Protein-protein interaction of detergent solubilized Ca^<2+>-ATPase during ATP hydrolysis analyzed by low-angle laser light scattering photometry coupled with high-performance gel chromatography.

为了阐明肌浆网（SR）钙泵ATP酶在酶反应过程中的分子间相互作用，我们<12>采用小角激光散射光度法结合高效凝胶色谱法分析了非离子去污剂C_ E_8增溶的ATP酶。洗涤剂溶解的ATP酶出现为一个单一的峰值与单体和二聚体之间的中间分子量，这表明两个组件的解离-缔合平衡。在无ATP存在下，在0 ℃，有50 μ g/ml磷脂酰胆碱和0.3mg/ml C_ E_8<12>存在时，钙泵ATP呈现为两种不同的组分，分子量分别为125，000 ± <plus-minus>2，100和211，300 ± <plus-minus>7，300，分别对应于单体和二聚体，表明两种形式之间没有快速的相互转化。加入ATP诱导两种组分融合。融合峰的表观分子量随着蛋白质量的增加从单体向二聚体转移。加入ADP或AMPPCP、不可水解的腺嘌呤核苷酸或存在5 mM CaCl_2（在此条件下ATP酶的转换非常缓慢），均不引起峰的融合。因此，溶解的Ca泵ATP酶在ATP水解的反应周期中经历了单体和二聚体之间的快速相互转化。这些结果表明，ATP酶多肽之间的蛋白质-蛋白质相互作用可能在Ca跨SR膜转运中起重要作用。

To elucidate the molecular interaction between Ca pump ATPases of sarcoplasmic reticulum (SR) during the enzyme reaction, we analyzed the solubilized ATPase with non-ionic detergent C_<12>E_8 using low-angle laser light scattering photometry coupled with high-performance gel chromatography. Detergent solubilized ATPase emerged as a single peak with an intermediate molecular weight between the monomer and dimer, suggesting a dissociation-association equilibrium of two components. In the presence of 50 ug/ml phosphatidylcholine and 0.3 mg/ml C_<12>E_8 at 0^ﾟC without ATP, the Ca pump ATP emerged as the two distinct components with molecular weights of 125,000 <plus-minus> 2,100 and 211,300 <plus-minus> 7,300, corresponding to the monomer and the dimer, indicating that there was no rapid interconversion between the two forms. Addition of ATP induced fusion of the two components. The apparent molecular weight of the fused peak shifted from the monomer to the dimer as the amount of the protein increased. Addition of ADP or AMPPCP, non-hydrolyzable adenine nucleotides, or the presence of 5 mM CaCl_2, the conditions in which the turn-over of the ATPase enzyme was extremely slow, did not induce the fusion of the peaks. Thus, the solubilized Ca pump ATPase underwent a rapid interconversion between the monomer and the dimer during the reaction cycle of ATP hydrolysis. These results indicate that the protein-protein interaction between ATPase polypeptides may play an important role in Ca translocation across SR membrane.