Basic proteinases from Bothrops moojeni (caissaca) venom--I. Isolation and activity of two 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 moojeni venom by chromatographies on Sephadex G-100, DEAE-Sephacel (pH 7.5) and SP-Sephadex C-50 (pH 7.5). Both enzymes are basic glycoproteins. On sodium dodecyl sulfate-polyacrylamide electrophoresis, MSP 1 presented 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 kallikrein substrate D-Val-Leu-Arg-Nan. MSP 1, in a concentration of 10(-8) M, causes platelet aggregation in platelet-rich plasma and washed platelets. It also enhances the ADP-induced platelet aggregation. Prostaglandin 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 amidolytic (Ki = 1.96 x 10(-7) M) and platelet-aggregating (Ki = 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 trypsin inhibitor (SBTI) and benzamidine and not affected by Trasylol. MSP 2 was also inhibited by TCTI (Ki = 0.7 x 10(-7) M).
Serviço de Bioquímica, Instituto Butantan, São Paulo, Brazil.
S M
SM
Serrano
M F
MF
Matos
F R
FR
Mandelbaum
C A
CA
Sampaio
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002364:Caseins / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology*; D006168:Guinea Pigs; D006470:Hemorrhage / Q000139:chemically induced / Q000473:pathology; D006736:Horses; D006801:Humans; D066298:In Vitro Techniques; D007705:Kinins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects*; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014106:Tosylarginine Methyl Ester / Q000378:metabolism
D000577:Amides; D002364:Caseins; D003435:Crotalid Venoms; D007705:Kinins; D015842:Serine Proteinase Inhibitors; D000244:Adenosine Diphosphate; D014106:Tosylarginine Methyl Ester; C081402:MSP 1 proteinase, Bothrops moojeni; C081403:MSP 2 proteinase, Bothrops moojeni; D012697:Serine Endopeptidases