Characterization of angiotensin I-converting enzyme from anterior gills of the mangrove crab Ucides cordatus

• Published in: International journal of biological macromolecules Vol. 74; pp. 304 - 309
• Main Authors: Bersanetti, Patrícia A., Nogueira, Regina F., Marcondes, Marcelo F., Paiva, Paulo B., Juliano, Maria A., Juliano, Luiz, Carmona, Adriana K., Zanotto, Flavia P.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-03-2015
• Subjects: ACE isoform; Angiotensin-Converting Enzyme Inhibitors - pharmacology; Animals; Brachyura - enzymology; Characterization; Crab gill; Enzyme Activation - drug effects; Enzyme Stability - drug effects; FRET peptides; Gills - enzymology; Hydrogen-Ion Concentration; Hydrolysis; Lisinopril - pharmacology; Peptidyl-Dipeptidase A - chemistry; Peptidyl-Dipeptidase A - classification; Peptidyl-Dipeptidase A - genetics; Peptidyl-Dipeptidase A - isolation & purification; Peptidyl-Dipeptidase A - metabolism; Phylogeny; Substrate Specificity; Temperature; Characterization; Crab gill; ACE isoform; FRET peptides
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2014.12.036

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates, invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE, here named crab-ACE, which presented catalytic properties closely resembling to those of mammalian ACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogeneity and a band of about 72kDa could be visualized after silver staining and Western blotting. Assays performed with fluorescence resonance energy transfer (FRET) selective ACE substrates Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH, allowed us to verify that crab-ACE has hydrolytic profile very similar to that of the ACE C-domain. In addition, we observed that crab-ACE can hydrolyze the ACE substrates, angiotensin I and bradykinin. The enzyme was strongly inhibited by the specific ACE inhibitor lisinopril (Ki of 1.26nM). However, in contrast to other ACE isoforms, crab-ACE presented a very particular optimum pH, being the substrate Abz-FRK(Dnp)-P-OH hydrolyzed efficiently at pH 9.5. Other interesting characteristic of crab-ACE was that the maximum hydrolytic activity was reached at around 45°C. The description of an ACE isoform in Ucides cordatus is challenging and may contribute to a better understanding of the biochemical function of this enzyme in invertebrates.