The major aims of this study were to determine tile degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DI, DNB) were used.
The rea'ction of hemoglobin, menrbrane protein and membrane PE and PS of erythrocytes with FDNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. "I'NBS reacts to an extremely small extent with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more lhan TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS wherea~, tire reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the crythrocyte membrane as compared to the outer surface.
TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5 2.0 haM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. ltence above 2 mM TNBS or FDNB a perturbation occurs in the menrbrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5c,;f of tire total menrbrahe PE is localized on tim outer surface of the erythrocyte membrane.
TNBS and FDNB were reacted with yeast, E. colt, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. colt, but not witlr erytlrrocytes or yeast cells, phospholipase A activity was very pronounced at pit 8.5 giving rise to a large anrount of '~