Reconstitution of the isolated phosphate-transport system of pig-heart mitochondria.
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.
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002308:Cardiolipins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D013552:Swine; D013696:Temperature
D002308:Cardiolipins; D008081:Liposomes; D010710:Phosphates