Differential reaction of cell membrane phospholipids and proteins with chemical probes

Abstract

The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (L DNB), a non-penetrating probe (TNBS) and a cross-linking probe (DU DNB) were used. The reaction of hemoglobin, membrane 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. TNBS reacts to an extremely small extent with hemoglobin over the concentration range 0.4 to 4 mD1 whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at l mM TNBS whereas the 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 erythrocyte membrane as compared to the outer surface. TN13S 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 mbl probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5–2.0 μM does not react with PS, but between 3 to LO mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane 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 5%, of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasnna 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. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPF from DNI'-PF. A phosplrodiestcrase was also present which lrvdrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear. however, that TNBS and I 'DNB react to a different extent Uvith IT in this cell. The Acholeplasma membrane had no detectable PF or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31%, being on the outer surface and 69°1, on the inner surface. 1-he reaction of ghosts and intact erythrocytes with varying concentrations of DFDNB show that optimal cross-linking of PE to PE, PF to PS and PS to PS occurs between 75–150 μM DFDNB with ghosts as compared to 500–800 μM DFDNB in erythrocytes. Appreciable crosslinking of PS to protein also occurs. Suberimidate is more effective than DI' DNB in protecting erythrocytes from osmotic lysis. In bicarbonate buffer erythrocytes are rendered refractory to hetnolysis by suberimidate at a much lower pH as compared to phosphate buffer. DFDNB is more effe,,tive in protecting cells from hemolysis in phosphate buffer than in bicarbonate buffer. A major goal in using chemical probes was to provide insight into the topology of phospholipids in cell membranes. This topology includes the asymmetric arrangement of phospholipids, sshich phospholipids are clustered about proteins and information on phospholipid neighbors. With the erythrocyte and Acholeplasma which contain only one type of membrane, the results are more definitive. However with E. coli and yeast cells which have several types of membranes. studies with whole cells are difficult to interpret and may be complicated by the presence of lipascs. With these latter cells, the different types of membranes can be isolated and studied individually.