Coelho-Sampaio T, Ferreira S T, Benaim G, Vieyra A
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil.
J Biol Chem. 1991 Nov 25;266(33):22266-72.
Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin in the presence of Ca2+ had no additional effect when compared to that observed in the presence of Ca2+ alone. This finding is interpreted in terms of the mechanism of calmodulin activation of ATPase catalysis.
通过荧光光谱法和高压技术相结合的方法，研究了红细胞质膜中Ca(2+)-ATP酶的亚基相互作用。在1巴至2.4千巴范围内施加静水压力促使ATP酶完全解离，这可通过内在荧光发射的光谱位移以及丹磺酰共轭ATP酶的荧光偏振变化来揭示。ATP酶的压力解离表现出对蛋白质浓度的依赖性，这与二聚体的解离情况相符。根据压力解离曲线计算，亚基缔合的标准体积变化dV0为43 - 50毫升/摩尔，大气压下的解离常数K0为6 - 9×10(-8) M。添加Ca2+可稳定二聚体ATP酶结构使其抵抗压力解离，而添加钒酸盐则促进压力解离。这些结果表明亚基间相互作用取决于ATP酶两种主要构象状态E1和E2之间的平衡。与仅存在Ca2+时相比，在Ca2+存在下添加钙调蛋白没有额外影响。这一发现根据钙调蛋白激活ATP酶催化的机制进行了解释。
