MOLECULAR-STRUCTURE OF HUMAN-ERYTHROCYTE SPECTRIN - BIOPHYSICAL AND ELECTRON-MICROSCOPIC STUDIES
MOLECULAR-STRUCTURE OF HUMAN-ERYTHROCYTE SPECTRIN - BIOPHYSICAL AND ELECTRON-MICROSCOPIC STUDIES
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DOI：
10.1016/0022-2836(79)90078-0
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发表时间：
1979-01-01
影响因子：
5.6
通讯作者：
BRANTON, D
中科院分区：
文献类型：
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作者：
SHOTTON, DM;BURKE, BE;BRANTON, D
用低角阴影法观察人红细胞血影蛋白分子。首先纯化Spectrin异源二聚体和四聚体，并通过聚丙烯酰胺凝胶电泳和分析性超离心在最小化蛋白水解和聚集的条件下进行表征。异源二聚体和四聚体分离的低角度阴影，在生理离子强度的乙酸铵缓冲液中，其中它们显示的沉降系数分别为8.9 S和12.5 S，类似于异源二聚体和四聚体在非挥发性缓冲液中报告的值。在氯化钠或氯化钾缓冲液中四聚体稳定的条件下，醋酸铵缓冲液促进血影蛋白四聚体解离成异二聚体。当通过低角度单向和旋转阴影可视化时，血影蛋白异源二聚体表现为平均阴影长度为97 nm的长柔性分子。每个异二聚体由2条多肽链组成，条带1（240，000 MW）和条带2（220，000 MW），通常表现为2条单独的链，彼此部分分离或以松散的双螺旋形式相互盘绕。这些多肽之间的关联似乎是弱的，除了在分子的两端有强结合位点。四聚体由2个血影蛋白异源二聚体分子的端对端缔合形成，没有可测量的重叠，并且具有194 nm的平均阴影长度。这种形成四聚体的关联可能涉及异二聚体的头对头结合，因为没有观察到预期来自头对头尾结合模式的更高的寡聚体。血影蛋白的分子形状与肌球蛋白的分子形状完全不同，后者经常被比作血影蛋白。
Molecules of human erythrocyte spectrin were examined by EM after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramers were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the 2 polypeptide chains, band 1 (240,000 MW) and band 2 (220,000 MW), often appeared as 2 separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appeared to be weak, except at both ends of the molecule where there were sites of strong binding. Tetramers were formed by the end-to-end association of 2 spectrin heterodimer molecules without measurable overlap, and had a mean shadowed length of 194 nm. This association to form tetramers probably involved head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode were not observed. The molecular shape of spectrin was quite distinct from that of myosin, to which it is often likened.