SECRETORY-CELL ACTIN-BINDING PROTEINS - IDENTIFICATION OF A GELSOLIN-LIKE PROTEIN IN CHROMAFFIN CELLS
SECRETORY-CELL ACTIN-BINDING PROTEINS - IDENTIFICATION OF A GELSOLIN-LIKE PROTEIN IN CHROMAFFIN CELLS
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DOI：
10.1083/jcb.102.2.636
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发表时间：
1986-02-01
影响因子：
7.8
通讯作者：
AUNIS, D
中科院分区：
文献类型：
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作者：
BADER, MF;TRIFARO, JM;AUNIS, D
嗜铬细胞是肾上腺髓质的分泌细胞，已被证明含有肌动蛋白和其他可能参与分泌过程的收缩蛋白。使用 DNase-I 作为配体，在亲和柱上从牛肾上腺髓质中纯化肌动蛋白和 Ca++ 敏感肌动蛋白结合蛋白。使用含有递减的 CA++ 浓度的缓冲液来洗脱 93、91 和 85 kD 的三种主要蛋白质。大部分肌动蛋白用盐酸胍缓冲液加上一些 93-和 91-kD 蛋白质洗脱。使用低剪切落球粘度计和电子显微镜显示，这些 Ca++ 敏感调节蛋白可抑制肌动蛋白的凝胶化。发现肌动蛋白丝因断裂而缩短。使用针对兔肺巨噬细胞凝溶胶蛋白的抗体、葡萄球菌 V8 蛋白酶的蛋白水解消化和二维凝胶电泳，91-kD 肌动蛋白结合蛋白被证明是一种凝溶胶蛋白样蛋白。 93-kD 肌动蛋白结合蛋白还显示出与抗凝溶胶蛋白抗体的交叉反应性、相似的肽图以及 pH1 的基本变化，表明该 93-kD 蛋白是一种短短病毒样蛋白，源自肾上腺髓质中大量存在的血液。从分离的嗜铬细胞中纯化证明不存在 91-kD 和 85-kD 蛋白质，而几乎检测不到 93-kD 蛋白质。 85-kD 蛋白质不是短列蛋白样或凝溶胶蛋白样蛋白质的分解产物。它不与抗凝溶胶蛋白抗体发生交叉反应，并且在用 V8 蛋白酶温和消化后显示出非常不同的肽图。针对 93-和 91-kD 肌动蛋白结合蛋白以及 85-kD 肌动蛋白结合蛋白产生抗体。针对 85-kD 蛋白的抗体不会与 93-和 91-kD 蛋白发生交叉反应，反之亦然。在体内，嗜铬分泌细胞的细胞骨架组织尚不清楚，但似乎受到细胞内游离钙浓度的控制。钙激活凝溶胶蛋白样蛋白的能力，以及如其他地方所示改变胞质蛋白定位的能力，提供了一种凝胶-溶胶转变的机制，这可能对于分泌过程中涉及的颗粒运动和膜-膜相互作用至关重要。
Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing CA++ concentration were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphyloccus V8 protease and two-dimensional gel electrphoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pH1 indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence nof 91- and 85-kD proteins whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton oraganization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.