Scofano H, Barrabin H, Inesi G, Cohen J A
Biochim Biophys Acta. 1985 Sep 25;819(1):93-104. doi: 10.1016/0005-2736(85)90199-3.
The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.
在天然肌浆网囊泡中，钙与特定位点（即那些产生酶激活作用的位点）结合的化学计量比为8 - 10 nmol/mg蛋白质，而通过非离子去污剂增溶和阴离子交换色谱法纯化的ATP酶为13.9 - 15.4 nmol/mg。对磷酸化酶的平行测量结果显示，两种制剂中蛋白质的磷酸化酶水平分别为4.0 - 4.9和6.0 - 7.7 nmol/mg，这表明每条115 kDa的ATP酶链包含一个催化位点和两个钙结合位点。当在天然肌浆网囊泡中进行测量时，以及当在中性pH值、存在10 mM MgCl₂和80 mM KCl的条件下，通过用磷脂酰胆碱或磷脂酰丝氨酸进行脂质替代来改变膜表面电荷时，表观缔合常数K = (6 ± 2)×10⁵ M⁻¹以及结合协同性nH = 1.9均未改变。另一方面，在不存在Mg²⁺时，或在较小程度上在不存在单价阳离子时，表观缔合常数会增加。还观察到，在低离子强度介质中，钙结合等温线的协同特性降低。对静电效应的分析表明，钙结合结构域因其位于ATP酶蛋白内部而免受膜磷脂表面电荷的影响。各种电解质的作用归因于钙结合结构域中的单价阳离子结合。在低离子强度下钙结合等温线协同性的明显丧失归因于滴定曲线的逐渐位移，这种位移在低饱和度时最小，而在高饱和度时变得更大。这种行为通过钙结合对由钙结合结构域内或其附近的局部蛋白质电荷密度产生的静电势的逐渐影响来定量描述。
