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J. Biochem, 1988, Vol. 104, No. 5 687-692
© 1988 Japanese Biochemical Society

research-article

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

Michihiro Sumida*,2, Minoru Hamada**, Akihiro Shimowake*, Chie Morimoto* and Hiromichi Okuda*

*Second Department of Medical Biochemistry, Ehime University School of Medicine Shigenobu, Ehime 791-02
**Department of Hygiene, Miyazaki Medical College Kiyotake, Miyazaki 889-16

2 To whom correspondence should be addressed

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 [GenBank] , but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100–110 kDa was phosphorylated by [{gamma}-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=0.2 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: kd=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.

1 Supported by the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan.


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