State of Spectrin Phosphorylation Does Not Affect Erythrocyte Shape or Spectrin Binding to Erythrocyte Membranes*

After equilibrating cells with [32P]phosphate there were 3.7 f 0.2 mol of [32P]phosphate/mol of Band 2 subunit; this is the total measured as nonradioactive phosphate in fresh cells (Harris, H. W., Wolfe, L. C., and Lux, S . E. (1978) Fed Roc. 37, 1507). This level can be reduced in intact cells by metabolic depletion, or on purified spectrin by tryptic removal of the terminal phosphorylated peptides, or by treatment with bacterial alkaline phosphatase. Dephosphorylation by these methods did not change either the affinity or saturation level for rebinding of spectrin to high affinity sites on spectin-depleted inside-out vesicles (lo” M and 125 pg/mg of membrane protein). The [32P]phosphate level was the same on spectrin extracted from membranes, residual membrane-bound spectrin, and on dimers and tetramers, suggesting that phosphorylation does not regulate the affinity of these binding interactions. After removal of glucose from red cell suspensions, cell ATP drops to one-half its initial level in about 1 h, followed by crenation of half of the cells in 3 t o 4 h; however, no detectable dephosphorylation of spectrin occurs until 4 t o 7 h. In addition, no dephosphorylation was observed during crenation and deformability loss induced by the calcium ionophore, A23187. We find no evidence for a causal relationship between spectrin phosphate levels and either red cell shape or spectrin binding to the membrane. These observations eliminate several simple mechanism by which spectrin phosphorylation-dephosphorylation might be involved in controlling cell membrane properties.