[32] Purification of phospholipid exchange proteins from beef heart

Publisher Summary This chapter describes the purification of phospholipid exchange proteins from beef heart. In the assays described, phosphatidylcholine is used to measure exchange activity. Transfer of 32 P-labeled phosphatidyleholine from artificial vesicles to mitochondria is measured with and without added exchange protein. After a 40-minute period for the exchange of phospholipids, the particles are separated by sedimentation of the mitochondria; exchange is measured as the appearance of radioactivity in the mitochondria or as its loss from the vesicles. In practice, the loss of 32 P from the vesicles is the more convenient measure of exchange activity. In the course of purification procedure, the postmitochondrial supernatant, containing heart muscle cytosol and microsomes (recovered during the preparation of mitochondria), is used as a source of exchange protein. About 900 ml of the ice-cold solution is adjusted to pH 5.1 with 3 N HCl. After 1 hour of flocculation the precipitate is removed by centrifugation for 15 minutes at 10,000 g. The pH is returned to 7.4 by titration with 18 N NaOH.