Hensley K, Postlewaite J, Dobbs P, Butterfield D A
Department of Chemistry, University of Kentucky, Lexington 40506-0055.
Biochim Biophys Acta. 1993 Feb 9;1145(2):205-11. doi: 10.1016/0005-2736(93)90290-g.
A fraction of band 3 protein, the major transmembrane protein of erythrocyte membranes, is held to the cytoskeletal protein spectrin via noncovalent interactions with the protein ankyrin (band 2.1). In this study, trypsin was used under defined conditions to selectively proteolyze ankyrin and thereby destroy the band 3-ankyrin linkage on the cytoplasmic side of erythrocyte ghost membranes. Electron paramagnetic resonance (EPR) spectroscopy, in conjunction with selective spin labeling methods, was used to monitor conformational changes occurring in cytoskeletal proteins or cell-surface carbohydrates as a result of this treatment. Treatment of RBC ghosts with TPCK-trypsin for 5 s at 0 degrees C caused an approx. 56% increase in the relevant EPR parameter of a maleimide spin label bound to spectrin (P < 0.004), indicative of increased segmental motion of the spin label and decreased protein-protein interactions. Analysis of the apparent rotational correlation time parameter tau of a spin label covalently and selectively bound to terminal sialic acid residues of glycophorin showed no significant effect from trypsin treatment. However, tau of spin label covalently and specifically bound to terminal galactose residues of cell-surface glycoconjugates of band 3 and other transmembrane glycoproteins significantly decreased with tryptic uncoupling of the ankyrin linkage (P < 0.005). These results suggest a marked conformational alteration in both cytoskeletal and transmembrane proteins as a result of uncoupling from ankyrin. Spermine (N,N'-bis(3-aminopropyl)tetramethylenediamine), a naturally occurring polyamine known to strengthen cytoskeletal protein-protein interactions (Wyse and Butterfield (1988) Biochim. Biophys. Acta 941, 141-149), was used to partially reverse the trypsin-induced cytoskeletal alterations. Addition of 2 mM spermine to ghosts previously treated with trypsin increased cytoskeletal protein-protein interactions as indicated by EPR (P < 0.002). SDS-PAGE was used to confirm the integrity of spectrin, band 3, and band 4.1 in all experiments. The results are discussed with reference to transmembrane signaling mechanisms and membrane-associated pathologies.
红细胞膜的主要跨膜蛋白带3蛋白的一部分，通过与锚蛋白（带2.1）的非共价相互作用与细胞骨架蛋白血影蛋白相连。在本研究中，在特定条件下使用胰蛋白酶选择性地对锚蛋白进行蛋白水解，从而破坏红细胞血影膜细胞质侧的带3 - 锚蛋白连接。电子顺磁共振（EPR）光谱结合选择性自旋标记方法，用于监测由于这种处理而在细胞骨架蛋白或细胞表面碳水化合物中发生的构象变化。在0℃下用TPCK - 胰蛋白酶处理红细胞血影5秒，导致与血影蛋白结合的马来酰亚胺自旋标记的相关EPR参数增加约56%（P < 0.004），这表明自旋标记的片段运动增加，蛋白质 - 蛋白质相互作用减少。对共价且选择性地结合到血型糖蛋白末端唾液酸残基的自旋标记的表观旋转相关时间参数tau的分析表明，胰蛋白酶处理没有显著影响。然而，随着锚蛋白连接的胰蛋白酶解偶联，共价且特异性地结合到带3和其他跨膜糖蛋白的细胞表面糖缀合物末端半乳糖残基的自旋标记的tau显著降低（P < 0.005）。这些结果表明，由于与锚蛋白解偶联，细胞骨架蛋白和跨膜蛋白都发生了显著的构象改变。精胺（N，N'-双（3 - 氨丙基）四亚甲基二胺）是一种天然存在的多胺，已知可增强细胞骨架蛋白 - 蛋白质相互作用（Wyse和Butterfield（1988）生物化学与生物物理学报941, 141 - 149），用于部分逆转胰蛋白酶诱导的细胞骨架改变。如EPR所示，向先前用胰蛋白酶处理过的血影中添加2 mM精胺可增加细胞骨架蛋白 - 蛋白质相互作用（P < 0.002）。在所有实验中，使用SDS - PAGE来确认血影蛋白、带3和带4.1的完整性。结合跨膜信号传导机制和膜相关病理学对结果进行了讨论。
