Open AccessReconstitution of the Isolated Phosphate‐Transport System of Pig‐Heart Mitochondria
Author(s) -
MENDE Peter,
KOLBE Hanno V. J.,
KADENBACH Bernhard,
STIPANI Italo,
PALMIERI Ferdinando
Publication year - 1982
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1982.tb06937.x
Subject(s) - phosphate , cardiolipin , chemistry , liposome , chromatography , arrhenius plot , ionic strength , mitochondrion , extraction (chemistry) , pi , kinetics , nuclear chemistry , biochemistry , membrane , phospholipid , activation energy , organic chemistry , aqueous solution , physics , quantum mechanics
The phosphate carrier of pig heart mitochondria has been isolated and reconstituted in liposomes. The highest specific activity for [ 32 P]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 32 P i × 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 E A was 64 kJ · mol −1 , between 27°C and 42°C 44 kJ · mol −1 . The exchange of P i followed a first‐order kinetic.