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Conversion of renin substrate tetradecapeptide to angiotensin II by rat MAB elastase-2. Can J Physiol Pharmacol 2004 Nov;82(11):1000-5 PMID: 15644940

Abstract

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.

Author List

Santos CF, Greene AS, Salgado MC, Oliveira EB

Author

Andrew S. Greene PhD Interim Vice Chair, Chief, Professor in the Biomedical Engineering department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Angiotensin II
Angiotensinogen
Animals
Dose-Response Relationship, Drug
Male
Mesenteric Arteries
Mesentery
Protease Inhibitors
Rats
Rats, Sprague-Dawley
Serine Endopeptidases



View this publication's entry at the Pubmed website PMID: 15644940
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