Phosphorylation of the Ca2+ pump intermediate in intact red cells, isolated membranes and inside-out vesicles

Ca2+-entry into intact red cells containing [32P]-ATP increases the phosphorylation of the 150 000 dalton polypeptide of the membrane. This phosphorylation occurs even in Mg2+-depleted red cells. Extracellular lanthanum applied during ATP-depletion further increases the Ca2+-induced phosphorylation. In fragmented membranes or resealed insideout vesicles (IOVs) membrane bound Mg2+ is sufficient to catalyze the phosphorylation of spectrin 2 and Band 3 polypeptides with low concentrations (less than micron of [32P]-ATP. In Ca-EDTA buffers one single polypeptide is phosphorylated which is located in the 150 000 molecular weight region. KmCa for phosphorylation is much lower (0.2 micron) than for active Ca2+ transport (40 micron) in IOVs. Lanthanum induced phosphorylation (up to 250 micron Lafree) is significantly greater than Ca2+-induced phosphorylation. Hg2+ inhibits both Ca2+ and La3+ induced phosphorylation. Ca2+-induced labelling can be rapidly "chased" by unlabelled ATP+Mg2+, but not with EGTA+Mg2+. Dephosphorylation in Ca2+ phosphorylated membranes and IOVs is significantly inhibited by La3+. It can be concluded that the mechanism of La3+ and Hg2+ inhibition of the Ca2+ pump is different in intact cells and isolated membranes or Iovs.