Ca2+ Uptake in Bovine Adrenocortical Microsomes: Formation of Phosphorylated Intermediate of Ca2+ Dependent ATPase1

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30 % sucrose solution, exhibited ATP dependent Ca2+ uptake. The Caz+ uptake was depen-dent on temperature and stimulated by free Ca24 (the concentration for half maximal activation = 1.0 pM) and Mg2+. The Caz+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accerelated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [/-32P]ATP in the presence of Caz+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 /uM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0°C in the presence of 1 juM ATP and 10 ftM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time=0.1 s). The rate of EP decomposition at 0"C after adding EGTA was about 6.7 times slower (rate constant: ka = 4.3xlO~3s~') than that of SR. It was suggested that adrenocortical microsomes contain