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.

为了阐明肌浆网钙泵ATPase在酶反应过程中的分子相互作用，我们用小角激光散射光度法结合高效凝胶色谱分析了非离子洗涤剂C&lt；12&Gt；E8增溶的肌浆网ATPase。洗涤剂增溶的ATPase在单体和二聚体之间呈现中等分子量的单峰，表明两个组分之间存在解离-缔合平衡。在不含三磷酸腺苷的0ﾟ℃，50 ug/ml磷脂酰胆碱和0.3 mg/mlC_(12&gt；E_8)存在下，钙泵ATP出现两种截然不同的组分，其相对分子质量分别为125,000&2,100和211,300&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。加入三磷酸腺苷可诱导两组分的融合。随着蛋白质含量的增加，融合峰的表观分子量由单体向二聚体转移。加入ADP或AMPPCP，非水解腺嘌呤核苷酸，或5 mM CaCl2存在时，ATPase酶的翻转非常缓慢，不能诱导峰的融合。因此，在ATP水解的反应循环中，溶解的钙泵ATPase在单体和二聚体之间经历了快速的相互转化。这些结果表明，ATPase多肽之间的蛋白质-蛋白质相互作用可能在钙跨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.