Conversion of renin substrate tetradecapeptide to angiotensin II by rat MAB elastase-2.
A new approach for the purification of rat mesenteric arterial bed (MAB) elastase-2 has been developed using the chromogenic substrates N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide to monitor the enzymatic activity during various stages of purification. The purified enzyme was evaluated in the presence of various inhibitors and confirmed to have angiotensin (Ang) II-forming ability. The active site-directed inhibitor acetyl-Ala-Ala-Pro-Leu-chloromethylketone (100 micromol x L(-1)), described for human pancreatic elastase-2, abolished the enzymatic activity, confirming that the enzyme is an elastase-2. Chymostatin (100 micromol x L(-1)), an inhibitor regarded as selective for chymases, also showed a remarkable inhibitory effect (94%), whereas captopril (100 micromol x L(-1)) had no effect at all on the Ang II-forming activity. The Ang II precursor renin substrate tetradecapeptide (RS-14P) was converted into Ang II by the rat MAB elastase-2 with the following kinetic constants: Km = 124 +/- 21 micromol x L(-1); Kcat = 629 min(-1); catalytic efficiency (Kcat /Km) = 5.1 min(-1) micro(mol/L)-1. In conclusion, the strategy for the purification of rat MAB elastase-2 with the chromogenic substrates proved to be simple, rapid, accurate, and highly reproducible; therefore, it can be reliably and conveniently used to routinely purify this enzyme. The kinetic parameters for the formation of Ang II from RS-14P by rat MAB elastase-2 emphasize differences in substrate specificity between this and other Ang II-forming enzymes.
Santos
Carlos F
CF
Discipline of Pharmacology, Bauru School of Dentistry, University of São Paulo, Brazil.
Greene
Andrew S
AS
Salgado
Maria Cristina O
MC
Oliveira
Eduardo B
EB
D000804:Angiotensin II / Q000378:metabolism*; D000808:Angiotensinogen / Q000378:metabolism*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D008643:Mesentery / Q000098:blood supply / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000378:metabolism*
D011480:Protease Inhibitors; D000808:Angiotensinogen; D000804:Angiotensin II; D012697:Serine Endopeptidases; C117309:pancreatic elastase II