KALLIKREIN-LIKE PROTEINASE FROM BUSHMASTER SNAKE VENOM

Vilela, L.F.(1), Souza, C.T.(1), Bello, C.A.(1), MagalhÃes, A.(1), Almeida, A.P.(2), Richardson, M.(1), Sanchez, E.F.(1)

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

Objectives: The experiments in this work were focused on the biochemical characterization of a kallikrein-like proteinase (LV-Ka) from Lachesis muta muta (bushmaster) snake venom. The isolated protein is a 33 kDa monomeric glycoprotein which Mr was reduced to 28 kDa after deglycosylation with PNGase F.  LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (1 mg/g) was shown to lower blood pressure in experimental rats. In vitro, the purified enzyme was shown to have neither fibrino(geno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity= 13 and 31.5 U/mg, respectively, crude venom= 0.25 and 6.0 U/mg), but had no proteolytic effect on dimethyl casein and insulin B chain. Its enzymatic activity was inhibited by NPGB, p-aminobenzamidine and PMSF, indicating that the enzyme is a serine proteinase. Approximately 76 % of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease.  Sequence studies on the NH₂-terminal region of the protein indicates that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with LMR-47 from L. m. rhombeata and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. Interestingly, one of the other reactions catalyzed by the venom enzyme, the activation of plasminogen was also exhibited by LV-Ka. In conclusion, these studies indicate that LV-Ka is a kallikrein-like enzyme from snake venom origin which may also participate in the extrinsic pathway of fibrinolysis.

Financial support: CNPq and FAPEMIG.

CORRESPONDENCE TO:

EladioOswaldo Flores Sanchez, Rua Furtado Nunes 110-101, Belo Horizonte, MG, CEP: 30730-090, Brasil, Email: eladio@funed.mg.gov.br