RESUMO
The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA2 from Agkistrodon contortrix laticinctus snake venom and Lys49PLA2-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA2-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.
Assuntos
Venenos de Crotalídeos/toxicidade , Neurotoxinas/toxicidade , Fosfolipases A/toxicidade , Bexiga Urinária/efeitos dos fármacos , Água/metabolismo , Agkistrodon , Animais , Transporte Biológico/efeitos dos fármacos , Bufo marinus , Venenos de Crotalídeos/enzimologia , Relação Dose-Resposta a Droga , Fosfolipases A2 do Grupo II , Técnicas In Vitro , Fragmentos de Peptídeos/toxicidade , Permeabilidade/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas de Répteis , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.
