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 [Pro 11 -D-Ala 12]-Ang I was converted into Ang II by the rat MAB elastase-2 with catalytic efficiency of 8.6 min-1 microM-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 microM-1 and 7.6 min-1 microM-1, respectively. The non-cleavable peptide inhibitor CH-5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50 = 49 microM) and N-succinly-Ala-Ala-Pro-Phe-p-nitroanilide (IC 50 = 4.8 microM), 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 (IC 50 = 4.5 microM). 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 [Pro 11-D-Ala 12]-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.

Functional role, cellular source, and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

“…Similarly, chymostatin inhibits both chymases (56,59) and elastase-2 (47), so the use of Ac-AAPL-CK and chymostatin cannot unequivocally indicate the relative contribution of different serine proteases in the generation of ANG II in the isolated rat MAB or elsewhere. However, as established in literature, rat chymase is mainly an angiotensinase (6,30,62), which argues in favor of elastase-2 being responsible for the ACE-independent pathway for ANG II generation in the rat MAB because it does not degrade ANG II (47,52 ]-ANG I are either homologous to human heart chymase (22,38,41,43,63) or rat elastase-2 (52), so this latter enzyme is the only known rat protease fitting the experimental evidence described for the ACE-independent pathway for ANG II generation in the rat MAB. ]-ANG I in the isolated MAB was abolished by the ANG II receptor antagonist saralasin (50 nM) in the perfusion solution (Fig.…”

“…MEC-conditioned media showed enzymatic activity toward the chromogenic substrate Nsuc-AAPL-pNA, a reaction strongly inhibited by 50 M Ac-AAPL-CK (data not shown). Rat MAB elastase-2 hydrolyzes N-suc-AAPL-pNA with a catalytic efficiency of 10.6 min Ϫ1 ⅐ M Ϫ1 and is inhibited by Ac-AAPL-CK at low micromolar concentrations (52). On the other hand, the substrate N-suc-AAPL-pNA is refractory to the action of both rat peritoneal mast cell chymase-like proteases (52) and human skin chymase (data not shown); this latter enzyme was shown to be inhibited by 50 M Ac-AAPL-CK when assayed with a convenient substrate, N-suc-AAPF-pNA.…”

“…Rat MAB elastase-2 hydrolyzes N-suc-AAPL-pNA with a catalytic efficiency of 10.6 min Ϫ1 ⅐ M Ϫ1 and is inhibited by Ac-AAPL-CK at low micromolar concentrations (52). On the other hand, the substrate N-suc-AAPL-pNA is refractory to the action of both rat peritoneal mast cell chymase-like proteases (52) and human skin chymase (data not shown); this latter enzyme was shown to be inhibited by 50 M Ac-AAPL-CK when assayed with a convenient substrate, N-suc-AAPF-pNA. Altogether, these data indicate that a functional elastase-2 is secreted by cultured MECs, but the lack of selectivity of the inhib- Fig.…”

“…We (47,52) have recently described the biochemical, enzymatic, and inhibitory properties of a chymostatinsensitive ANG II-forming elastase-2 found in the perfusate of the isolated rat mesenteric arterial bed (MAB). One of the most interesting findings was that purified elastase-2 from the rat MAB perfusate was also capable of efficiently forming ANG II from [Pro 11 ,D-Ala 12 ]-ANG I, suggesting that in vivo formation of ANG II ascribed to chymases may have been overestimated in previous investigations of ANG II-forming pathways.…”

“…One of the most interesting findings was that purified elastase-2 from the rat MAB perfusate was also capable of efficiently forming ANG II from [Pro 11 ,D-Ala 12 ]-ANG I, suggesting that in vivo formation of ANG II ascribed to chymases may have been overestimated in previous investigations of ANG II-forming pathways. It was also demonstrated that N-acetyl-Ala-AlaPro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of human pancreatic elastase-2 (29), efficiently blocked the ANG II-generating activity of the rat elastase-2 (52). The cloned and sequenced cDNA for this ANG II-generating elastase-2 was found to be identical to that for rat pancreatic elastase-2 (37), whose corresponding mRNA was shown to be expressed in the rat lung but not in the aorta (51).…”