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Hydrolysis of Peptide Hormones by Endothelin-converting Enzyme-1

1999; Elsevier BV; Volume: 274; Issue: 7 Linguagem: Inglês

10.1074/jbc.274.7.4053

ISSN

1083-351X

Autores

Gary D. Johnson, Tracy I. Stevenson, Kyunghye Ahn,

Tópico(s)

Renin-Angiotensin System Studies

Resumo

Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins. Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins. Endothelins (ETs) 1The abbreviations used are: ET, endothelin; ANP, atrial natriuretic peptide; C12E10, polyoxyethylene-10-lauryl ether; dansyl, 5-(dimethylamino)naphthalene-1-sulfonyl; ECE, endothelin-converting enzyme; HPLC, high performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; NEP, neprilysin, neutral endopeptidase 24.11; (pNO2)Phe, para-nitro-l-phenylalanine; solECE-1, soluble ECE-1.1The abbreviations used are: ET, endothelin; ANP, atrial natriuretic peptide; C12E10, polyoxyethylene-10-lauryl ether; dansyl, 5-(dimethylamino)naphthalene-1-sulfonyl; ECE, endothelin-converting enzyme; HPLC, high performance liquid chromatography; MES, 2-(N-morpholino)ethanesulfonic acid; NEP, neprilysin, neutral endopeptidase 24.11; (pNO2)Phe, para-nitro-l-phenylalanine; solECE-1, soluble ECE-1. are potent vasoconstrictive peptides of 21 amino acids produced by vascular endothelial cells (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar). Three ET isoforms, ET-1, ET-2, and ET-3, encoded by distinct genes, are known to exist in humans (2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar). Endothelins are involved in the regulation of vascular tone and may also play roles in various cardiovascular and renal diseases (3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar). ETs are also required during embryonic development for the intercellular signaling necessary for the proper development of neural crest-derived tissues (4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar). The final step in the biosynthesis of the endothelins is the conversion of 38–41 residue precursors (big ETs) to the active hormones via the cleavage of a Trp21-Val/Ile22 bond by endothelin-converting enzymes (ECEs (5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar)). ECE-1 has been purified from vascular endothelium, endothelial cell lines, and lung microsomes (6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar, 7Takahashi M. Matsushita Y. Iijima Y. Tanzawa K. J. Biol. Chem. 1993; 268: 21394-21398Abstract Full Text PDF PubMed Google Scholar, 8Ohnaka K. Takayanagi R. Nishikawa M. Haji M. Nawata H. J. Biol. Chem. 1993; 268: 26759-26766Abstract Full Text PDF PubMed Google Scholar). ECE-1 (EC 3.4.24.71) is a Type II integral membrane protein expressed by endothelial cells in tissues such as aorta, lung, ovary, and testis. It has also been reported to be expressed by endocrine cells such as adrenal chromaffin cells and pancreatic β cells (9Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike M. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar). Targeted disruption of the ECE-1 gene has shown that ECE-1 is the physiologically relevant activating enzyme for both ET-1 and ET-3in vivo (10Yanagisawa H. Yanagisawa M. Kapur R.P. Richardson J.A. Williams S.C. Clouthier D.E. de Wit D. Emoto N. Hammer R.E. Development. 1998; 125: 825-836Crossref PubMed Google Scholar). Molecular cloning of mammalian ECE-1 cDNAs has demonstrated the existence of three mRNAs transcribed from a single gene (11Shimada K. Takahashi M. Ikeda M. Tanzawa K. FEBS Lett. 1995; 371: 140-144Crossref PubMed Scopus (116) Google Scholar, 12Schweizer A. Valdenaire O. Nelböck P. Deuschle U. Dumas Milne Edwards J.-B. Stumpf J.G. Löffler B.-M. Biochem. J. 1997; 328: 871-877Crossref PubMed Scopus (191) Google Scholar). The proteins encoded by these RNAs have identical catalytic domains but differ only in their NH2-terminal amino acid sequence. Two of the ECE-1 isoforms are expressed on the cell surface; the other is localized in the trans-Golgi network (12Schweizer A. Valdenaire O. Nelböck P. Deuschle U. Dumas Milne Edwards J.-B. Stumpf J.G. Löffler B.-M. Biochem. J. 1997; 328: 871-877Crossref PubMed Scopus (191) Google Scholar) An additional isoform (ECE-2), 59% identical in amino acid sequence to ECE-1, that appears to be expressed in the trans-Golgi network has also been identified (13Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Crossref PubMed Scopus (430) Google Scholar). The endothelin-converting enzymes belong to a family of metallopeptidases including neprilysin (neutral endopeptidase 24.11, NEP) and Kell, an antigen expressed on the surface of erythrocytes (14Turner A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (377) Google Scholar). These proteins have the greatest amount of sequence identity in their COOH-terminal regions, especially in residues involved in zinc binding and catalysis, indicating a similar structure and catalytic mechanism for all. NEP is the most extensively characterized enzyme of this group (15Roques B.P. Noble F. Dauge V. Fournie-Zaluski M.-C. Beaumont A. Pharmacol. Rev. 1993; 45: 87-146PubMed Google Scholar). NEP cleaves a variety of biologically active peptides, usually at the amino side of hydrophobic residues. Studies using big ETs and peptides derived from endothelins have led to the conclusions that hydrolysis of substrates by ECE-1 may be highly dependent on substrate conformation and that the enzyme may have a narrow substrate specificity (8Ohnaka K. Takayanagi R. Nishikawa M. Haji M. Nawata H. J. Biol. Chem. 1993; 268: 26759-26766Abstract Full Text PDF PubMed Google Scholar, 16Corder R. Biochem. Pharmacol. 1996; 51: 259-266Crossref PubMed Scopus (5) Google Scholar). However, previous studies of ECE-1 peptidase activity have been hampered by the limited availability of the pure enzyme and have rarely used peptides other than big endothelins as substrates. A more thorough examination of ECE-1 activity and substrate specificity is required to better understand its mechanism of action and to identify additional in vivo substrates. As an initial step in the systematic examination of ECE-1 substrate specificity, we have purified recombinant soluble ECE-1 (solECE-1) to homogeneity and examined its activity against a number of biologically active peptides. The results indicate that ECE-1 can hydrolyze a broad spectrum of peptide substrates with a specificity similar to that of NEP. The human ECE-1a cDNA was modified so that the extracellular domain (amino acids 78–758) was fused in-frame to a DNA sequence encoding the signal sequence of human alkaline phosphatase. The modified cDNA was subcloned into the mammalian expression vector pSG5 (Stratagene, La Jolla, CA) with protein expression driven by a SV40 promoter. A stably transfected Chinese hamster ovary K1 cell line harboring the resultant plasmid secreted solECE-1 into the culture medium. The purification of solECE-1 from this conditioned medium has been recently described in detail (32Ahn K. Herman S.B. Fahnoe D.C. Arch. Biochem. Biophys. 1998; 359: 258-268Crossref PubMed Scopus (35) Google Scholar). Briefly, the purification involved successive chromatographic steps utilizing the binding of solECE-1 to DEAE-agarose, wheat germ agglutinin-agarose, and alkyl-Superose resins. The purification of solECE-1 was monitored by assaying the conversion of human big ET-1 to ET-1 using an enzyme-linked immunosorbent assay kit for the quantitation of ET-1 (Amersham Pharmacia Biotech). The solECE-1 was judged to be homogeneous when analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue staining. Arg-Pro-Pro-Gly-Phe-Ser-Pro (bradykinin (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar, 5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar, 7Takahashi M. Matsushita Y. Iijima Y. Tanzawa K. J. Biol. Chem. 1993; 268: 21394-21398Abstract Full Text PDF PubMed Google Scholar)) and Asp-Arg-Val-Tyr (angiotensin I (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar)) were purchased from American Peptide Co. (Sunnyvale, CA). Arg-Pro-Lys-Pro-Gln-Gln (substance P (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar, 5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar)), pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro (neurotensin (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar, 5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar, 7Takahashi M. Matsushita Y. Iijima Y. Tanzawa K. J. Biol. Chem. 1993; 268: 21394-21398Abstract Full Text PDF PubMed Google Scholar, 8Ohnaka K. Takayanagi R. Nishikawa M. Haji M. Nawata H. J. Biol. Chem. 1993; 268: 26759-26766Abstract Full Text PDF PubMed Google Scholar, 9Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike M. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 10Yanagisawa H. Yanagisawa M. Kapur R.P. Richardson J.A. Williams S.C. Clouthier D.E. de Wit D. Emoto N. Hammer R.E. Development. 1998; 125: 825-836Crossref PubMed Google Scholar)), and Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (neurotensin (3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar, 5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar, 7Takahashi M. Matsushita Y. Iijima Y. Tanzawa K. J. Biol. Chem. 1993; 268: 21394-21398Abstract Full Text PDF PubMed Google Scholar, 8Ohnaka K. Takayanagi R. Nishikawa M. Haji M. Nawata H. J. Biol. Chem. 1993; 268: 26759-26766Abstract Full Text PDF PubMed Google Scholar, 9Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike M. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 10Yanagisawa H. Yanagisawa M. Kapur R.P. Richardson J.A. Williams S.C. Clouthier D.E. de Wit D. Emoto N. Hammer R.E. Development. 1998; 125: 825-836Crossref PubMed Google Scholar, 11Shimada K. Takahashi M. Ikeda M. Tanzawa K. FEBS Lett. 1995; 371: 140-144Crossref PubMed Scopus (116) Google Scholar, 12Schweizer A. Valdenaire O. Nelböck P. Deuschle U. Dumas Milne Edwards J.-B. Stumpf J.G. Löffler B.-M. Biochem. J. 1997; 328: 871-877Crossref PubMed Scopus (191) Google Scholar, 13Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Crossref PubMed Scopus (430) Google Scholar)) were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). Oxidized insulin B chain, dansyl-d-Ala-Gly-(pNO2)Phe-Gly, leucine aminopeptidase, and phosphoramidon were obtained from Sigma. Human big ET-1 was purchased from Peptides International (Louisville, KY). All other peptides were purchased from Bachem Bioscience, Inc. (Torrance, CA). Polyoxyethylene-10-lauryl ether (C12E10) was from Calbiochem (La Jolla, CA). Peptides (0.25 mm) were incubated with solECE-1 (83 nm) at 37 °C in 50 mm MES-KOH, 0.01% C12E10, pH 6.5, for 2 to 16 h. Reaction products were separated by reversed-phase high performance liquid chromatography (HPLC) using pumps, ultraviolet detector, and software from Rainin Instrument Co. (Emeryville, CA). Peptides were bound to a 0.46 × 25-cm C18 column (Vydac, Hesperia, CA) and eluted by a gradient of 0 to 60% (v/v) acetonitrile in 0.1% trifluoroacetic acid. Peptides were detected by absorbance at 215 nm. Products were collected and analyzed by matrix-assisted laser desorption/ionization mass spectrometry. When necessary, products were further subjected to liquid chromatography-mass spectrometry or NH2-terminal amino acid sequence analysis to confirm their identity. SolECE-1 hydrolysis of dansyl-d-Ala-Gly-(pNO2)Phe-Gly and glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide were assayed fluorimetrically at concentrations of 0.1 mm as described previously for the assay of neprilysin (17Li C. Hersh L.B. Methods Enzymol. 1995; 248: 253-263Crossref PubMed Scopus (41) Google Scholar). Matrix-assisted laser desorption/ionization mass spectra were acquired on a PerSeptive Biosystems, Inc. (Framingham, MA) Voyager Elite-Delayed-Extraction time-of-flight mass spectrometer. Radiation from a Laser Science, Inc. (Newton, MA) nitrogen laser (337 nm, 3-ns pulse width) was used to desorb ions from the target. All linear delayed extraction experiments were performed using an extraction grid voltage of 23.125 kV and a pulse delay of 150 ns. Twenty-five to 100 laser shots were averaged for each spectrum. Electrospray ionization mass spectra were acquired with a Finnigan MAT 900 double-focusing mass spectrometer. Analyses were performed at 5 kV full accelerating potential. Tandem mass spectra were acquired by scanning the magnet and the electrical analyzer simultaneously at a constant B/E ratio. The rates of substrate hydrolysis were determined by measuring the appearance of products by HPLC under initial rate conditions (less than 10% hydrolysis of substrate, except for big ET-1). To detect products of big ET-1 hydrolysis at low substrate concentrations, it was necessary to digest up to 20% of the substrate. Depending on the substrate, solECE-1 concentrations ranging from 4.2 to 250 nm were used. Reactions were carried out in 50 mm MES-KOH, 0.01% C12E10, pH 6.5, at 37 °C. Reaction volumes ranging from 0.10 to 1.0 ml were analyzed by HPLC, depending on the detection limits of the products of interest. Product peak areas determined using Dynamax software (Rainin) were converted to mol of product using standard curves generated from known amounts of the peptide products. Big ET-1 hydrolysis was quantitated by measuring the formation of the COOH-terminal fragment, big ET-1 (22–38). The rates of hydrolysis of angiotensin I, angiotensin I (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar, 5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar), and bradykinin were measured by quantitating the appearance of the products angiotensin I (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1995; 51: 91-102Crossref Scopus (221) Google Scholar, 4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada M. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (885) Google Scholar, 5Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (854) Google Scholar, 6Ahn K. Beningo K. Olds G. Hupe D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8606-8610Crossref PubMed Scopus (59) Google Scholar, 7Takahashi M. Matsushita Y. Iijima Y. Tanzawa K. J. Biol. Chem. 1993; 268: 21394-21398Abstract Full Text PDF PubMed Google Scholar), angiotensin I (1Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10157) Google Scholar, 2Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2551) Google Scholar, 3Turner A.J. 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