Intermolecular interactions in the mechanism of skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1): evidence for a triprotomer.
Intermolecular interactions in the mechanism of skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1): evidence for a triprotomer.
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骨骼肌肌浆网 Ca(2)-ATP 酶 (SERCA1) 机制中的分子间相互作用：三旋体的证据。
DOI：
10.1021/bi801024a
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Froehlich,JeffreyP
中科院分区：
文献类型：
--
作者：
Mahaney,JamesE;Thomas,DavidD;Farrance,IainK;Froehlich,JeffreyP
从骨骼肌分离的天然膜肌浆网（SR）Ca 2 +-ATP酶（SERCA 1）表现出寡聚动力学行为[Mahaney，J.E.，托马斯，D. D、和Froehlich，J.P.（2004）Biochemistry 43，4400 - 4416]。在本研究中，我们使用淬灭流混合，电子顺磁共振（EPR），和化学交联探针在生理（0.1 M）和高（0.4 M）氯化钾的分子间相互作用。SR膜暴露于水溶性和脂溶性交联剂揭示了主要由二聚体和三聚体组成的SERCA 1低聚物种类的混合物。在10 μM Ca 2+和0.1 M KCl存在下，用AMPPCP滴定碘乙酰胺自旋标记的SERCA 1，分别显示高亲和力（KD= 45 μM）和低亲和力（KD= 315 μM）核苷酸结合位点，比例为2：1。将[KCl]提高到0.4M，增加了弱结合位点的分数，降低了高亲和力组分（20 μM）的KD。在21 °C和0.1 M KCl条件下，10 μM ATP的磷酸化作用导致Pi的早期爆发，而稳态磷酸酶（EP）水平没有相应的下降。稳态EP水平是Piburst的两倍，E1 P和E2 P的贡献相等。在0.4 M KCl和2 °C下用ADP追踪磷酸酶，发现E1 P形成的双相时间过程具有慢相，与自旋标记的Ca 2 +-ATP酶的瞬时EPR信号动力学相匹配。EPR信号中快速成分的缺乏排除了E1 P作为其来源。相反，它产生于一个缓慢的，KCl依赖的转换在开始的周期，控制下游中间体的形成与旋转限制探针的摩尔分数增加。我们用SERCA 1三聚体模拟了这种行为，其中E1 ATP/E2 P/E1 P形成E1 P/E2/E2 P是由于亚基偶联磷酸化（E1 ATP → E1 P + ADP）到去磷酸化（E2 P → E2 + Pi）以及E1 P转化为E2 P的协同转化。
Native membrane sarcoplasmic reticulum (SR) Ca2+-ATPase isolated from skeletal muscle (SERCA1) exhibits oligomeric kinetic behavior [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004)Biochemistry 43, 4400−4416]. In the present study we used quenched-flow mixing, electron paramagnetic resonance (EPR), and chemical cross-linking to probe for intermolecular interactions at physiological (0.1 M) and high (0.4 M) KCl. Exposure of SR membranes to water- and lipid-soluble cross-linking reagents revealed a mixture of SERCA1 oligomeric species consisting mainly of dimers and trimers. Titration of iodoacetamide spin-labeled SERCA1 with AMPPCP in the presence of 10 μM Ca2+and 0.1 M KCl revealed high- (KD= 45 μM) and low-affinity (KD= 315 μM) nucleotide binding sites in a 2:1 ratio, respectively. Raising the [KCl] to 0.4 M increased the fraction of weak binding sites and lowered theKDof the high-affinity component (20 μM). Phosphorylation by 10 μM ATP at 21 °C and 0.1 M KCl produced an early burst of Piproduction without a corresponding decline in the steady-state phosphoenzyme (EP) level. The steady-state EP level was twice as large as the Piburst and received equal contributions from E1P and E2P. Chasing the phosphoenzyme at 0.4 M KCl and 2 °C with ADP revealed a biphasic time course of E1P formation with a slow phase that matched the kinetics of the transient EPR signal from the spin-labeled Ca2+-ATPase. The absence of a fast component in the EPR signal excludes E1P as its source. Instead, it arises from a slow, KCl-dependent transformation at the start of the cycle which controls the formation of downstream intermediates with an increased mole fraction of rotationally restricted probes. We modeled this behavior with a SERCA1 trimer in which the formation of E1P/E2/E2P from E1ATP/E2P/E1P results from concerted transformations in the subunits coupling phosphorylation (E1ATP → E1P + ADP) to dephosphorylation (E2P → E2 + Pi) and the conversion of E1P to E2P.