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J. Biochem, 1984, Vol. 96, No. 5 1365-1374
© 1984 Japanese Biochemical Society

research-article

Ca2+,Mg2+-ATPase of Microsomal Membranes from Bovine Aortic Smooth Muscle. Identification and Characterization of an Acid- Stable Phosphorylated Intermediate of the Ca2+,Mg2+-ATPase1

Michihiro SUMIDA*, Hiromichi OKUDA* and Minoru HAMADA**,2

*Second Department of Medical Biochemistry Ehime University School of Medicine, Shigenobu, Onsen-gun, Ehime 791-02
**Department of Hygiene Ehime University School of Medicine, Shigenobu, Onsen-gun, Ehime 791-02

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1–10µM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 33P in the presence of 10 µM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 µM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 µM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15°C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/ mol between 0 and 16°C and 14.6kcal/mol between 16 and 30°C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.

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

2 Present address: Department of Hygiene, Miyazaki Medical College, Miyazaki 889-16.


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