PURIFICATION, CHARACTERIZATION, AND AMINO-ACID-SEQUENCE OF A SERINE PROTEINASE, PA-BJ, WITH PLATELET-AGGREGATING ACTIVITY FROM THE VENOM OFBOTHROPS-JARARACA

A platelet-aggregating enzyme, PA-BJ, was isolated from the venom of t he snake Bothrops jararaca. PA-BJ in a concentration of 3.2 x 10(-8) M promoted 95% platelet aggregation in platelet-rich plasma. SDS-polyac rylamide gel electrophoresis under reducing conditions showed a single protein band with an M(r) of 30 000. PA-BJ catalyzed the hydrolysis o f several p-nitroanilide peptide substrates containing Arg or Lys at t he scissile bond; among these the most sensitive were the thrombin sub strates D-Phe-Pip-Arg-pNA and Tos-Gly-Pro-Arg-pNA. Both the platelet-a ggregating and amidolytic activities of PA-BJ were abolished by reacti on with phenylmethanesulfonyl fluoride. Several benzamidine derivative s, 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 [((alpha)-[(2-naphthylsulfonyl)- glycyl]-4-amidinophenylalanine piperidide] showed the strongest inhib itor activity on PA-BJ. The complete amino acid sequence of PA-BJ, whi ch, to the best of our knowledge, is the first of a platelet-aggregati ng enzyme from snake venom, was deduced from the N-terminal sequencing of overlapping fragments cleaved from the reduced and S-pyridylethyla ted protein by chemical and enzymatic methods. PA-BJ is composed of 23 2 amino acid residues and contains one N- and one O-glycosidically lin ked carbohydrate moiety at residues Asn(20) and Ser(23). Sequence comp arison to other venom serine proteinases revealed significant homology , mainly in regions around the catalytic triad and conserved cysteine residues.