A CEREBELLAR PURKINJE-CELL MARKER P400 PROTEIN IS AN INOSITOL 1,4,5-TRISPHOSPHATE (INSP3) RECEPTOR PROTEIN - PURIFICATION AND CHARACTERIZATION OF INSP3 RECEPTOR COMPLEX
A CEREBELLAR PURKINJE-CELL MARKER P400 PROTEIN IS AN INOSITOL 1,4,5-TRISPHOSPHATE (INSP3) RECEPTOR PROTEIN - PURIFICATION AND CHARACTERIZATION OF INSP3 RECEPTOR COMPLEX
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DOI：
10.1002/j.1460-2075.1990.tb08080.x
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发表时间：
1990-01-01
期刊：
影响因子：
11.4
通讯作者：
MIKOSHIBA, K
中科院分区：
文献类型：
--
作者：
MAEDA, N;NIINOBE, M;MIKOSHIBA, K
P400蛋白是一种分子量为250 kd的糖蛋白，是小脑特有的糖蛋白，主要分布于内质网、质膜和浦肯野细胞的突触后致密区。本研究从小鼠小脑中分离纯化了1，4，5-三磷酸肌醇（InsP 3）受体，并研究了P400蛋白与InsP 3受体蛋白相同的可能性。InsP 3受体用Triton X-100从ddY小鼠小脑的核后部分溶解，并通过DE 52、肝素-琼脂糖、扁豆凝集素-琼脂糖和羟基磷灰石上的连续柱层析以高产率纯化。在这些色谱中，P400蛋白与InsP 3结合活性完全共迁移。纯化的受体是250 kd蛋白，Bmax为2.1 pmol/μ g，KD为83 nM。它与三种不同的抗P400蛋白的单克隆抗体反应，表明P400蛋白与InsP 3受体（P400/InsP 3受体蛋白）是同一物质。纯化的受体的电子显微镜显示出边长为apprx的正方形。25 nm长。浦肯野细胞变性（pcd）小鼠小脑与[~ 3 H] InsP 3的结合试验表明，小脑中的InsP 3结合位点仅分布在浦肯野细胞上。免疫组化结果显示，P400/InsP 3受体分布于浦肯野细胞的树突、胞体、轴突和突触终末。
P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the postsynaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/.mu.g and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides .apprx. 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.