Phospholipid asymmetry in plasma membrane vesicles derived from BHK cells

The transbilayer distribution of phospholipids in plasma membrane vesicles derived from BHK cells by treatment with iodoacetamide or fluoride and merocyanine 540 has been examined by exposing the vesicles to bee venom phospholipase A2 (PLA2) or to Bacillus cereus sphingomyelinase. The results show that almost all of the phosphatidylserine (PS) is on the inner lipid leaflet and most of the sphingomyelin is on the outer lipid leaflet. In contrast, about 50% of the phosphatidylcholine (PC) and 30-40% of the phosphatidylethanolamine (PE) is rapidly degraded by PLA2 and thus appears to be present on the surface of the vesicles. The pools of PC and PE which are accessible only slowly to PLA2 are degraded with halftimes of about 5 h and 2 h, respectively, and it is suggested that this rate reflects the rate of transbilayer migration of these lipids. We conclude that the profound energy depletion caused by treatment with iodoacetamide or fluoride does not alter the asymmetric distribution of PS across the plasma membrane but does have a marked effect on the transbilayer distribution of PE. Residual cells after treatment with fluoride and MC540 were also exposed to PLA2. The results were broadly in agreement with those obtained with vesicles, suggesting that the vesicles were representative of the BHK cell plasma membrane in terms of phospholipid asymmetry. Fluoride or MC540 added separately caused little vesicle release but did lead to significant loss of phospholipid asymmetry. When centrifuged on a sucrose density gradient, vesicles were separated into two major fractions accounting for about two thirds and about 20%, respectively, of total phospholipid but no significant differences were seen in the transbilayer phospholipid asymmetry of the two fractions.