Davis J Q, Bennett V
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1990 Oct 5;265(28):17252-6.
This report demonstrates that the high affinity binding of ankyrin to two well characterized ankyrin-binding proteins, the erythrocyte anion exchanger and kidney Na+K(+)-ATPase, requires interaction of these proteins with unique sites on the ankyrin molecule. Binding of 125I-labeled erythrocyte ankyrin and ankyrin proteolytic domains was measured to the anion exchanger and Na+K(+)-ATPase incorporated into phosphatidylcholine liposomes. 125I-Labeled ankyrin associated with both anion exchanger and Na+K(+)-ATPase liposomes with a high affinity (KD ranging from 10 to 25 nM), and a capacity approaching 1 mol of ankyrin/2 mol of ATPase and 1 mol of ankyrin/8 mol of anion exchanger. The 43 kDa cytoplasmic domain of the erythrocyte anion exchanger inhibited binding of ankyrin to both the anion exchanger and Na+K(+)-ATPase liposomes with a 50% reduction at approximately 90 nM for both proteins. Further binding experiments using proteolytic domains derived from ankyrin demonstrated the following differences between the anion exchanger and Na+K(+)-ATPase in interactions with ankyrin: 1) 125I-Labeled Na+K(+)-ATPase associated with both the 89-kDa domain as well as the spectrin binding domain of ankyrin, while the anion exchanger only associated with the 89-kDa domain. 2) The 125I-labeled 89-kDa domain of ankyrin associated with Na+K(+)-ATPase liposomes with at least a 20-fold lower affinity compared with intact ankyrin while this domain associated with the anion exchanger with a 2-3-fold increase in affinity compared with intact ankyrin. 3) The 125I-labeled spectrin-binding domain of ankyrin associated with the Na+K(+)-ATPase liposomes to at least an 8-fold greater extent than to anion exchanger liposomes. The data are consistent with an independent acquisition of high affinity ankyrin binding activity for the anion exchanger and Na+K(+)-ATPase proteins through a convergent evolutionary process.
本报告表明，锚蛋白与两种特性明确的锚蛋白结合蛋白（红细胞阴离子交换蛋白和肾钠钾ATP酶）的高亲和力结合，需要这些蛋白与锚蛋白分子上的独特位点相互作用。测定了125I标记的红细胞锚蛋白及其蛋白水解结构域与掺入磷脂酰胆碱脂质体中的阴离子交换蛋白和钠钾ATP酶的结合情况。125I标记的锚蛋白与阴离子交换蛋白脂质体和钠钾ATP酶脂质体均具有高亲和力结合（解离常数KD范围为10至25 nM），结合容量接近1摩尔锚蛋白/2摩尔ATP酶和1摩尔锚蛋白/8摩尔阴离子交换蛋白。红细胞阴离子交换蛋白43 kDa的胞质结构域抑制锚蛋白与阴离子交换蛋白脂质体和钠钾ATP酶脂质体的结合，对这两种蛋白而言，在约90 nM时结合减少50%。使用源自锚蛋白的蛋白水解结构域进行的进一步结合实验表明，阴离子交换蛋白和钠钾ATP酶在与锚蛋白相互作用方面存在以下差异：1）125I标记的钠钾ATP酶与锚蛋白的89 kDa结构域以及血影蛋白结合结构域均有结合，而阴离子交换蛋白仅与89 kDa结构域结合。2）125I标记的锚蛋白89 kDa结构域与钠钾ATP酶脂质体结合的亲和力比完整锚蛋白至少低20倍，而该结构域与阴离子交换蛋白脂质体结合的亲和力比完整锚蛋白高2至3倍。3）125I标记的锚蛋白血影蛋白结合结构域与钠钾ATP酶脂质体的结合程度至少比与阴离子交换蛋白脂质体的结合程度高8倍。这些数据与阴离子交换蛋白和钠钾ATP酶蛋白通过趋同进化过程独立获得高亲和力锚蛋白结合活性一致。
