The proposed investigation is to further elucidate the molecular mechanisms and the related functions of Ca+Mg-ATPase activities in human erythrocytes. Recent investigations by the principal investigator have led to the hypothesis that Ca+Mg-ATPase activity not associated with the Ca pump is an expression of coupled Mg2+ kinase and Ca2+ phosphatase activities. Preliminary rheological and phosphorylation studies indicate that these coupled Mg2+ kinase; Ca2+ phosphatase activities are involved in the regulation of membrane shape of human erythrocytes. We intend to explore this hypothesis further by identifying the unknown membrane component which is phosphorylated in the presence of Mg2+ and dephosphorylated in the presence of low concentrations of Ca2+ (1-10 mu M). Phosphorylation of proteins and phospholipids will be investigated. We wish to investigate the role of activator protein(s) of Ca+Mg-ATPase activity on the Mg2+, ATP and Ca2+ regulation of rheological properties of erythrocyte membrane suspensions. Lastly, we will attempt to separate and purify proteins associated with the Ca pump and those involved in the regulation of membrane shape and to reconstitute these proteins in an active form into liposomes prepared from phospholipids. It is expected that the results of this investigation will throw light on the mechanisms by which erythrocytes maintain low intracellular concentrations of Ca2+ and regulate their shape which is essential to the survival of erythrocytes in the circulation.