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

Summary 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 M) of ³²P]-ATP. In Ca-EDTA buffers one single polypeptide is phosphorylated which is located in the 150 000 molecular weight region. K_mCa for phosphorylation is much lower (0.2 m) than for active Ca²⁺ transport (40 M) in IOVs. Lanthanum induced phosphorylation (up to 250 m La_free) 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.Abbreviations EDTA ethylenediamine tetraacetate - EGTA ethyleneglycol-bis(2-aminoethylether)-N-N-tetraacetate - IOV inside-out vesicle - SDS sodium dodecylsulfate - TCA trichloroacetic acid