Phosphorylation and dephosphorylation of spectrin from human erythrocyte ghosts under physiological conditions: autocatalysis rather than reaction with separate kinase and phosphatase.

The mechanism of phosphosylation and dephosphorylation of spectrin from human erythrocyte membranes has been examined under closely physiological conditions. The results support the hypothesis that spectrin is an autophosphorylating and dephosphorylating system. (i) Extraction from ghosts of up to 85% of the kinase (casein kinase) suggested to catalyze the reaction [see Fairbanks, G., Avruch, J., Dino, E. J. & Patel, V. P. (1978) J. Supramol. Struct. 9, 97--112] only slightly reduced spectrin component 2 phosphorylation and did not affect ATP-induced changes in the ghosts' shapes. (ii) A spectrin--actin complex isolated from endocytotic inside-out vesicles under hyperteonic conditions contained virtually no casein kinase activity and still exhibited a largely intact phosphorylation machinery. (iii) Photoaffinity labeling experiments indicated that spectrin component 2 fulfills the necessary prerequisite of the hypothesis--i.e., it contains its own ATP-binding site. (iv) Under various conditions, spectrin phosphorylation and dephospohrylation seem to be tightly coupled. The implications of these findings for the understanding of spectrin function and the maintenance of erythrocyte shape are discussed.

ATP-induced endocytosis in human erythrocyte ghosts. Characterization of the process and isolation of the endocytosed vesicles.

ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.