Birchmeier W, Singer S J
J Cell Biol. 1977 Jun;73(3):647-59. doi: 10.1083/jcb.73.3.647.
In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.
在前一篇论文中（Sheetz, M. 和 S.J. Singer. 1977. 《细胞生物学杂志》73:638 - 646）已表明，红细胞血影在Mg - ATP存在的情况下会发生显著的形状变化。本文研究了ATP作用的生化效应。已知红细胞膜的血影蛋白成分2会被ATP磷酸化。我们已表明，在产生形状变化的条件下，只有膜蛋白会被显著磷酸化。这种磷酸化程度随ATP浓度增加而升高，在8mM ATP时，每摩尔成分2接近1摩尔磷酸基团。根据溴化氰肽裂解实验，这种磷酸化大部分似乎发生在蛋白质分子的单个位点上。成分2的磷酸化程度显然也受一种膜结合蛋白磷酸酶的调节。在用[(32)P]磷酸盐预标记的完整细胞制备的红细胞血影中可证明这种活性。除了成分2的磷酸化外，还已知一些脂质（主要是磷脂酰肌醇）也会发生磷酸化。然而，血影形状变化与成分2的磷酸化程度相关。在这样的实验中，将外源磷酸酶掺入血影可逆转由ATP或膜嵌入药物氯丙嗪产生的形状变化。在本文和前一篇论文中获得的结果与以下观点一致：红细胞膜具有激酶和磷酸酶活性，它们使血影蛋白成分2分子上的特定位点发生磷酸化和去磷酸化；这种磷酸化的稳态水平调节膜细胞质表面血影蛋白复合物的结构状态，进而对细胞形状施加重要控制。
