De Meis L, Suzano V A, Caldeira T, Mintz E, Guillain F
Departamento Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Eur J Biochem. 1991 Aug 15;200(1):209-13. doi: 10.1111/j.1432-1033.1991.tb21069.x.
The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.
肌浆网Ca2-ATP酶合成ATP的第一步是该酶被无机磷酸（Pi）磷酸化。磷酸化酶的形成需要Pi和Mg2+。在35摄氏度时，囊泡膜两侧存在Ca2+梯度会使ATP酶对Pi的表观亲和力增加10倍以上，而对Mg2+的表观亲和力没有影响。在没有Ca2+梯度的情况下，在所有使用的Mg2+浓度下，磷酸化反应都受到K+和Na+的抑制。然而，在存在1 mM Mg2+和跨膜Ca2+梯度的情况下，反应仍受到Na+的抑制，但K+促进的抑制作用大大降低。当Mg2+浓度提高到2 mM以上时，该酶不再区分K+和Na+，磷酸化反应受到这两种阳离子同等程度的抑制。发现三氟拉嗪、钌红和亚精胺通过不同机制抑制磷酸化反应。在没有Ca2+梯度的情况下，三氟拉嗪与Pi和Mg2+与酶的结合竞争，而发现亚精胺和钌红仅与Mg2+竞争。所提供的数据表明该酶对Mg2+和Pi具有不同的结合位点。
