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Phosphatidylserine (PS) containing a 7-nitrobenz-2-oxa-1, 3-diazol-4-yl- (NBD-) hexanoyl residue, like native PS, preferentially distributes into the inner membrane leaflet of human erythrocytes. In the case of NBD-PS, this preference results from two opposite active processes, an inward translocation mediated by the aminophospholipid flippase and an outward translocation mediated by an ill-defined floppase. Selective inhibition of this floppase by alkylating reagents or cationic and anionic drugs increases the extent of accumulation of NBD-PS in the inner membrane leaflet from about 70% in control cells to about 90%. Different inhibitor sensitivities of the flippase and the floppase strongly suggest that both represent different entities. The floppase was characterized in further detail by comparing inhibitory effects of various compounds on this translocase with their effects on known primary active transport systems for amphiphilic compounds. The inhibitory effects of various drugs, glutathione conjugates and GSSG on the floppase activity closely correlate with those reported for the active transport by the multidrug resistance protein (MRP) while only poorly going parallel with those for the active transport by the low affinity pump for glutathione conjugates and the multidrug resistance MDR1 P-glycoprotein. The NBD-phospholipid floppase activity of the erythrocyte is thus probably a function of MRP.

SUMMARYThe physical and barrier properties of the phospholipids phosphatidylglycerol and lysylphosphatidylglycerol were studied in membrane model systems. Packing in monolayers at the air-water interface showed a larger area per molecule for lysylphosphatidylglycerol than for phosphatidylglycerol. The non-electrolyte permeability of liposomes prepared with lysylphosphatidylglycerol was higher than of those prepared with phosphatidylglycerol. On the other hand, the permeability of SeRb+ was higher for liposomes of phosphatidylglycerol than for those of lysylphosphatidylglycerol. Valinomycin was able to increase the permeability of this cation in the phosphatidylglycerol liposome only.Studies on the effect of the environmental pH on the lysylphosphatidylglycerol to phosphatidylglycerol ratio in intact cells of staphylococcus aureus showed that the total amount of lysylphosphatidylglycerol, cardiolipin and neutral lipids in the membrane did not change when the pH of the medium was varied between pH 6.5 and 5.0; but the total amount of phosphatidylglycerol decreased when the pH of the medium was lowered. The permeability of the intact cells for erythritol appeared to increase with increasing lysylphosphatidylglycerol to phosphatidylglycerol ratio; whereas the valinomycin mediated exchange of 86Rb+ over the cell membrane appeared to decrease when this ratio was increased.From the correlation between the permeability properties of cells and liposomes the conclusion is drawn that in S. aureus the chemical nature of the phospholipids determines to a great extent the properties of the permeability barrier.

An increase of the intracellular Ca(2+) concentration in erythrocytes is known to activate rapid nonspecific bidirectional translocation of membrane-inserted phospholipid probes and to decrease the asymmetric distribution of endogenous membrane phospholipids. These scrambling effects are now shown to be suppressed by pretreatment of cells with the essentially impermeable reagents 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 2,4,6-trinitrobenzenesulfonate. The inhibitory effects are no longer observed during renewed activation of scrambling following a first transient activation by Ca(2+). Assuming the involvement of the human scramblase, this suggests a conformational alteration of this protein during activation by Ca(2+). Marked suppression of scrambling activity is also observed in cells pretreated with the disulfide reducing agent dithioerythritol which can be reverted by the SH oxidizing agent diamide. This indicates the importance of intramolecular and/or intersubunit disulfide bonds for the function of the scramblase. On the other hand, treatment of cells with the SH reagents N-ethylmaleimide and phenylarsine oxide enhances Ca(2+)-activated scrambling and diminution of asymmetry of membrane phospholipids. This suggests an allosteric connection of several protein SH groups to the translocation mechanism. The inhibitors retain their strong suppressive effects. Besides covalent modification, addition of oligomycin highly stimulates and addition of clotrimazole suppresses the Ca(2+)-activated translocation. No evidence for a role of the ATP-binding cassette transporter ABCA1 in the Ca(2+)-activated outward translocation is obtained. Suppression of phospholipid scrambling by dithioerythritol inhibits Ca(2+)-induced spheroechinocytosis and reduces the extent of subsequent microvesiculation. Scrambling of endogenous phospholipids is proposed to induce echinocytosis and to have only a stimulatory effect on microvesiculation.