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Hydrolysis of Bradykinin by Angiotensin-Converting Enzyme By Frederic E. Dorer. Joseph R. Kahn, Kenneth E. Lentz, Melvin Levine, and Leonard T. Skeggs ABSTRACT Two dipeptides, phenylalanylarginine (Phe-Arg) and serylproline (Ser-Pro), are released sequentially from bradykinin by angiotensin-converting enzyme purified from hog lungs; chloride increases the rate of release of both dipeptides. Using an automated ninhydrin-reagent method, we studied the kinetics of bradykinin hydrolysis. The reaction proceeded in the absence of chloride; however, the addition of chloride increased the rate of hydrolysis by decreasing Km and increasing Vm . The Km values for bradykinin were 3.9 x 10~6M in the absence of chloride and 0.85 x 10~6M in the presence of 0.01M NaCl (optimal concentration). Both of these Km values were well below the value of 30 x 10" 6 M determined for angiotensin I at its optimal chloride concentration of 0.1M. Hydrolysis of bradykinin had a pH optimum of 7 and was inhibited by low concentrations (10~ 6 M) of ethylenediaminetetraacetic acid or the nonapeptide pyroglutamyl (Pyr)-Trp-ProArg-Pro-Gln-Ile-Pro-Pro. It is concluded that one enzyme, acting as a dipeptidyl carboxypeptidase, catalyzes both the conversion of angiotensin I to angiotensin II and the hydrolysis of bradykinin. Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 KEY WORDS phenylalanylarginine enzyme kinetics dipeptidyl carboxypeptidase chloride activation serylproline hog lung Bothrops jararaca nonapeptide • Extracts of mammalian lung contain an enzyme or enzymes capable of converting angiotensin I to angiotensin II and of destroying the biological activity of bradykinin (1). (Hydrolysis of any peptide bond in bradykinin [Arg-Pro-Pro-GlyPhe-Ser-Pro-Phe-Arg] abolishes biological activity [2].) Recently, an enzyme purified from lung has been shown to possess both of these activities (3). Bradykinin is a competitive inhibitor of angiotensin conversion (4), implying a common enzyme. However, the hydrolysis of bradykinin by angiotensin-converting enzyme preparations is reported to be independent of the presence of chloride; this finding leads to speculation that the enzyme activities must differ in some way (4, 5). The availability of a purified enzyme preparation (6) and the development of a quantitative chemical assay for measuring the hydrolysis of bradykinin enabled us to investigate whether bradykinin and angiotensin are hydrolyzed by the same enzyme and whether chloride participates in the bradykininase reaction. Methods Angiotensin I, hippuryl (Hip)-Gly-Gly, and His-Leu were synthesized as described previously (6, 7). Bradykinin was purchased from Schwarz/Mann, and N-2From the Department of Medicine and Surgery, Veterans Administration Hospital, Cleveland, Ohio, and the Departments of Biochemistry and Pathology, Case Western Reserve University, Cleveland, Ohio 44106. Received June 15, 1973. Accepted for publication March 19, 1974. 824 hydroxyethylpiperazine - N' - 2 - ethanesulfonic acid (HEPES) was purchased from Calbiochem. The dipeptides, serylproline (Ser-Pro), phenylalanylarginine (PheArg), and Gly-Phe, were purchased from Fox Chemical Company. The concentrations of standard solutions of the peptides were determined by amino acid analysis following acid hydrolysis. The synthetic peptide pyroglutamyl (Pyr)-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro was a gift.1 The angiotensin-converting enzyme purified from hog lung was obtained from fraction G or fraction H as described previously (6). Fraction H has been shown to be homogeneous by disk gel electrophoresis at pH 9.5 (6). These two fractions are identical with regard to relative rates of bradykinin, angiotensin I, and Hip-Gly-Gly hydrolysis, chloride activation, and similarity of dipeptide formed from bradykinin. Enzyme assays were performed by a modification of the automated method of Skeggs et al. (8) in which the rate of peptide bond hydrolysis is measured continuously by the ninhydrin reaction. Increased sensitivity was obtained by using the Technicon AutoAnalyzer II colorimeter, which permitted a twenty-fold electrical range expansion so that 6 x 10" 6 M leucine gave a full-scale reading. The incubation mixtures (final volume 5 ml) contained enzyme in 0.025M HEPES buffer (pH 7.5) with 0.04% Brij-35 (a synthetic detergent [7]) and NaCl at the indicated concentration. After a 10-minute incubation at 37°C, the substrate was added, and the mixture was sampled continuously for 8 minutes at 37°C. Enzyme velocities were calculated directly from the slopes of the recordings and were expressed as nanomoles of dipeptide formed per minute in the 5-ml incubation mixture. The analytical system was calibrated with His-Leu when angiotensin I was the substrate, with Gly-Gly when 1 Generously supplied by Dr. J. W. Ryan. University of Miami School of Medicine. Circulation Research. Vol. XXXIV. June 1974 825 BRADYKININ HYDROLYSIS BY CONVERTING ENZYME Hip-Gly-Gly was the substrate, and with Ser-Pro or Phe-Arg (identical ninhydrin color values. 83^ of leucine) when bradykinin was the substrate. Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Results The automated enzyme assay method used in this study for bradykinin hydrolysis yielded velocities that were linear with time during the 8-minute sampling period and enzyme concentrations that were in the range of 0.1 to 1.0 Mg/ml. At pH 7.5, the Km and Vm values for bradykinin hydrolysis (Fig. 1) were 3.9 x 10' 6 M and 0.81 /umoles/min mg~' protein, respectively, in the absence of chloride, and 0.85 x 10"6M and 1.4 ^moles/min mg"' protein in the presence of 0.01M NaCl. Similarly, the Km and Vm values for angiotensin I hydrolysis were 30 x 10" 6 M and 2.1 /umoles/min mg" 1 protein, respectively, in the presence of 0.1M NaCl. The relationship between bradykinin hydrolysis 4.5-, and chloride concentration is shown in Figure 2. The optimal chloride concentration is about onetenth of that previously found for the angiotensinconversion reaction (phosphate buffer, pH 8) (6). The observed velocity in the absence of added chloride did not appear to result from the presence of trace amounts of chloride in the reagents, because there was no hydrolysis of Hip-Gly-Gly, a reaction which absolutely requires chloride (6) under these conditions. Also, bradykinin that had been treated by chromatography on BioGel P-2 or with an ion-exchange resin (AG 2-X8 acetate) to remove any traces of chloride was still hydrolyzed at the same rate in the absence of added NaCl. The pH-activity relationships for bradykinin and angiotensin I hydrolysis are shown in Figure 3. The shapes of the curves for bradykinin hydrolysis in the presence and the absence of chloride are similar. Figure 4 shows the time course of bradykinin hydrolysis with and without chloride. Chloride increased the rate of hydrolysis without changing the final value attained. The hydrolysis of bradykinin (4 x 10" 6 M) both in the presence and the absence of NaCl was inhibited more than 50% by 10" 6M ethylenediaminetetraacetic acid (EDTA) or 10" 6M Pyr-Trp-Pro-Arg3.0 0.5 1.0 1.5 2.0 Double reciprocal plot of bradykinin hydrolysis showing the effect of chloride on Km and Vm . Velocities are expressed as nmoles dipeptide formed/min. and substrate concentrations are expressed as nmoleslml. Assays were carried out as described in the text in the absence of NaCl with 2.5 ng of enzyme {solid circles) and 5.0 ng of enzyme {triangles) and in the presence of 0.01 M NaCl with 1.0 fig of enzyme (crosses) and 2.5 fig of enzyme (open circles). Circulation Research. Vol. XXXIV. June 1974 10" 10 10" 10" NaCl concentration (M) FIGURE 2 Bradykinin hydrolysis as a function of NaCl concentration. Assays were carried out as described in the text using 2.5 ng of enzyme and 20 nmoles of bradykinin. 826 DORER. KAHN. LENTZ, LEVINE, SKEGGS Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 FIGURE 3 Bradykinin hydrolysis and angiotensm-converting enzyme activity as a function of pH. Assays were carried out with 2-5 ng of enzyme as described in the text except that HEPES buffers of varying pH were used. The pH values were measured at 25°C at the end of the incubations. Circles - hydrolysis of 20 nmoles of bradvkinin in the presence of 0.01M NaCl. triangles = hydrolysis of 20 nrrwles of bradykinin in the absence of NaCl, and crosses hydrolysis of 200 nmoles of angiotensm 1 in the presence of 0.1 M NaCl. Pro-Gln-Ile-Pro-Pro, a bradykinin-potentiating peptide found in Bothrops jararaca venom (9). The demonstration by chemical methods of the hydrolysis of bradykinin by angiotensin-converting enzyme was confirmed by the loss of biological activity of bradykinin. Incubation for 1 hour with an excess of enzyme (with or without NaCl) completely abolished the depressor effect of bradykinin (1 nmole, iv) injected into an anesthetized rat (10). To determine which peptide bonds were hydrolyzed, bradykinin (260 nmoles) was incubated for 1 hour with an excess of converting enzyme in a volume of 0.7 ml at pH 7.5; this procedure was followed by identification of products on the amino-acid analyzer. The results were the same with and without chloride; two peaks were observed that coincided with Ser-Pro (240 nmoles) and Phe-Arg (280 nmoles). No free arginine or Gly-Phe was found. The effect of NaCl on the initial rate of release of Phe-Arg and Ser-Pro is shown in Figure 5. The bradykinin concentration used in this experiment 2.4- 2.0- 1.6- 1.2- o.e0.4- 10 20 30 40 Time (min) 50 60 FIGURE 6 0 20 40 60 Time (min) FIGURE 4 Time course of the hydrolysis of bradykinin. Twenty nmoles of bradykinin were incubated with 5 ng of enzyme as described in the text except that sampling was done discontinuously at the indicated times from an incubation volume of 10 ml. Circles indicate the absence of NaCl, and crosses indicate the presence of 0.01M NaCl. Release of Phe-Arg and Ser-Pro from bradykinin. Bradykinii (100 nmoles) was incubated with 1 fig of enzyme at pH 7.5 in i total volume of 25 ml in the presence and the absence of NaC for time intervals up to 1 how. The reaction was stopped b boiling, and the entire incubation mixture was pumped onto th column of the amino-acid analyzer, modified (11) with gradient to elute Phe-Arg. Solid circles - Phe-Arg formed in th presence of O.OlM NaCl, triangles - Phe-Arg formed in th absence of NaCl. open circles - Ser-Pro formed in the presenc of 0.01M NaCl, and crosses =• Ser-Pro formed in the absence < NaCl. Circulation Ratarth, Vol. XXXIV, June IS, 827 BRADYKININ HYDROLYSIS BY CONVERTING ENZYME was 4 x 10 6M, the same as that used in the experiments considered in Figures 2 and 3 and twice that in the experiments considered in Figure 4. The rate of release of both dipeptides depended on chloride (Fig. 5); in the case of Phe-Arg, the stimulation by chloride was about 2.5-fold. hydrolysis of bradykinin has been presented. This conclusion is compatible with the dipeptidyl carboxypeptidase type of specificity previously described for this enzyme (3). Discussion 1. BAKHLE, Y.S.: Conversion of angiotensin I to angiotensin II by cell-free extracts of dog lung. Nature (Lond) 220:919-921, 1968. Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Most of the data presented in this paper represent a mixed reaction, i.e., the actual quantity measured was the sum of Phe-Arg and Ser-Pro. Nevertheless, the initial mixed rate, at least under the experimental conditions of Figure 5, appears to represent the initial reaction, which was the release of Phe-Arg. Evidence that chloride participates in the hydrolysis of bradykinin was found. Although it is not an absolute requirement, chloride does affect both Km and Vm . The data presented in Figure 5 show that chloride is required for maximal rate of release of both dipeptides. Since the bradykinin molecule becomes inactive when the Phe-Arg moiety is removed, the rate of Phe-Arg release should parallel the loss of biological activity (bradykininase activity). Thus, it is difficult to understand why the chloride effect has not been observed by other investigators (4, 5) using biological assays. It is not known whether the pulmonary converting enzyme-bradykininase described in this paper is the enzyme responsible for bradykinin inactivation under physiological conditions in the intact animal. Bradykinin is hydrolyzed at several bonds during perfusion through the blood-free rat lung (2). However, the inactivation of bradykinin in the pulmonary circulation is inhibited by Bothrops jararaca peptides (5, 12). Evidence that one enzyme, which requires chloride for maximal activity, catalyzes both the conversion of angiotensin I to angiotensin II and the References 2. RYAN, J.W., ROBLERO. J., AND STEWART, J.M.: Inactivation of bradykinin in rat lung. Adv Exp Med Biol 8:263-271, 1970. 3. IGIC, R., ERDOS, E.G., YEH, H.S.J., SORRELLS, K., AND NAKAJIMA, T.: Angiotensin I converting enzyme of the lung. Circ Res 31(suppl. II):II-51-61. 1972. 4. SANDER, G.E., WEST, D.W., AND HUGGINS, C.G.: Peptide inhibitors of pulmonary angiotensin I converting enzyme. Biochim Biophys Acta 242:662-667, 1971. 5. ALABASTER, V.A., AND BAKHLE, Y.S.: Converting enzyme and bradykininase in the lung. Circ Res 31(suppl. II): 11-72-81, 1972. 6. DORER, F.E., KAHN, J.R., LENTZ, K.E., LEVINE, M., AND SKEGGS, L.T.: Purification and properties of angiotensinconverting enzyme from hog lung. Circ Res 31:356-366, 1972. 7. DORER, F.E., SKEGGS, L.T., KAHN, J.R., LENTZ, K.E., AND LEVINE, M.: Angiotensin converting enzyme: Method of assay and partial purification. Anal Biochem 33:102-113, 1970. 8. SKEGGS, L.T., LENTZ, K.E., KAHN, J.R., AND HOCHSTRASSER, H.: Kinetics of the reaction of renin with nine synthetic peptide substrates. J Exp Med 128:13-34, 1968. 9. FERREIRA, S.H., BARTELT, D.C., AND GREENE, L.J.: Isolation of bradykinin-potentiating peptides from Bothrops jararaca venom. Biochemistry 9:2583-2593, 1970. 10. SKEGCS, L.T., JR., KAHN, J.R., AND MARSH, W.H.: Method of assaying small amounts of Hypertensin. Lab IrTvest 2:109-114, 1953. 11. MITCHELL, A.R., AND ROESKE, R.W.: Chromatography of im-benzyl-L-histidine. J Chromatogr 43:266-267, 1969. 12. GREENE, L.J., CAMARCO, A.CM., KRIECER, E.M., STEWART, J.M., AND FERREIRA, S.H.: Inhibition of the conversion of angiotensin I to II and potentiation of bradykinin by small peptides present in Bothrops jararaca venom. Circ Res 31(suppl. II):II-62-71, 1972. Correction Vol. 34, p 66 Table 3, line five, should read TABLE 3 Author Present study Circulation Research. Vol. XXXIV. June 1974 Reference Compliance (ml/mm Hg kg ' body wt) Animal 2.3 Man Hydrolysis of Bradykinin by Angiotensin-Converting Enzyme FREDERIC E. DORER, JOSEPH R. KAHN, KENNETH E. LENTZ, MELVIN LEVINE and LEONARD T. SKEGGS Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Circ Res. 1974;34:824-827 doi: 10.1161/01.RES.34.6.824 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1974 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/34/6/824 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. 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