Cohen C M, Langley R C
Biochemistry. 1984 Sep 11;23(19):4488-95. doi: 10.1021/bi00314a039.
Human erythrocyte spectrin alpha and beta chains were purified by preparative sodium dodecyl sulfate gel electrophoresis and also by DEAE-cellulose chromatography in the presence of urea. The purified chains behaved as individual monomers on sucrose gradients and did not form homodimers. Recombination of the chains led to the formation of alpha-beta heterodimers with sedimentation characteristics identical with native alpha-beta dimers. The binding of 125I-labeled band 4.1 to alpha and beta chains was measured by sucrose gradient rate zonal sedimentation and by quantitative immunoassay. It was found that both alpha and beta chains associated with 125I-labeled band 4.1 in a nearly identical manner over the range of band 4.1 concentration studied. The association was abolished by heat denaturation of the spectrin chains or by denaturation of band 4.1 with a 40-fold molar excess of N-ethylmaleimide. As expected, purified beta chains but not alpha chains bound to 125I-labeled ankyrin as measured by a quantitative radioimmunoassay. The binding of purified alpha chains, beta chains, and recombinant alpha-beta heterodimers to F-actin was measured in the presence of band 4.1. We found that alpha or beta chains separately exhibited no band 4.1 dependent association with F-actin but that alpha-beta heterodimers formed by recombination of the chains did. We conclude that spectrin binding to F-actin in the presence of band 4.1 requires the participation of both of spectrin's polypeptide chains.
人红细胞血影蛋白α链和β链通过制备型十二烷基硫酸钠凝胶电泳以及在尿素存在下的二乙氨基乙基纤维素色谱法进行纯化。纯化后的链在蔗糖梯度中表现为单个单体，未形成同二聚体。链的重组导致形成α-β异二聚体，其沉降特性与天然α-β二聚体相同。通过蔗糖梯度速率区带沉降和定量免疫测定法测量了125I标记的带4.1与α链和β链的结合。发现在所研究的带4.1浓度范围内，α链和β链与125I标记的带4.1的结合方式几乎相同。血影蛋白链的热变性或用40倍摩尔过量的N-乙基马来酰亚胺使带4.1变性可消除这种结合。正如预期的那样，通过定量放射免疫测定法测量，纯化的β链而非α链与125I标记的锚蛋白结合。在存在带4.1的情况下，测量了纯化的α链、β链和重组α-β异二聚体与F-肌动蛋白的结合。我们发现，α链或β链单独与F-肌动蛋白没有带4.1依赖性结合，但由链重组形成的α-β异二聚体有。我们得出结论，在存在带4.1的情况下，血影蛋白与F-肌动蛋白的结合需要血影蛋白的两条多肽链都参与。
