Nakamura J
Biological Institute, Faculty of Science, Tohoku University, Miyagi, Japan.
J Biol Chem. 1994 Dec 9;269(49):30822-7.
The first part of this study reported the existence of two different conformations of chemically equivalent Ca(2+)-ATPase molecules from the sarcoplasmic reticulum, which were in pH-dependent equilibrium between E1 (high affinity state for calcium) and E2 (low affinity state for calcium) and predominantly in E2 independent of pH, respectively, before calcium binding (Nakamura, J., and Furokohri, T. (1994) J. Biol. Chem, 269, 30818-30821). Here, calcium bindings at the ATPase molecules were further studied by varying [Ca2+] and pH at 0 degree C. The molecule, which pre-existed in equilibrium between E1 and E2, noncooperatively (the Hill value (nH) approximately 1) bound calcium with an apparent calcium affinity (K0.5) of 2-6 microM independent of pH. nH of the pH profile of the pre-existing equilibrium was about 2, suggesting participation of two protons in the equilibrium. On the other hand, the molecule, which predominantly pre-existed in E2, cooperatively (nH approximately 2) bound calcium with K0.5, which was increased from about 7 microM to about 0.2 microM by increasing pH from 6.23 to 7.40. Analysis of pH dependency of the K0.5 showed participation of 1 proton/calcium in competition for the binding sites. Calcium bindings at the two ATPase molecules seem to be differently modulated by the proton(s).
本研究的第一部分报告称，肌浆网中化学性质相同的Ca(2+)-ATP酶分子存在两种不同的构象，在钙结合之前，它们在E1（对钙的高亲和力状态）和E2（对钙的低亲和力状态）之间处于pH依赖性平衡，且分别主要处于与pH无关的E2状态（中村，J.，和古森，T.（1994年）《生物化学杂志》，269，30818 - 30821）。在此，通过在0℃下改变[Ca2+]和pH，进一步研究了ATP酶分子上的钙结合情况。预先存在于E1和E2平衡状态的分子，以非协同方式（希尔系数（nH）约为1）结合钙，其表观钙亲和力（K0.5）为2 - 6 microM，与pH无关。预先存在的平衡的pH曲线的nH约为2，表明有两个质子参与该平衡。另一方面，主要预先存在于E2状态的分子，以协同方式（nH约为2）结合钙，其K0.5随着pH从6.23增加到7.40而从约7 microM增加到约0.2 microM。对K0.5的pH依赖性分析表明，在结合位点竞争中有1个质子/钙参与。两个ATP酶分子上的钙结合似乎受到质子的不同调节。
