Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid.
Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid.
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γ-羧基谷氨酸的作用。
DOI：
10.1016/s0021-9258(17)33107-1
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发表时间：
1976
影响因子：
4.8
通讯作者：
G. Nelsestuen
中科院分区：
文献类型：
--
作者：
G. Nelsestuen
一级钙依赖的转变可以通过凝血酶原分离的“原”(片段1)区域的固有荧光的减少来监测。片段1的最大荧光变化为-40%，整个凝血酶原的荧光变化仅为-6%左右。这种转变最显著的特征是它的速度和活化能。0度相变的半衰期约为100min，温度依赖关系的活化能为21kcal/mol。正向反应的速率常数在钙中为零级，不受磷脂膜存在的影响。然而，转变的平衡受到磷脂的影响。在30度时，[Ca]eq(一半蛋白质发生转变的钙浓度)为0.4 mm，希尔系数为2.6。在相同条件下，磷脂存在时，[Ca]eq为0.24 mm，Hill系数约为4.5。这种转变是通过结合3或4个钙离子来触发的。用凝胶过滤技术检测片段1在0度时与磷脂微囊的结合率。这一过程的速率常数、活化能和[Ca]eq值与荧光变化的性质相对应。整个凝血酶原与磷脂结合的速率常数、活化能和Hill系数对应于片段1的相同参数，但[Ca]eq值较低。在0度时，凝血酶原转变的[Ca]eq为0.19 mm，磷脂存在时的转变为0.1 mm。这些结果表明，当接触钙离子时，片段1和凝血酶原经历了转变，这必然先于蛋白质-磷脂相互作用。除了确定正确的蛋白质结构外，钙还在凝血酶原-磷脂相互作用中发挥第二作用，这是在蛋白质-磷脂键的实际形成中。蛋白质与磷脂结合的[Ca]eq约为0.06 mM。
A first order calcium-dependent transition can be monitored by a decrease in the intrinsic fluorescence of the isolated "pro" (Fragment1) region of prothrombin. The maximum fluorescence change is -40% for Fragment 1, and only about -6% for whole prothrombin. The most remarkable features of this transition are its rate and activation energy. The half-life for the transition at 0 degrees is about 100 min, and the temperature dependence shows an activation energy of 21 kcal/mol. The rate constant for the forward reaction is zero order in calcium and is not affected by the presence of phospholipid membranes. The equilibrium for the transition, however, is affected by phospholipid. At 30 degrees, [Ca]eq (the calcium concentration where half of the protein has undergone the transition) is 0.4 mM and the Hill coefficient is 2.6. Under the same conditions but in the presence of phospholipid [Ca]eq is 0.24 mM and the Hill coefficient is about 4.5. The transition is triggered by binding 3 or 4 calcium ions. The rate of Fragment 1 binding to phospholipid vesicles was tested using gel filtration techniques at 0 degrees. The rate constants, activation energy, and [Ca]eq values for this process were shown to correspond to the properties of the fluorescence change. The rate constants, activation energy, and Hill coefficients for binding of whole prothrombin to phospholipid correspond to the same parameters for Fragment 1 but the [Ca]eq values are lower. At 0 degrees, the [Ca]eq is 0.19 mM for the prothrombin transition and 0.1 mM for the transition in the presence of phospholipid. These results demonstrate that Fragment 1 and prothrombin undergo a transition when exposed to calcium ions which necessarily precedes protein-phospholipid interactions. In addition to its role in determining the correct protein structure, calcium plays a second role in prothrombin-phos-pholipid interaction which is in the actual formation of the protein-phospholipid bond. The [Ca]eq for binding protein (after its transition) to phospholipid is about 0.06 mM.