Comparison of hydrolysis of atriopeptin II stand-in substrate by atrial dipeptidyl carboxyhydrolase and angiotension I-converting enzyme

We recently found and partially purified a new membrane-bound metallo dipeptidyl dipeptidase from bovine atrial tissue homogenates (Harris, R.B. & Wilson, I.B. (1984) Arch. Biochem. Biophys. 233, 667-675). We suggested that this enzyme was capable of cleaving the dipeptide, phenylalanyl-arginine from the C-terminus of atriopeptin II to give atriopeptin I. The atriopeptins are two atrial natriuretic peptides and the existence of the atrial peptide system has implicated the mammalian heart as an endocrine organ. The tetrapeptide benzoyl-glycyl-seryl-phenylalanyl-arginine was synthesized because it contains the C-terminal tripeptide sequence of atriopeptin II and should be useful to test the roles of the atrial enzyme and angiotensin I-converting enzyme in processing the atrial peptides. We found that for the atrial enzyme, Vmax was 13-fold higher and Km 7-fold-lower for this stand-in substrate than for benzoyl-glycyl-histidyl-leucine, a standard substrate used to measure converting enzyme activity. The ratio of Vmax/Km as a measure of substrate specificity indicates that the stand-in substrate is 86-fold better than benzoyl-glycyl-histidyl-leucine. In contrast, the stand-in substrate is a 20-fold poorer substrate for the converting enzyme than benzoyl-glycyl-histidyl-leucine. With the stand-in substrate, the converting enzyme showed pronounced substrate inhibition. An effective Vmax and Km were calculated using only concentrations of S below the optimum substrate concentration. These results confirm that the atrial enzyme is distinct from the converting enzyme. They also suggest that the conversion of atriopeptin II to atriopeptin I is a physiological process that is mediated by this enzyme.     

Atrial tissue contains a metallo dipeptidyl carboxyhydrolase not present in ventricular tissue: partial purification and characterization

A new membrane-bound dipeptidyl carboxyhydrolase has been identified in bovine atrial tissue, and has been partially purified after extraction with Triton X-100. This enzyme, found in quantities of 0.01-0.03 units/g tissue assayed with Bz-Gly-His-Leu, is potentially capable of hydrolyzing atriopeptin II to atriopeptin I. The enzyme is located in the microsomal fraction and in sucrose density fractions enriched for atrial granules. The enzyme is completely inhibited by reagents for heavy metals such as EDTA, o-phenanthroline, dithiothreitol, and mercaptoethanol. The latter two compounds are also disulfide reagents. The atrial enzyme is also inhibited by D-2-methyl-3-mercaptopropanoyl-L-Pro(Captopril), 3-mercaptopropanoyl-L-Pro, 2-D-methylsuccinyl-L-Pro, and bradykinin potentiating factor, all inhibitors of the angiotensin I-converting enzyme. However, the atrial enzyme differs from the converting enzyme in a number of kinetic and molecular properties. Its activity increases with ionic strength, but the atrial enzyme does not have a chloride dependence for Bz-Gly-His-Leu hydrolysis; the pH optimum, 7.3, is slightly lower, and it is 5500 times less sensitive to the very potent converting enzyme inhibitor, D-Cys-L-Pro. The strokes radius of the atrial enzyme is 5.00 nm as compared to 4.10 nm, and its molecular weight is 240,000 compared to 145,000. Ventricular tissue, which does not contain the atrial peptides, does not contain the dipeptidyl carboxyhydrolase enzyme.     

Atrial dipeptidyl carboxyhydrolase is a zinc-metallo proteinase which possesses tripeptidyl carboxyhydrolase activity

Atrial dipeptidyl carboxyhydrolase readily converts one atrial natriuretic peptide, atriopeptin II (Ser103-Arg125 peptide), to another, atriopeptin I (Ser103-Ser123 peptide), by selective removal of the C-terminal dipeptide, Phe-Arg. The atrial peptides possess natriuretic, diuretic, smooth muscle relaxant, and cardiodynamic properties and their existence has shown the mammalian heart to be an endocrine organ. After inactivating the bovine atrial enzyme with EDTA, activity is restored by the addition of Co+2, Zn+2 and Mn+2 but not by Cu+2, Mg+2, Ca+2, or Cd+2. The enzyme is thus likely to be a zinc-metallo proteinase. In addition to its dipeptidyl activity, the enzyme also displays tripeptidyl carboxyhydrolase activity with atriopeptin III (Ser103-Try126 peptide) as substrate. The hydrolytic products resulting from tripeptidyl cleavage are atriopeptin I and Phe-Arg-Tyr. However, with [mercaptopropionyl105,(D)Ala107]-atriopeptin III-NH2 peptide (a potent agonist of atriopeptin III) as substrate, the enzyme acts exclusively as a tripeptidyl carboxyhydrolase. To examine the basis for this shift in cleavage point, pentapeptides based on the C-terminal sequence of atriopeptin III were prepared; a C-terminal Tyr or Tyr-NH2 residue is not sufficient to cause the change in cleavage point. The amidated pentapeptide is not a substrate but is a competitive inhibitor of hydrolysis of the corresponding free-acid peptide.     

A peptidyl dipeptidase-4 from Pseudomonas maltophilia: purification and properties

A peptidyl dipeptidase-4 (bacterial PDP-4) was purified to near homogeneity from a supernatant of Pseudomonas maltophilia extracellular medium. Bacterial PDP-4 is a single-polypeptide-chain enzyme, 82 kDa, with an alkaline isoelectric point. Peptides susceptible to hydrolysis by bacterial PDP-4 include angiotensin 1, bradykinin, enkephalins, atriopeptin 2, and smaller synthetic peptides. N-acylated tripeptides are hydrolyzed, but free tripeptides are not. A free carboxy terminus is required for hydrolysis. Peptides with ultimate and penultimate Pro residues are not hydrolyzed. The enzyme does not require an anion for activity. Bacterial PDP-4 was inhibited by EDTA and the dipeptide Phe-Arg. Thiorphan was an inhibitor only at levels well above those required for inhibition of neutral metalloendopeptidase (NEP), an enzyme for which thiorphan is specific. A second NEP and thermolysin inhibitor, phosphoramidon, did not inhibit bacterial PDP-4. The potent angiotensin-converting enzyme inhibitor lisinopril was not inhibitory. Bacterial PDP-4 is distinguished from a similar enzyme from Escherichia coli, which is not susceptible to EDTA inhibition, and one from Corynebacterium equi, which hydrolyzes free tripeptides. These data indicate that the bacterial PDP-4 catalytic site is unlike those of other enzymes that function either wholly or in part as peptidyl dipeptidases.     

A distinct peptidyl dipeptidase that degrades enkephalin: Exceptionally high activity in rabbit kidney

Peptidyl dipeptidase activity distinct from the angiotensin converting enzyme (EC 3.4.15.1) was isolated from membrane fractions of rabbit kidney and lung. The enzyme cleaved Leu-enkephalin at the Gly-Phe bond, releasing Tyr-Gly-Gly and Phe-Leu, and also acted on bradykinin releasing the terminal dipeptide Phe-Arg. In contrast to the converting enzyme, however, this peptidyl dipeptidase did not act on angiotensin I, or on hippuryl His-Leu, nor was it inhibited by captopril (SQ 14225) or by SQ 20881. Kinetic studies indicated a K_m for the kidney enzyme of 80 μM with Leu-enkephalin as a substrate. Our findings indicate that more than one enzyme is present in membrane preparations of lung and kidney inactivating enkephalin, and suggest a role for these enzymes in the peripheral actions of opiate and related peptides.     

Characterization and localization of atriopeptin in rat atrium

An antiserum specific for atriopeptin was used to characterize and localize atriopeptin-like immunoreactive material in rat atrium by radioimmunoassay and immunohistochemical techniques. The antiserum recognizes atriopeptin I, atriopeptin III, and -human atrial natriuretic polypeptide, but does not recognize met-enkephalin, cholecystokinin, dynorphin A (1–13), bradykinin, substance P, or β-endorphin. A high content of atriopeptin was found in crude extracts of rat atria, as compared to ventricles, and the atriopeptin-like immunoreactive material was found to be located exclusively in granules within atrial cardiocytes. Fractionation of the immunoreactive material by gel filtration and reverse-phase HPLC revealed the presence of multiple atriopeptins.     

A continuous spectrophotometric assay for angiotensin converting enzyme.

Furanacryloyl tripeptides conforming to the known substrate specificity of the angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) have been employed to provide a continuous spectrophotometric assay for this peptidase in the visible region. The assay is based on a blue shift of the absorption spectrum that occurs upon hydrolysis of the substrate to produce a furanacryloyl-blocked amino acid and a dipeptide. Of the various furanacryloyl tripeptides tested, furanacryloyl-l-phenylalanylglyeylglycine exhibits the most suitable characteristics for routine assays of angiotensin converting enzyme.     

Release of atriopeptin in the rat by vasoconstrictors or water immersion correlates with changes in right atrial pressure

Vasopressin, its 1-deamino analog (dAVP), angiotensin II, and phenylephrine, administered intravenously, increased plasma atriopeptin immunoreactivity in chloral hydrate-anesthetized rats. A continuous one hour infusion of either dAVP or phenylephrine caused a sustained elevation in: a) systemic blood pressure; b) right atrial pressure; c) left ventricular end diastolic pressure; and d) plasma atriopeptin immunoreactivity. While continuous infusion of angiotensin II also produced a sustained elevation in left ventricular end diastolic pressure, the changes in right atrial pressure and plasma atriopeptin were only transient. These data suggest that plasma atriopeptin most closely correlates with right atrial pressure. Consistent with this hypothesis, we found that the release of atriopeptin directly correlated with changes in right atrial pressure in anesthetized, water-immersed rats.     

Substrate binding properties of converting enzyme using a series of p-nitrophenylalanyl derivatives of angiotensin I.

The binding properties of angiotensin I for the active site of rabbit lung converting enzyme (CE) have been investigated. A series of angiotensin I like substrates, all containing the C-terminal tripeptide, (NO2)Phe-His-Leu, were synthesized by increasing the length of the peptide at the N-terminal end. A total of eight peptides were studied, the largest being [Asn1, (NO2)Phe8]angiotensin I. As determined by thin-layer chromatography, all substrates were hydrolyzed only at the (NO2)Phe-His bond by purified converting enzyme, with the release of the dipeptide, His-Leu. By using an absorbance increase upon hydrolysis, the Michaelis constants and velocity maxima were determined and used to estimate those amino acids in the angiotensin I molecule that contribute significantly to binding to converting enzyme. It was hypothesized that, upon addition or substitution of one or more amino acids to the N-terminal end, a proportional decrease in both KM and Vm is needed in order to conclude that the substrate actually increases its affinity for the enzyme. A test of the proportionality for the variation of KM and Vm was found to be positive for all the substrates, except the N-terminal carbobenzoxy-blocked tripeptide, Z(NO2)Phe-His-Leu. Substitutions near the bond that is hydrolyzed (e.g., proline for the carbobenzoxy group) appear to alter the catalytic properties of CE, while additions far removed from the site of hydrolysis (e.g., the N-terminal tripeptide Asn-Arg-Val) may enhance binding affinity.     

Angiotensin II generated by a human renal carboxypeptidase

Angiotensin II, the potent hypertensive octapeptide, can be generated by a sequential cleavage of the carboxyl-terminal leucine and histidine from angiotensin I by a human renal extract. This extract does not hydrolyze further the resulting octapeptide. The more widely recognized biosynthetic pathway is by the extracellular dipeptide cleavage of angiotensin I by an enzyme which also degrades bradykinin, i.e., angiotensin converting enzyme. The presence of a carboxypeptidase activity capable of generating but not further hydrolyzing angiotensin II was observed in an ammonium sulfate fraction of a human renal extract. This novel enzymatic activity is distinct from angiotensin converting enzyme activity in that it is not dependent upon calcium and is not inhibited by known angiotensin converting enzyme inhibitors.     

Purification and characterization of a dipeptidyl carboxypeptidase from the polychaete Neanthes virens resembling angiotensin I converting enzyme

Dipeptidyl carboxypeptidase (DCP) is well known as a mammalian angiotensin I converting enzyme (ACE) which plays an important role in blood pressure homeostasis. DCP was purified from the whole body of a polychaete, Neanthes virens. The purified enzyme was homogeneous by SDS-PAGE, with a molecular mass of 71 kDa by SDS-PAGE and 69 kDa by gel filtration, indicating that it is monomeric. The isoelectric point was 4.5 and optimum pH for the activity was 8.0. It showed a specific activity of 466.8 U/mg, which is the highest of known DCPs. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin, but does not cleave imido-bonds. Activity was completely inhibited by 1 mM EDTA and 5 mM o-phenanthroline, but it was not affected by serine and aspartic protease inhibitors. The original activity of EDTA-inactivated DCP was restored by addition of cobalt, manganese or low concentrations of zinc. The Km and Vmax values of the enzyme for Bz-Gly-His-Leu were 0.56 mM and 600 mumol/min per mg, respectively. The Ki values for specific mammalian ACE inhibitors, such as captopril and lisinopril, were 1.38 and 2.07 nM, respectively. In conclusion, we have shown the existence of a DCP from the polychaete, N. virens, with similar properties to those of mammalian ACE.     

Characterization of a distinct membrane bound dipeptidyl carboxypeptidase inactivating enkephalin in brain

A dipeptidyl carboxypeptidase distinct from the angiotensin converting enzyme (EC 3.4.15.1) was isolated from membrane preparations of rabbit brain. The enzyme cleaved enkephalin at the Gly-Phe bond, releasing either Phe-Leu from Leu-enkephalin or Phe-Met from Met-enkephalin, and also acted on bradykinin, releasing the terminal dipeptide Phe-Arg. In contrast to the converting enzyme, however, this dipeptidyl carboxypeptidase did not act on angiotensin-1, and it did not degrade hippuryl-His-Leu. Chloride ions did not affect its activity, but the enzyme was inhibited by metal chelating agents. The enzyme was not inhibited by captopril (SQ 14225) or by SQ 20881. Kinetic studies indicated a Km for this enzyme of 0.14 mM with Leu-enkephalin and 0.12 mM with bradykinin as substrates. Present data indicate that more than one enzyme is present in brain membrane fractions acting as dipeptidyl carboxypeptidases inactivating enkephalin; these data suggest multiple roles for such enzymes in the regulation of peptide metabolism.     

New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture.

To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3H-atom on the para-position of its benzoyl moiety. All three of the acylated tripeptides are substrates for the enzyme. Substrate I exhibits the lowest Km (12.5 micrometer) and yields the most sensitive assay: the enzyme of 10(6) cells can be measured in a 30 min incubation at 37 degrees C. Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase. Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180--240 min) and high salt concentrations (0.75 M Na2SO4). Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells.     

Inhibition of angiotensin converting enzyme: mechanism and substrate dependence

The interaction of angiotensin converting enzyme with six metal-coordinating [(D-3-mercapto-2-methylpropanoyl)-L-Pro (captopril), N-[1(S)-carboxy-3-phenylpropyl]-L-Ala-L-Pro (MK-422), N-(phenylphosphoryl)-L-Phe-L-Phe, N alpha-(3-mercaptopropanoyl)-L-Arg, N alpha-[1(S)-carboxy-3-phenylpropyl]-Ala-L-Lys, and N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly] and three dipeptide inhibitors (Gly-L-Trp, L-Phe-L-Arg, and L-Ala-L-Pro) was examined at pH 7.5 in the presence of 300 mM NaCl. Inhibition modes, apparent Ki [Ki(app)] values, and shapes of 1/v vs. [I] plots were found to vary with the substrate employed. All inhibitors except Phe-Arg were competitive with the substrate furanacryloyl (Fa)-Phe-Gly-Gly, while five of seven tested with Fa-Phe-Phe-Arg as substrate produced mixed patterns. Ki-(app) values for N-[1(S)-carboxy-5-aminopentyl]-L-Phe-Gly, N-(phenylphosphoryl)-L-Phe-L-Phe, Gly-Trp, and MK-422 were 8.3-, 5.5-, 4.7-, and 2.6-fold lower, respectively, when Fa-Phe-Gly-Gly was substrate, compared with values measured with Fa-Phe-Phe-Arg. In contrast, Ki(app) values for Phe-Arg and (3-mercaptopropanoyl)-Arg were lower (2.8- and 2.2-fold, respectively) when Fa-Phe-Phe-Arg was the substrate. Plots of 1/v vs. [I] for most of the inhibitors were nonlinear, to an extent which was also substrate dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Dipeptidyl carboxypeptidase from human seminal plasma.

Dipeptidyl carboxypeptidase (angiotensin I converting enzyme) was purified from human seminal plasma. The apparent relative molecular mass determined by gel filtration on Sephadex G-200 was 330 000. The pI in isoelectric focusing was 4.6--5.0 and the optimum pH 7.7--8.0. The enzyme is activated by chloride. These properties are similar to those reported for the lung enzyme. The specificity is that of a carboxypeptidase releasing dipeptides. A study of different substrates showed the activity to be highest with Z-Leu-Gly-Gly, followed by Z-Phe-His-Leu greater than bradykinin greater than Bz-Gly-Gly-Gly greater than Boc-Phe-Ala-Pro greater than Bz-Gly-His-Leu greater than angiotensin I.     

Effect of hemorrhage on plasma atriopeptin levels in conscious dogs

An increase in atrial pressure has been shown to cause an increase in the concentration of atrial peptides (atriopeptin) in plasma. We therefore hypothesized that a reduction in atrial pressure would decrease the concentration of atriopeptin in plasma. In formulating this hypothesis we assumed that changes in the concentration of other circulating hormones or changes in cardiac nerve activity during hemorrhage would not affect the secretion of atriopeptin. To test the hypothesis, we bled sham-operated conscious dogs at a rate of 0.8 ml.kg-1.min-1 to decrease right and left atrial pressures. Hemorrhage was continued until a total of 30 ml of blood per kilogram body weight had been removed. Identical experiments were performed on conscious cardiac-denervated dogs. The concentration of plasma atriopeptin was decreased in each group of dogs after 10 ml of blood per kilogram of body weight had been removed, but the decrease achieved statistical significance only in the cardiac-denervated dogs. Further hemorrhage, however, produced no further decreases in circulating atriopeptin in either group even though atrial pressures continued to decline as more blood was removed. A comparison of the atriopeptin response to hemorrhage revealed no significant difference between the sham-operated and cardiac-denervated dogs, thus providing no evidence for a specific effect of cardiac nerves on atriopeptin secretion during hemorrhage. Our results demonstrate that the relationship between atrial pressure and plasma atriopeptin that has been observed repeatedly during atrial stretch is not evident during relatively slow, prolonged hemorrhage. There is, however, a small decline in circulating atriopeptin during the initial stage of hemorrhage that could be of biological significance.     

Substrate specificity and kinetic characteristics of angiotensin converting enzyme

Furanacryloyl-Phe-Gly-Gly has been shown to be a convenient substrate for angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). A detailed kinetic analysis of the hydrolysis of this substrate indicates normal Michaelis-Menten behavior with kcat = 19000 min-1 and KM = 3.0 x 10(-4) M determined at pH 7.5, 25 degrees C. The enzyme is inhibited by phosphate and activated by chloride; maximal activity is observed with 300 mM NaCl. In the absence of added zinc, activity is lost rapidly below pH 7.5 due to spontaneous dissociation of the metal, but in the presence of zinc, the enzyme remains fully active to about pH 6. The pH-rate profile indicates two groups on the enzyme with apparent pK values of 5.6 and 8.4. The substrate specificity of the enzyme has been examined in terms of the fundamental specificity quantity kcat/KM as well as the separate constants by using a series of furanacryloyl-tripeptides. The activity toward furanacryloyl-Phe-Gly-Gly has been compared with that toward the physiological substrates angiotensin I and bradykinin.     

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.