Steiner J P, Walke H T, Bennett V
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1989 Feb 15;264(5):2783-91.
Brain spectrin, through its beta subunit, binds with high affinity to protein-binding sites on brain membranes quantitatively depleted of ankyrin (Steiner, J., and Bennett, V. (1988) J. Biol. Chem. 263, 14417-14425). In this study, calmodulin is demonstrated to inhibit binding of brain spectrin to synaptosomal membranes. Submicromolar concentrations of calcium are required for inhibition of binding, with half-maximal effects at pCa = 6.5. Calmodulin competitively inhibits binding of spectrin to protein(s) in stripped synaptosomal membranes, with Ki = 1.3 microM in the presence of 10 microM calcium. A reversible receptor-mediated process, and not proteolysis, is responsible for inhibition since the effect of calcium/calmodulin is reversed by the calmodulin antagonist trifluoperazine and by chelation of calcium with sodium [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The target of calmodulin is most likely the spectrin attachment protein(s) rather than spectrin itself since: (a) membrane binding of the brain spectrin beta subunit, which does not associate with calmodulin, is inhibited by calcium/calmodulin, and (b) red cell spectrin which binds calmodulin very weakly, is inhibited from interacting with membrane receptors in the presence of calcium/calmodulin. Ca2+/calmodulin inhibited association of erythrocyte spectrin with synaptosomal membranes but had no effect on binding of erythrocyte or brain spectrin to ankyrin in erythrocyte membranes. These experiments demonstrate the potential for differential regulation of spectrin-membrane protein interactions, with the consequence that Ca2+/calmodulin can dissociate direct spectrin-membrane interactions locally or regionally without disassembly of the areas of the membrane skeleton stabilized by linkage of spectrin to ankyrin. A membrane protein of Mr = 88,000 has been identified that is dissociated from spectrin affinity columns by calcium/calmodulin and is a candidate for the calmodulin-sensitive spectrin-binding site in brain.
脑血影蛋白通过其β亚基与定量去除锚蛋白的脑细胞膜上的蛋白质结合位点高亲和力结合（施泰纳，J.，和贝内特，V.（1988年）《生物化学杂志》263卷，14417 - 14425页）。在本研究中，已证明钙调蛋白可抑制脑血影蛋白与突触体膜的结合。抑制结合需要亚微摩尔浓度的钙，在pCa = 6.5时达到半数最大效应。钙调蛋白竞争性抑制血影蛋白与去除蛋白的突触体膜中蛋白质的结合，在存在10微摩尔钙的情况下，Ki = 1.3微摩尔。钙/钙调蛋白的作用是由可逆的受体介导过程而非蛋白水解引起的，因为钙调蛋白拮抗剂三氟拉嗪以及用[乙二胺双（氧乙基腈）]四乙酸钠螯合钙可逆转钙/钙调蛋白的作用。钙调蛋白的靶标很可能是血影蛋白附着蛋白而非血影蛋白本身，原因如下：（a）不与钙调蛋白结合的脑血影蛋白β亚基的膜结合被钙/钙调蛋白抑制；（b）与钙调蛋白结合非常弱的红细胞血影蛋白在存在钙/钙调蛋白时被抑制与膜受体相互作用。Ca2 + /钙调蛋白抑制红细胞血影蛋白与突触体膜的结合，但对红细胞血影蛋白或脑血影蛋白与红细胞膜中锚蛋白的结合没有影响。这些实验证明了血影蛋白 - 膜蛋白相互作用存在差异调节的可能性，结果是Ca2 + /钙调蛋白可在局部或区域解离血影蛋白与膜的直接相互作用，而不会拆散通过血影蛋白与锚蛋白连接而稳定的膜骨架区域。已鉴定出一种分子量为88,000的膜蛋白，它可被钙/钙调蛋白从血影蛋白亲和柱上解离，是脑中钙调蛋白敏感的血影蛋白结合位点的候选蛋白。
