Abstract
Degradation of the biologically potent octapeptide angiotensin Ang II-(1-8) is mediated by the activities of several peptidases. The conversion of Ang II to the septapeptide Ang-(1-7) is of particular interest as the latter also confers organ protection. The conversion is catalyzed by angiotensin-converting enzyme 2 and other enzymes that selectively cleave the peptide bond between the proline and the phenylalanine at the carboxyl terminus of Ang II. The contribution of various enzyme activities that collectively lead to the formation of Ang-(1-7) from Ang II, in both normal conditions and in disease states, remains only partially understood. This is largely due to the lack of a reliable and sensitive method to detect these converting activities in complex samples, such as blood and tissues. Here, we report a fluorometric method to measure carboxypeptidase activities that cleave the proline-phenylalanine dipeptide bond in Ang II. This method is also suitable for measuring the conversion of apelin-13. The assay detects the release of phenylalanine amino acid in a reaction with the yeast enzyme of phenylalanine ammonia lyase (PAL). When used in cell and mouse organs, the assay can robustly measure endogenous Ang II and apelin-13-converting activities involved in the renin-angiotensin and the apelinergic systems, respectively.
Highlights
To circumvent these problems, we have developed an assay to evaluate conversion of Ang II to Ang-(1-7), as compared to that driven by recombinant mouse ACE2 as an exogenous control and the combined activities www.nature.com/scientificreports/
Because the amino acid phenylalanine is stable in any tissue lysis conditions, the method is amenable for experiments designed to screen for new enzymes that degrade Ang II and form Ang-(1-7)
Since apelin-13 can be degraded through proteolytic removal of the carboxyl terminal phenylalanine (Phe13/F) residue30, we reasoned that the proposed phenylalaninine assay should be able to detect the cleavage of apelin-13 by ACE2
Summary
We have developed an assay to evaluate conversion of Ang II to Ang-(1-7), as compared to that driven by recombinant mouse ACE2 as an exogenous control and the combined activities www.nature.com/scientificreports/. Of endogenous Ang-(1-7) forming enzymes naturally expressed in organs This method takes advantage of the fact that Ang II can only be converted to Ang-(1-7) by splitting phenylalanine (Phe) from the carboxyl end of Ang II. Because the amino acid phenylalanine is stable in any tissue lysis conditions, the method is amenable for experiments designed to screen for new enzymes that degrade Ang II and form Ang-(1-7). This fluorescence-based assay is time-saving, quantitative and reliable to measure specific Ang II to Ang-(1-7) converting activity in complex biological samples. Since apelin-13 can be degraded through proteolytic removal of the carboxyl terminal phenylalanine (Phe13/F) residue, we reasoned that the proposed phenylalaninine assay should be able to detect the cleavage of apelin-13 by ACE2
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