doi:10.1016/j.bbadis.2004.05.005 
Copyright © 2004 Elsevier B.V. All rights reserved.
Rapid report
Interaction of ACE2 and integrin β1 in failing human heart
a The Center for Functional Genomics, Proteomics Core Facility, University at Albany, Rensselaer, NY, USA
b The Center for Cardiovascular Sciences, Albany Medical College, Albany, NY, USA
c Myomatrix Therapeutics, One University Place, Rm DB224, Rensselaer, NY 12144, USA
Received 8 March 2004;
accepted 14 May 2004.
Available online 11 June 2004.
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Abstract
ACE2 purified from failing human heart was found to form a complex with integrin β1 by immunoprecipitation, Western blotting, activity assay, and ESI tandem mass spectroscopy. The ACE2/integrin complex showed a Km of 6.8 μM and a Vmax of 2.13 pmol/min/μl purified enzyme. Activity was optimal at pH 7.5 with Ang II substrate.
Author Keywords: Angiotensin-converting enzyme 2; Angiotensin-(1–7); Heart Failure; Cardiomyopathy; Integrin β1
Article Outline
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Fig. 1. Tandem mass spectroscopy performed on the SEC fraction containing 3000-fold purified ACE2 complex demonstrated the presence of integrin β1 in addition to ACE2. (A) CID fragmentation of tryptic peptide demonstrating the sequence ISFNFFVTAPK from ACE 2 (AB046569). (B) CID fragmentation of tryptic peptide demonstrating the sequence WDTQENPIYK from integrin β (NM_033668).
Fig. 2. Western blots of the ACE 2/integrin complex. (A) Western blot with the ACE2 antibody. (B) Western blot with the anti-integrin β1 D antibody. Lane 1, extraction; lane 2, Q-sepharose; lane 3, phenyl-sepharose; lane 4, DEAE-sepharose; lane 5, SP-sepharose. Each lane was loaded with 2.5 μg of protein.
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Fig. 3. Immunoprecipitation of the ACE2/integrin complex purified from human heart. The immunoprecipitation was performed with the anti-integrin β1 antibody, P5D2. (A) Q-sepharose-purified ACE2 was immunoprecipitated with P5D2. Lane 1: Western blot with the anti-ACE2 antibody in the presence of scrambled peptide; 2: Western blot with anti-ACE2 antibody in the presence of blocking peptide showing specificity of the ACE2 antibody; 3: Western blot with the anti-integrin β1D specific antibody showing the presence of integrin β1D in the immunoprecipitate. (B) Direct immunoprecipitation of a solubilized membrane preparation from a failing human heart right ventricle. Lanes 1 and 3: supernatant after immunoprecipitation. Lanes 2 and 4: immunoprecipitate with the P5D2 antibody. Western blots were performed with either the ACE2 antibody (lanes 1 and 2) or the anti-integrin β1D antibody (lanes 3 and 4). Immunoprecipitation with the anti-integrin antibody pulled down both ACE2 and integrin β 1D from a failing human heart membrane preparation.
Fig. 4. HPLC chromatograms showing ACE2 activity in the anti-integrin β1 antibody immunoprecipitate (lanes 1 and 2 are duplicate experiments). Lanes 3 and 4 show chromatograms of residual ACE2 activity in the supernatant after immunoprecipitation. Lane 5 shows that all 125I-Ang-(1-7) forming activity in the immunoprecipitate was inhibited by the ACE2 specific inhibitor C16. These data indicate that ACE2 formed a complex with integrin β1D, and that ACE2 in this complex was catalytically active.
Table 1. Purification of the ACE2/integrin complex from failing human heart
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