Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human alpha2-macroglobulin and rabbit immunoglobulin.
Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.
Souza
C T
CT
Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, 30510-010 Belo Horizonte, MG, Brazil.
Moura
M B
MB
Magalhaes
A
A
Heneine
L G
LG
Olortegui
C C
CC
Diniz
C R
CR
Sanchez
E F
EF
D000818:Animals; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D011817:Rabbits; D013997:Time Factors; D014757:Viper Venoms / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D017819:Viperidae; D000511:alpha-Macroglobulins / Q000378:metabolism / Q000494:pharmacology*
D002364:Caseins; D004791:Enzyme Inhibitors; D005338:Fibrin Fibrinogen Degradation Products; D007074:Immunoglobulin G; C002087:N,N-dimethylcasein; D014757:Viper Venoms; D000511:alpha-Macroglobulins; D005337:Fibrin; D005340:Fibrinogen; D008666:Metalloendopeptidases; C068795:hemorrhagic factor LHFII