Reconstitution of the Isolated Phosphate‐Transport System of Pig‐Heart Mitochondria

Abstract

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 μmol 32Pi× min−1× 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°C. Between 0°C and 27°C the EA was 64 kJ · mol−1, between 27°C and 42°C 44 kJ · mol−1. The exchange of Pi followed a first‐order kinetic. Copyright © 1982, Wiley Blackwell. All rights reserved