Cooperative calcium binding and ATPase activation in sarcoplasmic reticulum vesicles.
Cooperative calcium binding and ATPase activation in sarcoplasmic reticulum vesicles.
复制标题
肌浆网囊泡中钙结合和 ATP 酶协同激活。
DOI：
--
复制
发表时间：
1980
影响因子：
4.8
通讯作者：
D. Lewis
中科院分区：
文献类型：
--
作者：
G. Inesi;M. Kurzmack;C. Coan;D. Lewis
高亲和力的钙结合，这是参与atp酶激活后测量肌浆网囊泡与Ca2+缓冲液在色谱柱的平衡。结合发生在ir = 2.3 × 1.6 M”，最高水平为每mg蛋白质8 ~ 10 μ l。这个值对应于每个ATP酶磷酸化位点2个钙离子，因为在ATP存在下获得的磷酸化酶中间体的最大水平是每毫克蛋白质4至5 nmol。分析钙离子浓度对钙离子结合的影响，发现了一种合作机制，包括与第一个位点结合，随后发展出更高的亲和力，并与第二个位点结合。因此，平衡结合曲线的最佳拟合需要两个常数，第二个常数比第一个常数增加2个数量级。Scatchard和Hill图也显示出高度合作（n ~ =; 1.82）的结合机制。高亲和力钙结合伴随着蛋白质构象变化，这是由自旋标记制剂的EPR光谱揭示的，并且与平衡结合数据的分析一致。很明显，这种构象变化是协同结合机制和钙结合后酶活化的组成部分。在Ca2+预培养的酶中添加ATP后酶磷酸化的动力学实验，与在Ca2+缺失的酶中添加ATP和Ca ' +相比，揭示了Ca2+诱导的酶活化是一个缓慢的步骤，受底物（如ATP）结合的影响。综上所述，平衡和动力学分析表明，高亲和力钙结合和atp酶活化的机制如下：E + Caa+。+ E -Ca + E -Ca + Ca " -+ E " - ca2。
High affinity calcium binding which is involved in ATPase activation was measured following equilibration of sarcoplasmic reticulum vesicles with Ca2+ buffers in chromatography columns. Binding occurred with ir,,, = 2.3 X lo6 M” and with a maximal level of 8 to 10 m o l per mg of protein. This value corresponds to 2 calcium ions per ATPase phosphorylation site, since the maximal level of phosphorylated enzyme intermediate obtained in the presence of ATP was 4 to 5 nmol per mg of protein. Analysis of calcium binding as a function of Ca” concentration demonstrates a cooperative mechanism including binding to a first site, followed by development of higher affinity and binding to a second site. Accordingly, the best fit of the equilibrium binding curve requires two constants, with a 2 orders of magnitude increase in the second as compared to the first constant. Scatchard and Hill plots also demonstrate a highly cooperative ( n ~ =; 1.82) binding mechanism. High affinity calcium binding is accompanied by a protein conformational change which is revealed by EPR spectroscopy of spin-labeled preparations and is consistent with the analysis developed for the equilibrium binding data. It is apparent that such a conformational change is an integral part of the cooperative binding mechanism and of the enzyme activation following calcium binding, Kinetic experiments on enzyme phosphorylation upon addition of ATP to enzyme preincubated with Ca2+, as compared to addition of ATP and Ca’+ to enzyme deprived of Ca2+, revealed that the Ca2+-induced enzyme activation is a slow step which is influenced by substrate (e.g. ATP) binding. In conclusion, equilib~um and kinetic analysis indicate the following mechanism for high affinity calcium binding and ATPase activation: E + Caa+ .+ E.Ca + E’oCa + Ca” -+ E”-Ca2.