Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster).
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-alpha-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-alpha and B-beta 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.
Giovanni-De-Simone
S
S
Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, RJ, Brazil.
Aguiar
A S
AS
Gimenez
A R
AR
Novellino
K
K
de Moura
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000139:chemically induced; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism; D007022:Hypotension / Q000139:chemically induced; D007610:Kallikreins / Q000737:chemistry* / Q000302:isolation & purification*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012911:Snakes; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity
D003435:Crotalid Venoms; C072644:Lachesis venom; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D010450:Endopeptidases; D007610:Kallikreins