title: 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.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, University of São Paulo, Ribeirão Preto, Brazil.
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Santos
authors.forename: Carmem A
authors.initials: CA
authors.lastname: Paula
authors.forename: Maria Cristina O
authors.initials: MC
authors.lastname: Salgado
authors.forename: Eduardo Brandt
authors.initials: EB
authors.lastname: Oliveira
mesh_terms: D000804:Angiotensin II / Q000096:biosynthesis; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008638:Mesenteric Arteries / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D010455:Peptides; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D000804:Angiotensin II; D007703:Peptidyl-Dipeptidase A; D012697:Serine Endopeptidases; C117309:pancreatic elastase II