DETECTION AND CHARACTERIZATION OF METALLOPROTEINASES WITH GELATINOLYTIC, FIBRONECTINOLYTIC AND FIBRINOGENOLYTIC ACTIVITIES IN BROWN SPIDER (LOXOSCELES INTERMEDIA) VENOM

By studying Loxosceles intermedia (Brown spider) venom we were able to detect a proteolytic action on fibronectin and fibrinogen but an inab ility to degrade full length laminin, type I and type IV collagens. By studying enzyme inhibitors we observed that divalent metal chelators as EDTA and 1,10-phenanthroline completely blocked this cleaving actio n whereas serine-protease inhibitors, thiol-protease inhibitor and aci d-protease inhibitor showed little or no effect on the proteolytic act ivity of the venom indicating involvement of a metalloproteinase. Zymo gram analysis of venom detected a 35 kDa molecule with gelatinolytic a ctivity. The metalloproteinase nature was further supported by its sen sitivity to 4-aminophenyl mercuric acetate (APMA) treatment which decr eased its molecular weight to 32 kDa, inhibition of its gelatinolytic effect by 1,10-phenanthroline and its elution from gelatin-sepharose a ffinity beads. In addition, zymogram experiments using fibronectin and fibrinogen as substrates detected a fibronectinolytic and fibrinogeno lytic band at 28 kDa which changed its electrophoretic mobility to 20 kDa band after organomercurial treatment. The inhibitory effect of 1,1 0 phenanthroline and APMA sensitivity on this proteolytic effect confi rmed the presence of a second metalloproteinase in the venom. The data presented herein describe two invertebrate metalloproteinases in L. i ntermedia venom with different specificities one gelatinolytic and ano ther, fibronectinolytic and fibrinogenolytic, probably involved in the harmful effects of the venom. (C) 1998 Elsevier Science Ltd. All righ ts reserved.