POLYAMINE INHIBITION OF TRANSBILAYER MOVEMENT OF PLASMA-MEMBRANE PHOSPHOLIPIDS IN THE ERYTHROCYTE GHOST

The resting plasma membrane of circulating blood cells demonstrates an asymmetric distribution of the phospholipid classes across the bilaye r that is altered during cellular activation. To better understand the mechanisms governing transbilayer distribution of phospholipids, stud ies were conducted using the erythrocyte ghost, in which plasma membra ne leaflet distribution of phospholipids can be readily probed. Prepar ation of ghosts by hypotonic lysis at increasingly high dilution marke dly enhanced (up to 10-fold) calcium-induced (50-500 mu M) transbilaye r movement of phospholipids. The enhanced transbilayer movement was as sessed by translocation of exogenously added sn-2-[6[(7- obenz-2-oxa-1 ,3-diazol-4-yl)amino]caproyl]-labeled phosphatidylcholine and phosphat idylserine from the plasma membrane outer leaflet to the inner leaflet and vice versa, as well as transbilayer movement of endogenous phosph atidylserine to the outer leaflet. It was found that phospholipid move ment was bidirectional and also nonspecific with regard to polar head group. It was further demonstrated that preparation of ghosts at incre asing dilution resulted in depletion of cellular polyamines and that p hysiologic replenishment of spermine, and to a lesser extent spermidin e, resulted in significant inhibition (50 and 25%, respectively) of tr ansbilayer movement of phospholipids. Replenishment of other di- and p olyamines demonstrated that inhibition was not simply dependent on tot al cationic charge but rather on charge density and suggestive of spec ific interaction of the polyamines, particularly spermine, with the pl asma membrane. As most cells demonstrate both a high degree of regulat ion in maintenance of polyamine levels and the means for facile shifts within cellular polyamine pools, it is suggested that loss of membran e asymmetry during cellular activation may be mediated in part through enhanced transbilayer movement of phospholipids due to altered polyam ine-membrane associations.