Mitochondrial swelling and oxygen consumption during respiratory state 4 induced by phospholipase A2 isoforms isolated from the South American rattlesnake (Crotalus durissus terrificus) venom.
The non-covalent interaction between two molecular entities namely, phospholipase A2 and crotapotin, results in the main toxin, crotoxin, present in the venom of the South American rattlesnake Crotalus durissus terrificus. High performance liquid chromatography has enabled us the isolation of three phospholipase A2 isoforms (F1, F2 and F3), characterized through denaturing and non-denaturing polyacrylamide gel electrophoresis and also through the N-terminal amino acid sequence analysis. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined through 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 0.5 mM EGTA in the incubation medium. Taking the mitochondrial swelling as an activity parameter, all of them presented the same behaviour at different intensities, leading to permeabilization of the mitochondrial membrane. In this case, addition of EGTA prevented it 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 observed effects induced by phospholipase A2 isoforms on oxygen consumption experiments. The protection conferred by cyclosporin-A on swelling induced by the isoforms, when present in low concentrations, may suggest that cyclosporin-A binds to a mitochondrial membrane site protecting the membrane against the phospholipase A2 attack.
Valente
R H
RH
Departamento de Bioquímica, IB UNICAMP, Campinas, SP, Brasil.
Novello
J C
JC
Marangoni
S
S
Oliveira
B
B
Pereira-da-Silva
L
L
Macedo
D V
DV
D000595:Amino Acid Sequence; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology*; D017839:Crotalus; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D007527:Isoenzymes / Q000302:isolation & purification; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008933:Mitochondrial Swelling; D008969:Molecular Sequence Data; D010101:Oxygen Consumption / Q000187:drug effects; D010741:Phospholipases A / Q000302:isolation & purification / Q000633:toxicity*; D054467:Phospholipases A2; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D003435:Crotalid Venoms; D007527:Isoenzymes; D012710:Serum Albumin, Bovine; D004533:Egtazic Acid; D010741:Phospholipases A; D054467:Phospholipases A2