Kinetic characterization and inhibition of the rat MAB elastase-2, an angiotensin I-converting serine protease.

Abstract

An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursor [Pro11-D-Ala12]-Ang I was converted into Ang II by the rat MAB elastase-2 with a catalytic efficiency of 8.6 min–1·mM–1, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro- Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min–1·mM–1 and 7.6 min–1·mM–1, respectively. The non-cleavable peptide inhibitor CH-5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50 = 49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50 = 4.8 mM), whereas N-acetyl-Ala- Ala-Pro-Leu-chloromethylketone, an effective active site-directed inhibitor of pancreatic elastase-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50 = 4.5 mM). Altogether, the data presented here confirm and extend the enzymological similarities between pancreatic elastase-2 and its rat MAB counterpart. Moreover, the thus far unrealized interaction of elastase-2 with [Pro11-D-Ala12]-Ang I and CH-5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways.