On the interrelation between calmodulin and EGTA in the regulation of the affinity to Ca2+ and the maximal activity of the erythrocyte-membrane calcium pump.

1. Calmodulin distribution in rat erythrocytes and the interrelation between calmodulin and EGTA in regulation of the rate of 45Ca accumulation by erythrocyte-membrane inside-out vesicles were studied. 2. The total content of calmodulin in rat erythrocytes is about 24 mumol/l of cells. About 60% of calmodulin is localized in cytoplasm and 40% (10 mumol/l of cells) of calmodulin is loosely bound to membranes; after subsequent washing by hypotonic solution (membranes A) the content of membrane-bound calmodulin decreases up to 0.1 mumol/l of cells. 3. The addition of exogenous calmodulin to membranes A results in increase of the maximal activity of the Ca-pump and does not influence its affinity to Ca2+. Troponin I (30 microM) completely abolishes the calmodulin effect on the Ca-pump activity without significant alterations in its basal activity. 4. The addition of EGTA in the membrane-washing solution results in decrease of the membrane-found pool calmodulin up to 0.01 mumol/l of cells (membranes B). This procedure is accompanied by decrease of the affinity of Ca-pump to Ca2+ and does not influence its maximal rate. 5. The effect of EGTA treatment (membranes B) on the affinity of Ca-pump to Ca is abolished after addition of micromolar concentrations of calmodulin or millimolar concentration of EGTA in the incubation medium. The increase of EGTA concentration in the incubation medium results in decrease of the affinity of Ca-pump to calmodulin. 6. It is assumed that two essentially different pools of calmodulin participate in the regulation of the activity of Ca-pump. (a) The pool of calmodulin which is loosely bound to membrane (this size is dependent on calmodulin concentration in cytoplasm) determines the maximal activity of Ca-pump. The effect of this calmodulin pool is blocked by troponin I. (b) The tightly bound pool of calmodulin which is removed by EGTA treatment determines the affinity of Ca-pump to Ca. 7. In this connection the reasons for contradictory data on estimation of calmodulin effect on the kinetic parameters of plasma membrane Ca-pump are discussed.