Use of enzymatic and electron microscopy (freeze-etching) methods for studying ATP-dependent masking of erythrocyte membrane phospholipids

Abstract

Membrane phospholipids of ATP-depleted chicken, rat, and toad erythrocytes are more susceptible than fresh cells to hydrolysis by phospholipase C (Bacillus cereus), phospholipase A (bee venom), or the combination of these enzymes and sphingomyelinase. ATP depletion of chicken and rat erythrocytes greatly increased the membrane phospholipid fraction, which can be extracted by dry ether. Trinitrobenzene sulfonic acid attacked about 20% of the phosphatidylethanolamine of fresh chicken erythrocytes and 45% of ATP-depleted chicken erythrocytes. The intramembranous particles on the PF face of fresh chicken, rat, and toad erythrocytes are evenly distributed, while those in the same ATP-depleted erythrocytes are significantly clustered. The average distance between the intramembranous particles in the PF face of fresh chicken erythrocytes is about 13 nm, while in ATP-depleted erythrocytes it is about 30 nm. Studies with chicken erythrocytes revealed that ATP depletion is accompanied by dephosphorylation of certain membrane polypeptides. The correlation of dephosphorylation of membrane polypeptides, exposure of membrane phospholipids, and clustering of intramembranous particles is discussed.