Weaver D C, Pasternack G R, Marchesi V T
J Biol Chem. 1984 May 25;259(10):6170-5.
Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.
人红细胞锚蛋白在0℃下经有限蛋白酶解作用被切割成两个不同的化学结构域。通过血影蛋白亲和层析以及在蔗糖梯度中与血影蛋白共沉降，发现锚蛋白上与血影蛋白结合的位点位于一个55,000道尔顿的结构域内。制备了该结构域的一个32,000道尔顿的片段（胰蛋白酶消化，0℃，24小时），通过凝胶过滤分离，结果表明它能抑制血影蛋白与膜的结合。通过与先前的二维肽图比较，血影蛋白结合位点位于该32,000道尔顿片段内靠近分子末端的位置。通过与带3亲和柱结合，在一个82,000道尔顿的结构域内鉴定出了带3结合位点。在无去污剂条件下的凝胶电泳证实了这些结果，并表明来自带3细胞质部分的一个肽段保留了与82,000道尔顿结构域结合的能力。锚蛋白结构域的结合特性与完整分子亲和常数的测定相关。锚蛋白与带3的细胞质部分具有高亲和力结合（KD = 8×10⁻⁸ M），与血影蛋白四聚体具有高亲和力结合（KD = 1×10⁻⁷ M），但与血影蛋白二聚体的结合亲和力较低（KD = 1×10⁻⁶ M）。这些发现总结在一个关于锚蛋白在将血影蛋白连接到膜上所起作用的初步结构和功能模型中。
