title: Biochemical properties of a bushmaster snake venom serine proteinase (LV-Ka), and its kinin releasing activity evaluated in rat mesenteric arterial rings.
abstract: 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.
authors.affiliation: Vascular Smooth Muscle Laboratory, Department of Physiology and Biophysics, ICB, Federal University of Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Maria L D
authors.initials: ML
authors.lastname: Weinberg
authors.forename: Liza F
authors.initials: LF
authors.lastname: Felicori
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Bello
authors.forename: Henrique P B
authors.initials: HP
authors.lastname: Magalhães
authors.forename: Alvair P
authors.initials: AP
authors.lastname: Almeida
authors.forename: Arinos
authors.initials: A
authors.lastname: Magalhães
authors.forename: Eladio F
authors.initials: EF
authors.lastname: Sanchez
mesh_terms: D000818:Animals; D002417:Cattle; D006801:Humans; D066298:In Vitro Techniques; D007705:Kinins / Q000378:metabolism*; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014757:Viper Venoms / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D017819:Viperidae
chemical_list: D007705:Kinins; D014757:Viper Venoms; D012697:Serine Endopeptidases