MITOCHONDRIAL SWELLING AND OXYGEN-CONSUMPTION DURING RESPIRATORY STATE-4 INDUCED BY PHOSPHOLIPASE A(2) ISOFORMS ISOLATED FROM THE SOUTH-AMERICAN RATTLESNAKE (CROTALUS-DURISSUS-TERRIFICUS) VENOM

The non-covalent interaction between two molecular entities namely, ph ospholipase A(2) and crotapotin, results in the main toxin, crotoxin. present in the venom of the South American rattlesnake Crotalus duriss us terrificus. High performance liquid chromatography has enabled us t he isolation of three phospholipase A(2) isoforms (F1, F2 and F3), cha racterized through denaturing and non-denaturing polyacrylamide gel el ectrophoresis and also through the N-terminal amino acid sequence anal ysis. The effect of each purified phospholipase A(2) isoform on isolat ed rat liver mitochondria was determined through mitochondrial swellin g and O-2 consumption during respiratory state 4. F1 showed a dose-dep endent stimulation of O-2 consumption while F2 and F3 caused stimulati on only at low doses and inhibition at high amounts. These effects wer e completely suppressed by the presence of 0.1% bovine serum albumin o r 0.5 mM EGTA in the incubation medium. Taking the mitochondrial swell ing as an activity parameter, all of them presented the same behaviour at different intensities, leading to permeabilization of the mitochon drial membrane. In this case, addition of EGTA prevented it whereas bo vine serum albumin was ineffective; indicating that the lipid microenv ironment was affected. These results suggest that free fatty acids are directly responsible for the observed effects induced by phospholipas e A(2) isoforms on oxygen consumption experiments. The protection conf erred by cyclosporin-A on swelling induced by the isoforms, when prese nt in low concentrations, may suggest that cyclosporin-A binds to a mi tochondrial membrane site protecting the membrane against the phosphol ipase A(2) attack. (C) 1998 Elsevier Science Ltd. All rights reserved.