Browsing by Author "Oliveira, Eduardo Brandt de"

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    Kinetic characterization and inhibition of the rat MAB elastase-2, an angiotensin I-converting serine protease.
    (2002) Santos, Carlos F.; Paula, Carmem Aparecida de; Salgado, Maria Cristina de Oliveira; Oliveira, Eduardo Brandt de
    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.
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    Purification and substrate specificity of an angiotensin converting elastase-2 from the rat mesenteric arterial bed perfusate.
    (1998) Paula, Carmem Aparecida de; Sousa, Marcelo Valle de; Salgado, Maria Cristina de Oliveira; Oliveira, Eduardo Brandt de
    A soluble angiotensin (Ang) II-generating enzyme has been purified to homogeneity from the rat mesenteric arterial bed(MAB) perfusate by a combination of gel filtration and affinity chromatographies. The enzyme is a glycoprotein of 28.5 kDa(SDS-PAGE), whose N-terminal sequence is identical with that of the rat pancreatic elastase-2 ; therefore the enzyme willhenceforth be referred to as rat MAB elastase-2. When Ang I was used as the substrate, the enzyme specifically released AngII and the dipeptide His-Leu (Km=36WM;Kcat= 1530 min31). The catalytic efficiency (Kcat/Km= 42.5 min31WM31) of thisreaction was comparable to those of other known Ang I-converting enzymes. The proteolytic specificity of the purifiedenzyme toward mellitin, oxidized insulin B chain, somatostatin-14 and renin substrate tetradecapeptide suggested that theenzyme-substrate interaction was defined by an extended substrate binding site, typical of elastases-2 of pancreatic origin. According to the sensitivity of the rat MAB elastase-2 to various inhibitors this enzyme could be described as a member ofthe chymostatin-sensitive group of Ang II-forming serine proteases. The localization and biochemical properties of thisenzyme suggest that it might play a role in the regional control of vascular tonus.