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

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Abstract
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 Ca 2+ -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 (< 1 μM) 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 μm) than for active Ca2+ transport (40 μM) in IOVs. Lanthanum induced phosphorylation (up to 250 μm 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 Ca 2+ phosphorylated membranes and IOVs is significantly inhibited by La 3+. It can be concluded that the mechanism of La and H g2+ inhibition of the Ca 2+ pump is different in intact cells and isolated membranes or IOVs.