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- Title
Cloning, Characterization and Site-Directed Mutagenesis of Canine Renin.
- Authors
Bukhtiyarov, Yuri; Zecher, Marianne; Panemangalore, Reshma; Zhongren Wu; Bruno, Joseph G.; Jing Yuan; Zhenrong Xu; Dillard, Lawrence W.; McGeehan, Gerard M.; Harrison, Richard K.; Scott, Boyd B.
- Abstract
Inhibition of renin has been shown to be successful in managing hypertension and maintaining cardiac health. Canine models have played a key role in preclinical assessment of renin inhibitors. Here we report the cloning of canine prorenin gene. The amino acid sequence of mature canine renin was ∼70% identical to that of human renin. The full-length prorenin was expressed in HEK 293 cells, purified and converted to its active form by trypsin-mediated cleavage of the 43 residue propeptide. The mature enzyme was characterized by steady-state kinetics using a peptide corresponding to the canine angiotensinogen sequence, Ac–Asp–Arg–Val–Tyr–Ile–His–Pro–Phe–His–Leu–Leu–Val–Tyr–Ser–OH (cleavage between Leu10–Leu11). The reaction followed Michaelis–Menten kinetics with a KM of 120 µM and a second-order rate constant (kcat/KM) of 1.7 × 105 M−1s−1. The enzyme was inhibited by various human renin inhibitors, but at reduced potency compared to the human renin. The basis of the species specificity was investigated by mutagenesis. Based on primary sequence and structural alignments, three mutants were prepared (G149S-S150T, V286L, G149S-S150T-V286L). Each mutant yielded catalytically active enzymes with lower specific activities than native canine renin. V286L had the greatest effect on substrate specificity, while G149S, S150T mutations produced enzymes with inhibitor profiles similar to human renin.
- Subjects
CLONING; MUTAGENESIS; RENIN; GENE expression; LABORATORY dogs
- Publication
Journal of Biochemistry, 2007, Vol 142, Issue 6, p671
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvm182