HALOTHANE AND CYCLOPIAZONIC ACID MODULATE CA-ATPASE OLIGOMERIC STATE AND FUNCTION IN SARCOPLASMIC-RETICULUM

被引:41
作者
KARON, BS [1 ]
MAHANEY, JE [1 ]
THOMAS, DD [1 ]
机构
[1] UNIV MINNESOTA, SCH MED, DEPT BIOCHEM, MINNEAPOLIS, MN 55455 USA
关键词
D O I
10.1021/bi00250a048
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have studied the effects of cyclopiazonic acid (CPA) and halothane on the enzymatic activity, oligomeric state, and conformational equilibrium of the Ca-ATPase in skeletal muscle sarcoplasmic reticulum (SR). CPA is a potent inhibitor of Ca-ATPase activity, and this inhibition is competitive with respect to ATP concentration. Time-resolved phosphorescence anisotropy was used to detect the fraction of Ca-ATPase monomers, dimers, and larger aggregates in the absence and presence of CPA. CPA increased the fraction of dimers and larger aggregates of the Ca-ATPase. Addition of halothane to SR, or detergent solubilization of the Ca-ATPase, increased the apparent K-I of CPA inhibition, and increased the fraction of Ca-ATPase present as monomers. CPA stabilized the E2 conformational state of the Ca-ATPase relative to the E1 and E2-P states, as measured by fluorescein 5-isothiocyanate fluorescence and enzyme phosphorylation from inorganic phosphate. E2-P formation in the presence of CPA was partially restored by halothane and solubilization. We conclude that CPA inhibits the Ca-ATPase in part by overstabilizing dimers or small oligomers of the Ca-ATPase, which is correlated with stabilization of the E2 conformation of the enzyme.
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页码:13928 / 13937
页数:10
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