

The angiotensin I converting enzyme.

The angiotensin I converting enzyme (kininase II; peptidyl dipeptidase; EC3.4.15.1) has a dual function: it converts angiotensin I to angiotensin II and it inactivates bradykinin. Lung, kidney, guinea pig plasma and testicles are among the richest sources of the enzyme. Vascular endothelial cells and bursh borders of renal proximal tubular cells contain high concentrations of the enzyme. The availability of synthetic peptide inhibitors was a great help in establishing the function of converting enzyme in normal and pathological conditions.     

Comparison of ACE activity in amphibian tissues: Rana esculenta and Xenopus laevis

Angiotensin converting enzyme (ACE) is the dipeptidyl-carboxypeptidase of the renin-angiotensin system involved in the control of blood pressure and hydromineral metabolism. It converts angiotensin I to angiotensin II, the biologically active octapeptide. Angiotensin converting enzyme-like activity has been demonstrated in a wide range of vertebrates. The presence of ACE was investigated in tissues of two amphibian species, the frog Rana esculenta and the toad Xenopus laevis. ACE activities were determined by specific substrate hydrolysis in gut, gonads, lung, kidney, heart, liver, skin, erythrocytes, and muscle homogenates and plasma by means of high performance liquid chromatography. Significant ACE activity was found in gut, gonads, lung and kidney, while that in heart, liver, skin, erythrocytes, muscle, and plasma was very low. Testis of toad contained the highest ACE activity, while that in erythrocytes of male and female frogs was notable.     

Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence.

The cellular and subcellular sites of angiotensin converting enzyme (kininase II) in lung tissue and endothelial cells in culture were examined by immunocytochemical and immunofluorescence techniques. Converting enzyme is capable of inactivating bradykinin and of converting angiotensin I to its potent lower homolog, angiotensin II. Immunocytochemistry at the electron microscope level used goat anti- (pig lung and angiotensin converting enzyme) coupled to 11-MP (11-microperoxidase) via glutaraldehyde or to 8-MP (8-microperoxidase) via a bifunctional active ester, bis-succinyl succinate. The latter conjugate, which does not contain complex polymers, has been characterized in detail in terms of immunoreactivity and peroxidase activity.     

Low molecular weight angiotensin I converting enzyme from rat lung.

A low molecular weight angiotensin I converting enzyme (light angiotensin enzyme) was isolated from a homogenate of rat lung subjected to dialysis against sodium acetate at pH 4.8. This enzyme has a molecular weight of 84 000 on Sephadex G-200 and a molecular weight of 91 000 on SDS-poly-acrylamide gel as compared with a molecular weight of 139 000 for angiotensin I converting enzyme on SDS-polyacrylamide. Light angiotensin enzyme was activated by NaCl and inhibited by EDTA, angiotensin II, and bradykinin potentiating factor nonapeptide. Light angiotensin enzyme cross-reacted with antibody prepared against angiotensin I converting enzyme and stained with periodic acid-Schiff reagent as a glycoprotein. The evidence suggests that light angiotensin enzyme is a fragment of the higher molecular weight enzyme.     

Conversion of angiotensin I to angiotensin II by cathepsin A isoenzymes of porcine kidney

We have reported the existence of a carboxypeptidase in a human renal extract that converts Angiotensin I (AI) to Angiotensin II (AII) in two steps with des-leu-AI (dl-AI) being formed as an intermediate. Since this carboxypeptidase had properties similar to cathepsin A, the ability of cathepsin A to metabolize AI was studied. Cathepsin A was purified from hog kidney with enzyme activity being monitored using both benzyloxycarbonyl-glutamyl-tyrosine (ZGT) and AI as substrates. The procedure separated the expected large and small molecular weight forms of cathepsin A as well as two additional isoenzymes. All of the isoenzymes had carboxypeptidase activity with ZGT, AI, and dl-AI. No detectable cleavage of AII was observed. Cathepsin A,S (small) activity with ZGT or AI as substrate was inhibited to a similar extent by diisopropylfluorophosphate, mersalyl acid, and a decapeptide renin inhibitor. It is concluded that the renal angiotensin carboxypeptidase activity is catalyzed by cathepsin A. By its ability to convert AI to AII, cathepsin A may be a component of the intrarenal renin-angiotensin system.     

Substance K and substance P as possible endogenous substrates of angiotensin converting enzyme in the brain

In the brain angiotensin converting enzyme is highly localized to a striatonigral pathway, which contains no endogenous angiotensin. Substance P, also localized to a striatonigral pathway, is degraded by ACE via two different pathways. The lung and striatal isozymes of angiotensin converting enzyme exhibit differential cleavage of substance P, with lung preferring an initial tripeptide cleavage, and striatum an initial dipeptide cleavage. Substance K is degraded by the striatal isozyme but is not cleaved by the lung isozyme. Substance P 5-11 is not cleaved by either form of angiotensin converting enzyme.     

Characterization of Angiotensin Converting Enzyme-2 (ACE2) in Human Urine

Angiotensin converting enzyme-2 (ACE2) is a recently described membrane-bound carboxypeptidase identified by its homology to ACE, the enzyme responsible for the formation of the potent vasoconstrictor angiotensin II (Ang II). ACE2 inactivates Ang II and is thus thought to act in a counter-regulatory fashion to ACE. ACE2 is highly expressed in epithelial cells of distal renal tubules, and recent evidence indicates that expression is increased in a range of renal diseases. A soluble form of ACE, generated by proteolytic cleavage of the membrane-bound form, has been shown to be present in urine; although evidence for a similar release of ACE2 has been reported in cell culture, it is not yet known whether this occurs in vivo. The present study has identified ACE2 in human urine, both by a sensitive fluorescence-based activity assay and by Western immunoblot. Levels of ACE2 were surprisingly higher than ACE, which may reflect preferential targeting of the enzyme to the luminal surface of the renal epithelium. Future studies will determine whether increased expression of ACE2 in renal diseases are reflected in higher urinary levels of this novel enzyme.Australian Peptide Conference Issue.     

Identification of a highly specific chymase as the major angiotensin II-forming enzyme in the human heart

Although angiotensin II (Ang II)-forming enzymatic activity in the human left cardiac ventricle is minimally inhibited by angiotensin I (Ang I) converting enzyme inhibitors, over 75% of this activity is inhibited by serine proteinase inhibitors (Urata, H., Healy, B., Stewart, R. W., Bumpus, F. M., and Husain, A. (1990) Circ. Res. 66, 883-890). We now report the identification and characterization of the major Ang II-forming, neutral serine proteinase, from left ventricular tissues of the human heart. A 115,150-fold purification from human cardiac membranes yielded a purified protein with an Mr of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based upon its amino-terminal sequence, the major human cardiac Ang II-forming proteinase appears to be a novel member of the chymase subfamily of chymotrypsin-like serine proteinases. Human heart chymase was completely inhibited by the serine proteinase inhibitors, soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, and chymostatin. It was partially inhibited by p-tosyl-L-phenylalanine chloromethyl ketone, but was not inhibited by p-tosyl-L-lysine chloromethyl ketone, and aprotinin. Also, human heart chymase was not inhibited by inhibitors of the other three classes of proteinases. Human heart chymase has a high specificity for the conversion of Ang I to Ang II and the Ang I-carboxyl-terminal dipeptide His-Leu (Km = 60 microM; Kcat = 11,900 min-1; Kcat/Km = 198 min-1 microM-1). Human heart chymase did not degrade several peptide hormones, including Ang II, bradykinin, and vasoactive intestinal peptide, nor did it form Ang II from angiotensinogen. The high substrate specificity of human heart chymase for Ang I distinguishes it from other Ang II-forming enzymes including Ang I converting enzyme, tonin, kallikrein, cathepsin G, and other known chymases.