This Article Full Text Full Text (PDF) All Versions of this Article: 47/5/1010    most recent 01.HYP.0000215588.38536.30v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Elased, K. M. Articles by Morris, M. Search for Related Content PubMed PubMed Citation Articles by Elased, K. M. Articles by Morris, M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Related Collections Cardiovascular Pharmacology ACE/Angiotension receptors Animal models of human disease Other hypertension Heart failure - basic studies Hypertension - basic studies Receptor pharmacology

(Hypertension. 2006;47:1010.)
© 2006 American Heart Association, Inc.

Original Articles

New Mass Spectrometric Assay for Angiotensin-Converting Enzyme 2 Activity

Khalid M. Elased; Tatiana S. Cunha; Susan B. Gurley; Thomas M. Coffman; Mariana Morris

From the Boonshoft School of Medicine (K.M.E., T.S.C., M.M.), Wright State University, Dayton, Ohio; Faculty of Dentistry of Piracicaba (T.S.C.), State University of Campinas, Piracicaba, Sao Paulo, Brazil; and Duke University and Durham VA Medical Center (S.B.G., T.M.C.), Durham, NC.

Correspondence to Khalid M. Elased, Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Hwy, Dayton, OH 45435. E-mail: Khalid.elased{at}wright.edu

A novel assay was developed for evaluation of mouse angiotensin-converting enzyme (ACE) 2 and recombinant human ACE2 (rACE2) activity. Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (MS) with ProteinChip Array technology, ACE1 and ACE2 activity could be measured using natural peptide substrates. Plasma from C57BL/6 mice, kidney from wild-type and ACE2 knockout mice, and rACE2 were used for assay validation. Plasma or tissue extracts were incubated with angiotensin I (Ang I; 1296 m/z) or angiotensin II (Ang II; 1045 m/z). Reaction mixtures were spotted onto the ProteinChips WCX2 and peptides detected using surface-enhanced laser desorption/ionization time of flight MS. MS peaks for the substrates, Ang I and Ang II, and the generated peptides, Ang (1-7) and Ang (1-9), were monitored. The ACE2 inhibitor MLN 4760 (0.01 to 100 µmol/L) significantly inhibited rACE2 activity (IC₅₀=3 nmol/L). Ang II was preferably cleaved by rACE2 (km=5 µmol/L), whereas Ang I was not a good substrate for rACE2. There was no detectable ACE2 activity in plasma. Assay specificity was validated in a model of ACE2 gene deletion. In kidney extract from ACE2-deficient mice, there was no generation of Ang (1-7) from Ang II. However, Ang (1-7) was produced when Ang I was used as a substrate. In conclusion, we developed a specific and sensitive assay for ACE2 activity, which used the natural endogenous peptide substrate Ang II. This approach allows for the rapid screening for ACE2, which has applications in drug testing, high-throughput enzymatic assays, and identification of novel substrates/inhibitors of the renin-angiotensin system.

Key Words: angiotensin • renin-angiotensin system • mice • angiotensin converting enzyme

This article has been cited by other articles:

				B. Dong, C. Zhang, J. B. Feng, Y. X. Zhao, S. Y. Li, Y. P. Yang, Q. L. Dong, B. P. Deng, L. Zhu, Q. T. Yu, et al. Overexpression of ACE2 Enhances Plaque Stability in a Rabbit Model of Atherosclerosis Arterioscler. Thromb. Vasc. Biol., July 1, 2008; 28(7): 1270 - 1276. [Abstract] [Full Text] [PDF]

				K. M. Elased, T. S. Cunha, F. K. Marcondes, and M. Morris Brain angiotensin-converting enzymes: role of angiotensin-converting enzyme 2 in processing angiotensin II in mice Exp Physiol, May 1, 2008; 93(5): 665 - 675. [Abstract] [Full Text] [PDF]

				C. Tikellis, K. Bialkowski, J. Pete, K. Sheehy, Q. Su, C. Johnston, M. E. Cooper, and M. C. Thomas ACE2 Deficiency Modifies Renoprotection Afforded by ACE Inhibition in Experimental Diabetes Diabetes, April 1, 2008; 57(4): 1018 - 1025. [Abstract] [Full Text] [PDF]

				Y. Feng, X. Yue, H. Xia, S. M. Bindom, P. J. Hickman, C. M. Filipeanu, G. Wu, and E. Lazartigues Angiotensin-Converting Enzyme 2 Overexpression in the Subfornical Organ Prevents the Angiotensin II-Mediated Pressor and Drinking Responses and Is Associated With Angiotensin II Type 1 Receptor Downregulation Circ. Res., March 28, 2008; 102(6): 729 - 736. [Abstract] [Full Text] [PDF]

				M. C. Chappell Emerging Evidence for a Functional Angiotensin-Converting Enzyme 2-Angiotensin-(1-7)-Mas Receptor Axis: More Than Regulation of Blood Pressure? Hypertension, October 1, 2007; 50(4): 596 - 599. [Full Text] [PDF]

				V. Farah, K. M. Elased, and M. Morris Genetic and dietary interactions: role of angiotensin AT1a receptors in response to a high-fructose diet Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1083 - H1089. [Abstract] [Full Text] [PDF]

				J. C. Q. Velez, A. M. Bland, J. M. Arthur, J. R. Raymond, and M. G. Janech Characterization of renin-angiotensin system enzyme activities in cultured mouse podocytes Am J Physiol Renal Physiol, July 1, 2007; 293(1): F398 - F407. [Abstract] [Full Text] [PDF]

				H. A. Shaltout, B. M. Westwood, D. B. Averill, C. M. Ferrario, J. P. Figueroa, D. I. Diz, J. C. Rose, and M. C. Chappell Angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ACE2-dependent processing of angiotensin II Am J Physiol Renal Physiol, January 1, 2007; 292(1): F82 - F91. [Abstract] [Full Text] [PDF]

				M. F. Doobay, L. S. Talman, T. D. Obr, X. Tian, R. L. Davisson, and E. Lazartigues Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R373 - R381. [Abstract] [Full Text] [PDF]