Characterization of ATP-dependent Ca2+ uptake by canine brain microsomes with saponin

Abstract

ATP-dependent Ca2+ uptake by brain microsomes was classified into 2 fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50% inhibition (IC50) of major saponin-sensitive Ca2+ uptake was 11 .mu.g/ml, and this uptake was enhanced by calmodulin. The minor saponin-insensitive Ca2+ uptake fraction (IC50; 90 .mu.g/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KCl. The IC50 of saponin for inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 .mu.g/ml, respectively. A characteristic ring-like saponin-cholesterol micellar structure was observed by EM in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. Saponin-sensitive and insensitive Ca2+ uptake was probably derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations.