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

Abstract

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 Ca²⁺ uptake was dependent on temperature and stimulated by free Ca²⁺ (the concentration for half maximal activation=1.0 μM) and Mg²⁺. The Ca²⁺ 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 [γ-³²P] ATP in the presence of Ca²⁺, and the Ca²⁺ dependency was over the same range as the Ca²⁺ uptake (the concentration for half maximal activation=3.0 μM). 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 μM ATP and 10 μM Ca²⁺ (half time=0.2s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time=0.1s). The rate of EP decomposition at 0°C after adding EGTA was about 6.7 times slower (rate constant: k_d=4.3×10^-3s^-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca²⁺ dependent ATPase which function as a Ca²⁺ pump with similar properties to that of SR.