J. Biochem, 1984, Vol. 96, No. 5 1343-1350
© 1984 Japanese Biochemical Society
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Characterization of the (Ca2+-Mg2+)ATPase Purified by Calmodulin-Affinity Chromatography from Bovine Aortic Smooth Muscle1
Department of Molecular Physiology, National Cardiovascular Center Research Institute Suita, Osaka 565
A clamodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into micorsomes prepared from bovine aortic smooth muscle. From this micro somal preparation which we expected to contain clamodulin-dependent Ca2+ transport ATPase [EC 3.6.1.3
[EC]
], we perfied (Ca2+-Mg2+) ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin dependent (Ca2+Mg2+) ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl suphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [
-32P]ATP in a Ca2+ dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+ Km was 7µM and the maximum ATPase activity was 1.4µmol/mg/min. These values were chnaged to 0.17 µM and 3.5 µmol/mg/min, respectively by the addition of clamodulin. The activity of the purified (Ca2+-Mg2+) ATPase was inhibited by orthovandae, and the concentration required for half-maximal inhibition was about 1.8µM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the moleculare weight, the pruified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentratin required for half-maximal inhibition was about 1.8µM which is close to that of plasma membrane ATPses. Judging from the effect of orthovanadate and the moleculare weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.
1This investigation was supported in part by grants from the Ministries of Health and Welfare and of Education, Science and Culture of Japan.
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