BASIC PROTEINASES FROM BOTHROPS-MOOJENI-(CAISSACA) VENOM .1. ISOLATION AND ACTIVITY OF 2 SERINE PROTEINASES, MSP-1 AND MSP-2, ON SYNTHETIC SUBSTRATES AND ON PLATELET-AGGREGATION

Two serine proteinases, MSP 1 and MSP 2, were isolated from Bothrops m oojeni venom by chromatographies on Sephadex G-100, DEAE-Sephacel (pH 7.5) and SP-Sephadex C-50 (pH 7.5). Both enzymes are basic glycoprotei ns. On sodium dodecyl sulfate-polyacrylamide electrophoresis, MSP 1 pr esented two close protein bands corresponding to the mol.wts of 34,000 and 32,500. MSP 2 behaved as a single-chain protein with a mol. wt of 38,000. Specific esterolytic activities of MSP 1 and MSP 2 on alpha-N -tosyl-L-arginine methyl ester (TAME) are 33 mumol min-1 mg-1 and 184 mumol min-1 mg-1, respectively. The most sensitive substrates for the amidolytic activity of both proteinases were the thrombin substrate D- Phe-pipecolyl(Pip)-Arg-4-nitroanilide(Nan) and the glandular kallikrei n substrate D-Val-Leu-Arg-Nan. MSP 1, in a concentration of 10(-8)M, c auses platelet aggregation in platelet-rich plasma and washed platelet s. It also enhances the ADP-induced platelet aggregation. Prostaglandi n E1 (PGE1), phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA) abolished completely the aggregation induced by MSP 1. Torresea cearensis trypsin inhibitor (TCTI) inhibited both ami dolytic (K(i) = 1.96 x 10(-7)M) and platelet-aggregating (K(i) = 1.66 x 10(-7)M) activities of MSP 1. The esterolytic activity of MSP 1 and MSP 2 was completely abolished by PMSF, only partially by soybean tryp sin inhibitor (SBTI) and benzamidine and not affected by Trasylol. MSP 2 was also inhibited by TCTI (K(i) = 0.7 x 10(-7)M).