Karon B S, Mahaney J E, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Biochemistry. 1994 Nov 22;33(46):13928-37. doi: 10.1021/bi00250a048.
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 KI 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.
我们研究了环匹阿尼酸（CPA）和氟烷对骨骼肌肌浆网（SR）中钙ATP酶的酶活性、寡聚状态和构象平衡的影响。CPA是钙ATP酶活性的强效抑制剂，这种抑制作用在ATP浓度方面具有竞争性。利用时间分辨磷光各向异性来检测在不存在和存在CPA的情况下钙ATP酶单体、二聚体和更大聚集体的比例。CPA增加了钙ATP酶二聚体和更大聚集体的比例。向SR中添加氟烷，或对钙ATP酶进行去污剂增溶，会增加CPA抑制作用的表观抑制常数（KI），并增加以单体形式存在的钙ATP酶的比例。通过异硫氰酸荧光素荧光和无机磷酸对酶的磷酸化作用测定，相对于E1和E2-P状态，CPA稳定了钙ATP酶的E2构象状态。在存在CPA的情况下，氟烷和增溶作用可部分恢复E2-P的形成。我们得出结论，CPA部分通过过度稳定钙ATP酶的二聚体或小寡聚体来抑制钙ATP酶，这与该酶E2构象的稳定相关。
