Hill J S, Sawyer W H, Howlett G J, Wiley J S
Biochem J. 1982 Feb 1;201(2):259-66. doi: 10.1042/bj2010259.
Human erythrocytes possess a lattice work of extrinsic proteins on the inner face of the membrane (;cytoskeleton') that maintains the shape and deformability of the cell. The major proteins of the cytoskeleton are spectrin and actin, which are attached to the membrane by protein bands 2.1 (;ankyrin') and 4.1. The interactions of spectrin/actin with erythrocyte membranes from normal subjects and from patients with hereditary spherocytosis (HS) have been studied by using an air-driven ultracentrifuge, which can rapidly separate membranes from soluble proteins (150000g for 30s). The total amount of spectrin/actin in HS and normal ghosts is similar. However, the rate of dissociation of spectrin and actin from HS erythrocyte membranes at low ionic strength is significantly lower than that observed for normal membranes. Spectrin and actin isolated from either HS or normal membranes re-associated in a similar manner to spectrin/actin-depleted vesicles prepared from normal cells. Scatchard analysis showed an average binding capacity of 278mug/mg of membrane protein. However, spectrin/actin-depleted vesicles prepared from HS cells bound significantly less spectrin/actin prepared from either the normal or abnormal cells (average binding capacity 158mug/mg of membrane protein). The defect was defined further by studying the cytoskeleton obtained by Triton X-100 extraction of membranes. Under conditions of low ionic strength cytoskeletons prepared from HS membranes dissociated more slowly than those prepared from normal membranes, and only 80% of the protein from HS cytoskeletons could be solubilized after 180min compared with 100% for normal cytoskeletons. The difference between HS and normal membranes, which persists in isolated cytoskeletons, suggests that alterations in either the primary structure or the degree of phosphorylation of protein bands 2.1 or 4.1 may be central to the molecular basis of hereditary spherocytosis.
人类红细胞在膜的内表面（“细胞骨架”）具有一层外在蛋白晶格结构，该结构维持细胞的形状和可变形性。细胞骨架的主要蛋白质是血影蛋白和肌动蛋白，它们通过蛋白带2.1（“锚蛋白”）和4.1附着于膜上。通过使用空气驱动的超速离心机研究了血影蛋白/肌动蛋白与正常受试者和遗传性球形红细胞增多症（HS）患者红细胞膜的相互作用，该超速离心机可快速将膜与可溶性蛋白分离（150000g离心30秒）。HS和正常红细胞影中血影蛋白/肌动蛋白的总量相似。然而，在低离子强度下，血影蛋白和肌动蛋白从HS红细胞膜上解离的速率明显低于正常膜。从HS或正常膜中分离出的血影蛋白和肌动蛋白以与从正常细胞制备的血影蛋白/肌动蛋白缺失囊泡相似的方式重新结合。Scatchard分析显示膜蛋白的平均结合能力为278μg/mg。然而，从HS细胞制备的血影蛋白/肌动蛋白缺失囊泡与从正常或异常细胞制备的血影蛋白/肌动蛋白的结合明显减少（膜蛋白的平均结合能力为158μg/mg）。通过研究用Triton X-100提取膜得到的细胞骨架进一步确定了该缺陷。在低离子强度条件下，从HS膜制备的细胞骨架比从正常膜制备的解离更慢，180分钟后HS细胞骨架中只有80%的蛋白质可溶解，而正常细胞骨架为100%。HS膜和正常膜之间的差异在分离的细胞骨架中仍然存在，这表明蛋白带2.1或4.1的一级结构或磷酸化程度的改变可能是遗传性球形红细胞增多症分子基础的核心。
