alexa Cloning, characterization and site-directed mutagenesis of canine renin.
Diabetes & Endocrinology

Diabetes & Endocrinology

Journal of Diabetic Complications & Medicine

Author(s): Bukhtiyarov Y, Zecher M, Panemangalore R, Wu Z, Bruno JG, , Bukhtiyarov Y, Zecher M, Panemangalore R, Wu Z, Bruno JG,

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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 approximately 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 Leu(10)-Leu(11)). The reaction followed Michaelis-Menten kinetics with a K(M) of 120 microM and a second-order rate constant (k(cat)/K(M)) of 1.7 x 10(5) M(-)(1)s(-)(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. This article was published in J Biochem and referenced in Journal of Diabetic Complications & Medicine

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