Purification and substrate specificity of an angiotensin converting elastase-2 from the rat mesenteric arterial bed perfusate.
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 will henceforth be referred to as rat MAB elastase-2. When Ang I was used as the substrate, the enzyme specifically released Ang II and the dipeptide His-Leu (Km=36 microM; Kcat=1530 min-1). The catalytic efficiency (Kcat/Km=42.5 min-1 microM-1) of this reaction was comparable to those of other known Ang I-converting enzymes. The proteolytic specificity of the purified enzyme toward mellitin, oxidized insulin B chain, somatostatin-14 and renin substrate tetradecapeptide suggested that the enzyme-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 of the chymostatin-sensitive group of Ang II-forming serine proteases. The localization and biochemical properties of this enzyme suggest that it might play a role in the regional control of vascular tonus.
Paula
C A
CA
Department of Biochemistry, Faculty of Medicine, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil.
Sousa
M V
MV
Salgado
M C
MC
Oliveira
E B
EB
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000096:biosynthesis; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007700:Kinetics; D008297:Male; D017538:Mesenteric Artery, Superior / Q000201:enzymology*; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D000806:Angiotensin-Converting Enzyme Inhibitors; D015842:Serine Proteinase Inhibitors; D000804:Angiotensin II; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; D010196:Pancreatic Elastase