Purification, characterization, and amino acid sequence of a serine proteinase, PA-BJ, with platelet-aggregating activity from the venom of Bothrops jararaca.
A platelet-aggregating enzyme, PA-BJ, was isolated from the venom of the snake Bothrops jararaca. PA-BJ in a concentration of 3.2 x 10(-8) M promoted 95% platelet aggregation in platelet-rich plasma. SDS-polyacrylamide gel electrophoresis under reducing conditions showed a single protein band with an M(r) of 30,000. PA-BJ catalyzed the hydrolysis of several p-nitroanilide peptide substrates containing Arg or Lys at the scissile bond; among these the most sensitive were the thrombin substrates D-Phe-Pip-Arg-pNA and Tos-Gly-Pro-Arg-pNA. Both the platelet-aggregating and amidolytic activities of PA-BJ were abolished by reaction with phenylmethanesulfonyl fluoride. Several benzamidine derivatives, which are competitive inhibitors of trypsin-like serine proteinases, also inhibited the amidolytic activity of PA-BJ. Among the compounds tested, the thrombin inhibitor NAPAP [N alpha-[(2-naphthylsulfonyl)-glycyl]-4-amidinophenylalanine piperidide] showed the strongest inhibitor activity on PA-BJ. The complete amino acid sequence of PA-BJ, which, to the best of our knowledge, is the first of a platelet-aggregating enzyme from snake venom, was deduced from the N-terminal sequencing of overlapping fragments cleaved from the reduced and S-pyridylethylated protein by chemical and enzymatic methods. PA-BJ is composed of 232 amino acid residues and contains one N- and one O-glycosidically linked carbohydrate moiety at residues Asn20 and Ser23. Sequence comparison to other venom serine proteinases revealed significant homology, mainly in regions around the catalytic triad and conserved cysteine residues.
Serrano
S M
SM
Instituto Butantan, São Paulo, S.P., Brazil.
Mentele
R
R
Sampaio
C A
CA
Fink
E
E
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D002852:Chromatography, Ion Exchange; D003435:Crotalid Venoms / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D003435:Crotalid Venoms; D015842:Serine Proteinase Inhibitors; C038929:Bothrops jararaca protease A; D012697:Serine Endopeptidases