A Fluorometric Method of Measuring Carboxypeptidase Activities for Angiotensin II and Apelin-13

Sci Rep. 2017 Apr 5;7:45473. doi: 10.1038/srep45473.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Angiotensin I / metabolism
  • Angiotensin II / metabolism*
  • Angiotensin-Converting Enzyme 2
  • Animals
  • Carboxypeptidases / metabolism*
  • Fluorometry*
  • HEK293 Cells
  • Humans
  • Immunoassay
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Kidney / enzymology
  • Mice
  • Peptide Fragments / metabolism
  • Peptidyl-Dipeptidase A / metabolism
  • Phenylalanine / metabolism
  • Substrate Specificity

Substances

  • Intercellular Signaling Peptides and Proteins
  • Peptide Fragments
  • apelin-13 peptide
  • Angiotensin II
  • Phenylalanine
  • Angiotensin I
  • Carboxypeptidases
  • Peptidyl-Dipeptidase A
  • ACE2 protein, human
  • Ace2 protein, mouse
  • Angiotensin-Converting Enzyme 2
  • angiotensin I (1-7)