Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme's activity.

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.

Large-scale isolation of human erythrocyte Ca2+-transport ATPase.

A rapid procedure for preparing large quantities of purified erythrocyte Ca2+-transport ATPase is presented. The method involves: (1) fast preparation of calmodulin-deficient, essentially haemoglobin-free, erythrocyte membranes by molecular filtration using Pellicon filters; (2) solubilization of membrane proteins by deoxycholate; and (3) a batch procedure using calmodulin-Sepharose 4B gel for purification of Ca2+-transport ATPase.