RESUMO
Mutalysin II, a zinc endopeptidase possessing direct-acting fibrinolytic activity has been previously purified from bushmaster (Lachesis muta muta) snake venom. We now report a method to isolate two isoforms of natural mutalysin II (mut IIa and mut IIb) using chromatographies on Sephacryl S-200, CM Sepharose CL 6B and Sephadex G-50. The two proteins are monomeric non-glycosylated proteinases with similar molecular masses of 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Both isoforms showed high similarity in all enzymatic properties using fibrinogen, fibrin and dimethylcasein as substrates. Thus, the specific fibrinolytic activity was estimated as 12+/-1.04 and 11.5+/-1.02 U/microg for mut IIa and mut IIb, respectively. The antigenic cross-reactivity of both isoforms was examined using rabbit hyperimmune serum or immunoglobulin G anti-mut IIa assays on immunodiffusion microscope slides, indirect enzyme-linked immunoabsorbent assay and western blots. From these experiments it was concluded that the two metalloproteinases mut IIa and mut IIb share identical antigenic structures. Since the stability of mutalysin II is dependent upon the presence of zinc, we examined the EDTA sensitivity of the isoforms of mutalysin II. Thus, the IC(50) values (concentration of EDTA to produce 50% inhibition of dimethylcasein hydrolysis) for mut IIa is 180 microM and 165 microM for mut IIb.
Assuntos
Glicoproteínas/metabolismo , Metaloendopeptidases/metabolismo , Venenos de Serpentes/isolamento & purificação , Venenos de Serpentes/metabolismo , Viperidae/metabolismo , Animais , Caseínas/efeitos dos fármacos , Caseínas/metabolismo , Cromatografia Líquida de Alta Pressão , Fibrina/efeitos dos fármacos , Fibrina/metabolismo , Fibrinogênio/efeitos dos fármacos , Fibrinogênio/metabolismo , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Imuno-Histoquímica , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Mapeamento de Peptídeos , Coelhos , Venenos de Serpentes/química , Venenos de Serpentes/enzimologia , Análise Espectral , Zinco/farmacologiaRESUMO
A serine proteinase with kallikrein-like activity (LV-Ka) has been purified to homogeneity from bushmaster snake (Lachesis muta muta) venom. Physicochemical studies indicated that LV-Ka is a single chain glycoprotein with a molecular mass (Mr) of 33 kDa under reducing conditions which was reduced to 28 kDa after treatment with N-Glycosidase F (PNGase F). LV-Ka can be bounded and neutralized by serum alpha2-macroglobulin (alpha2-M), a prevalent mammalian protease inhibitor that is capable of forming a macromolecular complex with LV-Ka (Mr >180 kDa). Cleavage of alpha2-M by the enzyme resulted in the formation of 90-kDa fragments. The proteolytic activity of LV-Ka against dimethylcasein could be inhibited by alpha2-M, and the binding ratio of the inhibitor:enzyme complex was found to be 1:1. The Michaelis constant, Km, and catalytic rate constant, kcat, of LV-Ka on four selective chromogenic substrates were obtained from Lineweaver-Burk plots. LV-Ka exhibits substrate specificities not only for the glandular kallikrein H-D-Val-Leu-Arg-pNA (S-2266) but also for the plasmin substrates S-2251 and Tos-Gly-Pro-Lys-pNA. Bovine kininogen incubated with LV-Ka generated a polypeptide that dose dependently contracted mesenteric arterial rings from spontaneously hypertensive rats (SHR) in a similar way as bradykinin (BK) does. As it happens with BK, LV-Ka generated polypeptide was inhibited by HOE-140, a bradykinin B2-receptor antagonist and by indomethacin, a cyclo-oxygenase inhibitor. These results strongly suggest that the polypeptide generated by LV-Ka by cleavage of bovine kininogen is bradykinin. In addition, our studies may help to understand the mechanism of action involved in hypotension produced by envenomation of bushmaster snake.