Voss J C, Mahaney J E, Jones L R, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biophys J. 1995 May;68(5):1787-95. doi: 10.1016/S0006-3495(95)80355-3.
We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.
我们直接测定了抑制性肽磷酸受磷蛋白（PLB）对心肌肌浆网（SR）钙泵（Ca-ATPase）旋转动力学的影响。这是通过将PLB水平与其他哺乳动物相似的小鼠心室SR，与实际上不含PLB且因此具有高得多（不受调节）的钙泵活性的小鼠心房SR进行比较来实现的。为了获得足够量的心房SR，我们从心房肿瘤细胞中分离出膜。我们使用选择性且牢固地附着在Ca-ATPase上的异硫氰酸藻红蛋白标记物的时间分辨磷光各向异性，来检测两种制剂中Ca-ATPase的微秒级旋转运动。由于存在不同的寡聚体种类，两种制剂在25℃下的时间分辨磷光各向异性衰减都是多指数的。两种制剂中不同寡聚体的旋转相关时间相似，但心房肿瘤SR的总衰减幅度明显更大，这表明作为大聚集体存在的Ca-ATPase分子比例较小。心室SR中PLB的磷酸化将大规模Ca-ATPase聚集体的数量降低到与心房肿瘤SR相似的水平。通过硬脂酸自旋标记物的电子顺磁共振检测到的脂质链流动性（流动性）在两种制剂中非常相似，这表明心房肿瘤SR中较高的蛋白质流动性不是由于较高的脂质流动性。我们得出结论，PLB通过诱导Ca-ATPase侧向聚集来发挥抑制作用，这种聚集可以通过使PLB磷酸化或去除PLB来解除。
