Negash S, Chen L T, Bigelow D J, Squier T C
Department of Biochemistry, University of Kansas, Lawrence 66045-2106, USA.
Biochemistry. 1996 Sep 3;35(35):11247-59. doi: 10.1021/bi960864q.
We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.
我们利用自旋标记电子顺磁共振波谱技术，研究了受磷蛋白（PLB）磷酸化后，心肌肌浆网（SR）Ca-ATP酶激活过程中蛋白质-蛋白质相互作用可能发生的变化。我们使用马来酰亚胺自旋标记（MSL）的放射性衍生物，开发了在天然心肌和骨骼肌肌浆网膜中对Ca-ATP酶进行选择性自旋标记的条件。利用饱和转移电子顺磁共振技术测量了天然SR膜中心肌和骨骼肌Ca-ATP酶同工型的旋转动力学。我们报告称，心肌SR中PLB的磷酸化导致Ca-ATP酶的整体旋转流动性降低了（1.8±0.2）倍。Ca-ATP酶旋转动力学的改变是PLB磷酸化的直接结果，与Ca-ATP酶或任何其他SR蛋白的磷酸化无关，因为在用ATP使心肌Ca-ATP酶磷酸化的条件下，未观察到ST-EPR谱的改变。在不表达PLB的骨骼肌SR中，使用激活心肌SR中Ca-ATP酶的条件也不会导致Ca-ATP酶的旋转动力学或催化特性发生改变。使用传统电子顺磁共振技术对在5位和12位带有氮氧化物的硬脂酸自旋标记（SASL）掺入心肌SR膜中的旋转动力学进行测量表明，PLB磷酸化后心肌SR膜中的脂质酰基链动力学几乎没有差异。这些结果表明，与PLB磷酸化相关的心肌SR膜中Ca-ATP酶旋转动力学的降低与单个Ca-ATP酶多肽链之间相互作用增强有关，这是由于（i）在确定的寡聚状态下心肌Ca-ATP酶多肽链的空间排列改变，或（ii）蛋白质-蛋白质结合增加。我们认为，Ca-ATP酶多肽链与PLB之间相互作用的改变有助于调节与心肌SR膜中Ca-ATP酶钙激活相关的激活屏障。
