versão ISSN 0104-7930
J. Venom. Anim. Toxins v. 5 n. 1 Botucatu 1999
THESIS: R. H. Valente submitted this thesis for the degree of Master of Science in Biochemistry, publicly examined at the Institute of Biology, Campinas State University (UNICAMP), Campinas, São Paulo, Brazil in March 1996.
Advisor: Prof. Dr. Benedito Oliveira Filho
ABSTRACT. The non-covalent interaction between the two molecular entities namely phospholipase A2 and crotapotin results in the main toxin, crotoxin, found in the venom of the South American rattlesnake Crotalus durissus terrificus. High-performance liquid chromatography has enabled us to isolate three phospholipase A2 isoforms (F1, F2, and F3), using denaturing and non-denaturing polyacrylamide gel electrophoresis and N-terminal amino acid sequence analysis. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined by mitochondrial swelling and O2 consumption during respiratory state 4. F1 showed a dose-dependent stimulation of O2 consumption, while F2 and F3 caused stimulation only at low doses and inhibition at high amounts. These effects were completely suppressed by the presence of 0.1% bovine serum albumin or 0.5mM EGTA in the incubation medium. With the mitochondrial swelling as an activity parameter, the three phospholipase isoforms showed the same behavior at different intensities, resulting in the permeabilization of the mitochondrial membrane. The addition of EGTA also prevented the mitochondrial swelling, whereas bovine serum albumin was ineffective, indicating that the lipid microenvironment was affected. These results suggest that free fatty acids are directly responsible for the effects induced by phospholipase A2 isoforms on oxygen consumption experiments. The protection afforded by cyclosporin-A (CSA) against swelling induced by the isoforms, when used in low concentrations, may suggest that CSA binds to a mitochondrial membrane site protecting the membrane against the phospholipase A2 attack. In contrast, the low level of protection given by carboxyatractylate (CAT), when used with both CSA and CAT, suggest that CAT binding to ADP/ATP carrier inhibits the protection previously afforded by CSA.
01 VALENTE RH., NOVELLO JC., MARANGONI S., OLIVEIRA B., PEREIRA DA SILVA L., MACEDO DV. Mitochondrial swelling and oxygen consumption during respiratory state 4 induced by phospholipase A2 isoforms isolated from the South American rattlesnake (Crotalus durissus terrificus) venom. Toxicon, 1998, 36, 901-13.
R. H. VALENTE - R. Francisco Sá, 88 apt. 802, Copacabana, CEP 22080-010, Rio de Janeiro, RJ, Brasil.