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Bowman–Birk serine protease inhibitors are a family of small plant pro-teins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. | ễFEBS Journal Inhibitory properties and solution structure of a potent Bowman-Birk protease inhibitor from lentil Lens culinaris L seeds Enzio M. Ragg1 Valerio Galbusera1 Alessio Scarafoni1 Armando Negri2 Gabriella Tedeschi2 Alessandro Consonni1 Fabio Sessa1 and Marcello Duranti1 1 Department of Agri-Food Molecular Sciences University degli Studi Milano Italy 2 Department of AnimalPathology Hygiene and Veterinary Public Health-Section of Biochemistry Universita degli Studi Milano Italy Keywords Bowman-Birk inhibitor antitryptic activity dicotyledonous plant Lens culinaris nuclear magnetic resonance Correspondence E. M. Ragg Department of Agri-Food Molecular Sciences University degli Studi via Celoria 2 20133 Milano Italy Fax 39 0250316801 Tel 39 0250316800 E-mail enzio.ragg@unimi.it Received 10 May 2006 revised 29 June 2006 accepted 5 July 2006 doi 10.1111 j.1742-4658.2006.05406.x Bowman-Birk serine protease inhibitors are a family of small plant proteins whose physiological role has not been ascertained as yet while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil Lens culinaris L. var. Macrosperma seed trypsin inhibitor LCTI and its functional and structural characterization. LCTI is a 7448 Da double-headed tryp-sin chymotrypsin inhibitor with dissociation constants equal to 0.54 nM and 7.25 nM for the two proteases respectively. The inhibitor is however hydrolysed by trypsin in a few minutes timescale leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k on values 1.1 pM_1-s-1 vs. 0.002 pM_1-s-1 respectively for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor BBI conformation with two antipodal b-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and .
