Structure and Function of Ion Transport Systems

1.本文对肌浆网Ca^<2+>转运ATP酶的结构和作用机制进行了研究。(1)随后通过HPLC的肌浆网膜的热处理消化被证明是有效的，在确定荧光和亲和标记试剂的靶位点。(2)IAEDANS和NEM（ANM）分别与Cys 674和Cys 344/364特异性结合。(3)时间分辨荧光各向异性测量显示独立的灵活运动的亚分子结构域的ATP酶标记IAEDANS和ANM。(4)对有限胰蛋白酶消化的产物进行纯化和分析。由于Lys 218、Lys 234和Arg 236残基周围的构象变化，Ca^2+和AMP-P（NH）P与ATP酶分子的结合对这些残基的敏感性产生了深刻的影响。这个特殊的区域似乎与Ca^<2+>-运输的能量耦合密切相关，也被认为是Ca^<2 +>-运输之间的接触部位。 ...更多信息 <2+>-结合和ATP-结合结构域。因此，目前的结果表明，亚分子结构域之间的相互作用可能是重要的ATP分裂和Ca^2+-运输之间的正确耦合。(5)亲和标记试剂，ATP-吡哆醛被用来定位ATP结合位点。Lys 684和Lys 492在无Ca^2+存在时被标记，但只有前者被标记，这表明ATP结合位点的构象变化依赖于Ca^2+。Asp 351，Lys 492和Lys 684也被建议为紧密相邻。(6)分析了共价连接的自旋标记物和Gd^3+（一种顺磁性Ca^2+类似物）之间的顺磁性相互作用，发现Ca^2+结合位点位于ATP酶分子的膜内区域，离膜表面不远.通过胆盐透析法将Na^+/H^+逆向转运蛋白重组为蛋白脂质体。用凝胶过滤和DEAE-Sephacel柱层析法从牛肾红缘膜中获得部分纯化的反向转运蛋白。少

1. The following results were obtained concerning the structure and the reaction mechanism of Ca^<2+>-transporting ATPase of sarcoplasmic reticulum. (1) Thermolytic digestion of sarcoplasmic reticulum membranes followed by HPLC was shown to be effective in identifying target sites of fluorescence- and affinity-labeling reagents. (2) IAEDANS and NEM(ANM) were shown to be specifically attached to Cys674 and Cys344/364, respectively. (3) Timeresolved fluorescence anisotropy measurements revealed independent flexible motion of submolecular domains of ATPase labeled with IAEDANS and ANM. (4) Products of limited tryptic digestion were purified and analyzed. Susceptibility of Lys218, Lys234 and Arg236 was profoundly affected by binding of Ca^<2+> and AMP-P(NH)P to the ATPase molecule due to a conformational change around these residues. This particular region seems to be intimately involved in energy coupling of the Ca^<2+>-transport, and also is supposed to be the site of contact between Ca^ … More <2+>-binding and ATP-binding domains. The present results thus suggest that interactions between submolecular domains may be important for proper coupling between ATP splitting and Ca^<2+>-transport. (5) An affinity labeling reagent, ATP-pyridoxal was utilized to map the ATP binding site. Lys684 and Lys492 were labeled in the absence of Ca^<2+>, but only the former was labeled in its presence, indicating a Ca^<2+>-dependent conformational change in the ATP-binding site. A close vicinity of Asp351, Lys492 and Lys684 was also suggested. (6) Paramagnetic interactions between covalently attached spin labels and Gd^<3+> (a paramagnetic Ca^<2+> analogue) was analyzed and the Ca^<2+>-binding site was located in the intramembranous region of the ATPase molecule, not being very far from the membrane surface.2. Na^+/H^+ antiporter was reconstituted into proteoliposome by cholatedialysis procedure. Partially purified preparation of the antiporter was obtained from the blush border membranes of bovine kidney through gel filtration and DEAE-Sephacel chromatography. Less