RESUMO
The phosphate carrier of pig heart mitochondria was solubilized with Triton X-100 and purified by chromatography on hydroxylapatite. Incubation of the phosphate carrier fraction with cardiolipin stimulated the reconstituted [32P]phosphate exchange activity in liposomes, whereas increased Triton X-100 concentrations inhibited it. The effects of cardiolipin and Triton X-100 are reversible. The activation by cardiolipin is highly specific and could not be obtained with any other applied phospholipid.
Assuntos
Cardiolipinas/farmacologia , Proteínas de Transporte/metabolismo , Detergentes/farmacologia , Mitocôndrias Cardíacas/metabolismo , Tensoativos/farmacologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Bovinos , Octoxinol , Proteínas de Ligação a Fosfato , Polietilenoglicóis/farmacologia , SuínosRESUMO
The phosphate carrier of pig heart mitochondria has been isolated and reconstituted in liposomes. The highest specific activity for [32P]phosphate exchange was obtained with hydroxyapatite eluate from mitochondria, extracted with Triton X-114 in the presence of cardiolipin. This fraction, which is free from the ADP ATP-carrier, had a specific activity of 30 mumol 32Pi x min-1 x mg protein -1. The following conditions were found to inactivate the phosphate carrier irreversibly in the solubilized state: high ionic strength, high detergent concentrations and a high pH. The decrease of the activity by high detergent concentrations can be largely prevented by cardiolipin, present in the extraction buffer, suggesting a specific removal of this lipid by the detergent. After reconstitution in liposomes, the phosphate carrier is rather stable. The Arrhenius plot of the temperature-dependence of the reconstituted phosphate exchange showed different slopes above and below 27 degrees C. Between 0 degrees C and 27 degrees C the EA was 64 kJ . mol-1, between 27 degrees C and 42 degrees C 44 kJ . mol-1. The exchange of Pi followed a first-order kinetic.