The reaction of chemical probes with the erythrocyte membrane

Abstract

Trinitrobenzenesulfonate (TNBS), fluorodinitrobenzene (FDNB) and suberimidate have been reacted with intact human erythrocytes. TNBS does not penetrate the cell membrane significantly at 23 degrees C in bicarbonate-NaCl buffer, pH 8.6, as estimated by the labeling of the N-terminal valine of hemoglobin. Hence, under these conditions it can be used as a vectorial probe. However, at 37 degrees C, especially in phosphate buffer, at pH 8.6, TNBS does penetrate the cell membrane. FDNB and suberimidate both penetrate the erythrocyte membrane. The time course reaction of TNBS with intact erythrocytes over a 24-hr period at 23 degrees C is complex and shows transition zones for both membrane phosphatidylethanolamine (PE) and membrane proteins. No significant cell lysis occurs up to 10 hr. The fraction of total PE or phosphatidylserine (PS) which reacts with TNBS by this time period can be considered to be located on the outer surface of the cell membrane. Under these conditions it can be located on the outer surface of the cell membrane. Under these conditions it can be shown that 10 to 20% of the total PE and no PS is located on the outer surface of the membrane and hence these amino phospholipids are asymmetrically arranged. The pH gradient between the inside and outside of the cell in our system is 0.4 pH units. Nigericin has no effect on the extent of labeling of PE or PS by TNBS. Isotonic sucrose gives a slight enhancement of the labeling of PE by TNBS. Hence, the inability of PE and PS to react with the TNBS is considered not due to the inside of the cell having a lower pH. The extent of reaction of TNBS with PE is not influenced by changing the osmolarity of the medium or by treatment of cells with pronase, trypsin, phospholipase A or phospholipase D. However, bovine serum albumin (BSA) does protect some of the PE molecules from reacting with TNBS. Cels treated with suberimidate were suspended in either isotonic NaCl or in distilled water. In both cases the suberimidate-treated cells became refractory to hypotonic lysis. Pretreatment of cells with TNBS did not prevent them from interacting with suberimidate and becoming refractory to lysis. However, pretreatment of cells with the penetrating probe FDNB abolished the suberimidate effect. Electron-microscopic analysis of the cells showed a continuous membrane in the case of cells suspended in isotonic saline. The cells suspended in water did not lyse but their membranes had many large holes, sufficient to let the hemoglobin leak out. Since the hemoglobin did not leak out we know that the hemoglobin is cross-linked into a large supramolecular aggregate.

Myoblast aminophospholipid asymmetry differs from that of fibroblasts

“…The contrast between the aminophospholipid asymmetry of the fibroblasts and myoblasts is striking and significant. As reported for all the other animal cell plasma membranes examined [8][9][10] the fibroblasts have ~20% of their PS and 35% of their PE externally disposed. The myoblast data is unique in this regard being enriched ~2-fold in externally disposed PS and PE ( fig.2).…”

“…The other major aminophospholipid, PE, forms a hexagonal II-type phase that is reported to be present in fusing cells and is postulated to be an intermediate in that process [7]. The hypothesized role in fusion of these two lipids poses an interesting problem since asymmetry studies O n erythrocytes [8,9] and a cultured mammalian cell line [ 10] indicate a predominantly internal location for these two classes of lipids.…”

Alteration of membrane phospholipid bilayer organization in human erythrocytes during drug-induced endocytosis.

“…Studies with both small molecular probes like trinitrobenzenesulfonate (4,5) and nonpenetrating enzymes (6)(7)(8) provide generally consistent data showing that the phospholipids in the bilayer are also asymmetrically arranged, with sphingomyelin (SM) and phosphatidylcholine (PC) being located primarily in the outer leaflet while the aminophospholipids, phosphatidylethanolamine (PE) and phosphatidylserine (PS), being located at the inner leaflet. The negatively charged PS, in particular, seems to be totally restricted to the inner half of the bilayer.…”

The Distribution of Lipids in the Protoplast Membranes of Bacillus subtilis

“…The reaction of phospholipases A and C, and trinitrobenzenesulphonic acid with the membrane lipids of the chicken erythrocyte is dependent upon the ATP status of the erythrocyte, the degree of hydrolysis, or labelling increasing with ATP depletion [12,31,32], although nigericin has no effect on the degree of labelling of phosphatidylethanolamine and phosphatidylserine by trinitrobenzenesulphonic acid in human erythrocytes [33]. Treatment of B. subtilis protoplasts with nigericin or gramicidin had no effect on the rate or extent of hydrolysis of any of the four phospholipids, by phospholipase C.…”