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

Publisher Website
Google Scholar
Abstract
The phosphate carrier of pig heart mitochondria was isolated and reconstituted in liposomes. The highest specific activity for [32P]phosphate exchange was obtained with hydroxylapatite 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 .mu.mol 32Pi .times. min-1 .times. mg protein-1. The following conditions inactivated 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 stable. The Arrhenius plot of the temperature-dependence of the reconstituted phosphate exchange showed different slopes above and below 27.degree. C. Between 0.degree. and 27.degree. C the EA was 64 kJ .cntdot. mol-1; between 27.degree. and 42.degree. C it was 44 kJ .cntdot. mol-1. The exchange of Pi followed a first orker kinetic.