Two-dimensional separation of erythrocyte membrane proteins.
Two-dimensional separation of erythrocyte membrane proteins.
复制标题
红细胞膜蛋白的二维分离。
DOI：
10.1016/0003-9861(79)90019-5
复制
发表时间：
1979
影响因子：
3.9
通讯作者：
M. Morrison
中科院分区：
文献类型：
--
作者：
D. Harell;M. Morrison
详细的二维分离程序专门设计的研究红细胞膜提出。在这种高重复性的方法中，膜蛋白溶解在十二烷基硫酸钠中，首先通过电荷等电聚焦分离。样品被加载在阴极（CIF）或阳极（AIF）。CIF样品对酸性蛋白的分离效果较好，而AIF样品对红细胞高分子量多肽的分离效果较好。该方法在pH值5 ~ 8范围内可检测到90多种多肽。特别注意的是高分子量组分。例如，在主要膜蛋白蛋白3的9万到10万分子量范围内，可以检测到6种组分。检测到与光谱带2具有相同或非常接近相同分子量的组分。它比spectrin带2更基本，并且spectrin带2和基本组分在完整细胞中都很容易被磷酸化。然而，带2的磷酸化与cAMP无关，而基本组分的磷酸化被cAMP增强。与谱质相比，0.1 mmEDTA不能从膜中提取碱性成分，尽管稀释的NaOH会将其从膜中去除。红细胞质的Ca2+激活的转移酶不会交联这一成分。然而，钙确实可以激活这种成分转化为较低的分子量。这种高分子量的基本组分具有费尔班克斯命名体系中标记为2.1的组分的性质。
The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mmEDTA, although dilute NaOH will remove it from the membrane. The Ca2+-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.