BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES  OF  A KALLIKREIN- LIKE ENZYME FROM BUSHMASTER SNAKE VENOM

BELLO,C.A¹; HERMÓGENES,A.L.N¹; FELICORI,L.F¹; WEINBERG, M. de L.D²; ALMEIDA,A.P²; MAGALHÃES,A¹; SANCHEZ,E.F¹

1 Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, 2 Dept. de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

Experiments were focused on several of the biochemical and pharmacological properties of a Kallikrein - Like proteinase ( LV- Ka ) from Lachesismuta muta (Bushmaster) snake venom. Physicochemical studies indicated that LV-Ka is a single chain glycoprotein with a Mr of 33 kDa under reducing conditions which was reduced to 28 kDa after treatment with PNGase F. LV-Ka can be bounded and neutralized by serum a2- macroglobulin (a2-M), a prevalent mammalian protease inhibitor which is capable of forming a macromolecular complex with LV-Ka (Mr> 180 kDa). Cleavage of a2-M by the enzyme resulted in the formation of 90 kDa fragments. The proteolytic activity of LV-Ka against dimethylcasein could be inhibited by a2-M, and the binding ratio of the inhibitor: enzyme complex was found to be 1:1. The Michaelis constant, Km and catalytic rate constant, kcat of LV-Ka on four selective chromogenic substrates were obtained from Lineweaver- Burk plots. LV- Ka was significantly more effective on the substrate for glandular kallikrein H-D-Val-Leu-Arg-pNA (S-2266). Bovine kininogen incubated with LV-Ka generated a polypeptide that, dose dependently, contracted mesenteric arterial rings from spontaneously hypertensive rats (SHR) in a similar way as bradykinin does. Such contraction was inhibited by HOE-140, a bradykinin B2 receptor inhibitor and by indomethacin, a cyclo- oxygenase inhibitor.

These results strongly suggest that the polypeptide generated by LV-Ka by cleavage of bovine kininogen is bradykinin.

Financial Support: CNPq and FAPEMIG.

Correspondence to: cynthiabello@yahoo.com.br