Purification, Properties, and N-Terminal Amino Acid Sequence of a Kallikrein-like Enzyme from the Venom of Lachesis muta rhombeata (Bushmaster) |
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Authors: | Salvatore Giovanni-De-Simone Aniesse S. Aguiar Anibal R. Gimenez Katia Novellino Roberto S. de Moura |
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Affiliation: | (1) Laboratório de Microseqüênciamento de Proteinas, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz-FIOCRUZ, 21040-900 Rio de Janeiro, RJ, Brazil;(2) Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil;(3) Instituto Vital Brazil, Niteról, RJ, Brazil;(4) Departamento de Farmacologia, Universidade Estadual do Rio de Janeiro, Rio de Janeiro, RJ, Brazil |
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Abstract: | Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0–6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N--tosyl-L-Arg methyl esterase (955.38 units/mg) and N-BZ-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A- and B- chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein. |
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Keywords: | Lachesis muta rhombeata serine proteinase kallikrein, purification sequence |
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