Shi Y, Karon B S, Kutchai H, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biochemistry. 1996 Oct 15;35(41):13393-9. doi: 10.1021/bi9614085.
We have studied the effects of the nonionic detergent C12E8 on Ca-ATPase enzymatic activity and oligomeric state (detected by time-resolved phosphorescence anisotropy, TPA) in skeletal and cardiac sarcoplasmic reticulum (SR). In skeletal, SR, C12E8 inhibits the CA-ATPase, both at high (micromolar and above) and low (submicromolar) Ca. In cardiac SR, C12E8 inhibits at high Ca but activates at low Ca. Thus C12E8 activates enzymatic activity only in cardiac SR and only under conditions (submicromolar Ca) where phospholamban (PLB) regulates (inhibits) the enzyme [Lu, Y.-Z., & Kirchberger, M.A. (1994) Biochemistry 33, 5056-5062]. TPA of skeletal SR at low and high Ca demonstrates that C12E8 induces aggregation of ATPase monomers and small oligomers. C12E8 also aggregates the Ca-ATPase in cardiac SR at high Ca. In cardiac SR at low Ca, the Ca-ATPase is already highly aggregated, and C12E8 partially dissociates these aggregates. Thus the TPA results provide a simple physical explanation for the functional effects: C12E8 inhibits the ATPase when it aggregates the enzyme (skeletal SR at high and low Ca; cardiac SR at high Ca), and the detergent activates when it dissociates ATPase oligomers (cardiac SR at low Ca). C12E8 stabilizes the E2P conformation of the Ca-ATPase with respect to the E2 conformation, and this stabilization is PLB-dependent. Both the physical and functional effects of C12E8 on the Ca-ATPase are PLB-dependent, with C12E8 reversing the effects of PLB. The results provide insight into the mechanism by which PLB regulates the Ca-ATPase in cardiac SR.
我们研究了非离子去污剂C12E8对骨骼肌和心肌肌浆网（SR）中Ca-ATP酶活性和寡聚状态（通过时间分辨磷光各向异性检测，TPA）的影响。在骨骼肌肌浆网中，C12E8在高钙（微摩尔及以上）和低钙（亚微摩尔）条件下均抑制Ca-ATP酶。在心肌肌浆网中，C12E8在高钙时抑制，在低钙时激活。因此，C12E8仅在心肌肌浆网中且仅在磷蛋白（PLB）调节（抑制）该酶的条件下（亚微摩尔钙）激活酶活性[Lu，Y.-Z.，& Kirchberger，M.A.（1994）Biochemistry 33，5056 - 5062]。低钙和高钙条件下骨骼肌肌浆网的TPA表明，C12E8诱导ATP酶单体和小寡聚体聚集。C12E8在高钙时也使心肌肌浆网中的Ca-ATP酶聚集。在低钙的心肌肌浆网中，Ca-ATP酶已经高度聚集，C12E8使这些聚集体部分解离。因此，TPA结果为功能效应提供了一个简单的物理解释：当C12E8使酶聚集时（高钙和低钙条件下的骨骼肌肌浆网；高钙条件下的心肌肌浆网），它抑制ATP酶，而当去污剂使ATP酶寡聚体解离时（低钙条件下的心肌肌浆网），它激活ATP酶。相对于E2构象，C12E8稳定了Ca-ATP酶的E2P构象，并且这种稳定作用依赖于PLB。C12E8对Ca-ATP酶的物理和功能效应均依赖于PLB，C12E8可逆转PLB的效应。这些结果为PLB调节心肌肌浆网中Ca-ATP酶的机制提供了深入见解。
