Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature.

Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.