Identification of proteases in the extract of venom glands from brown spiders.
In the present investigation, in order to dispute the rational criticism against the presence of proteolytic enzymes in the electrostimulated venom obtained from spiders of the genus Loxosceles, as a consequence of contamination with abdominal secretions, venoms of L. intermedia and L. laeta were directly collected from venom glands by microdissection and gentle homogenization. Gel electrophoresis stained by silver method carried out to compare L. intermedia electrostimulated venom and venom gland extract demonstrated no significant differences in protein profile. Zymogram analysis of L. intermedia venom gland extract detected a gelatinolytic activity in the 32-35 kDa region. The inhibitory effect of 1,10-phenanthroline on this proteolytic activity further supported its metalloprotease nature. In proteolytic digestion experiments L. intermedia venom gland extract was also able to cleave purified fibronectin and fibrinogen. The inhibitory effect of 1,10-phenanthroline on these degrading activities confirmed the presence of metalloproteases in the venom. In addition, when purified fibrinogen was incubated with L. intermedia abdominal extract, the fibrinogenolysis was completely different, generating low mass fragments that ran away from the gel, a proteolytic event not blocked by 1,10-phenanthroline. Zymogram experiments using L. laeta venom gland extracts further detected a gelatinolytic band at 32-35 kDa, also inhibited by 1,10-phenanthroline, confirming the presence of metalloproteases in both species.
da Silveira
Rafael B
RB
Department of Cell Biology, Federal University of Paraná, Jardim das Américas, 81531-990 Curitiba, Paraná, Brazil.
dos Santos Filho
José F
JF
Mangili
Oldemir C
OC
Veiga
Silvio S
SS
Gremski
Waldemiro
W
Nader
Helena B
HB
von Dietrich
Carl P
CP
D000818:Animals; D004210:Dissection; D004558:Electric Stimulation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005088:Exocrine Glands / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D005342:Fibrinolysis / Q000187:drug effects; D006801:Humans; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D010618:Phenanthrolines / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013111:Spider Venoms / Q000201:enzymology* / Q000378:metabolism; D013112:Spiders
D010618:Phenanthrolines; D011480:Protease Inhibitors; D013111:Spider Venoms; C055161:loxosceles venom; D010727:Phosphoric Diester Hydrolases; D008666:Metalloendopeptidases; C025205:1,10-phenanthroline