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牛肾上腺皮质肌醇1,4,5-三磷酸受体的增溶及部分特性揭示其与大鼠小脑受体相似。

Solubilization and partial characterization of inositol 1,4,5-trisphosphate receptor of bovine adrenal cortex reveal similarities with the receptor of rat cerebellum.

作者信息

Guillemette G, Favreau I, Boulay G, Potier M

机构信息

Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Québec, Canada.

出版信息

Mol Pharmacol. 1990 Dec;38(6):841-7.

PMID:2174504
Abstract

Inositol 1,4,5-trisphosphate (InsP3) is an intracellular messenger generated upon stimulation of a wide variety of cells by Ca2(+)-mobilizing stimuli. Specific binding sites for InsP3 have been identified in the adrenal cortex and many other tissues. The purpose of the present study was to solubilize and further characterize InsP3 receptors of bovine adrenal cortex. When adrenal cortex microsomes were incubated with Triton X-100 (1%) for 45 min and centrifuged at 100,000 x g for 1 hr, substantial InsP3-binding activity was recovered in the pellet fraction (82 +/- 46 fmol/mg of protein; Kd of 2.7 +/- 1.2 nM), suggesting a possible association with the cell skeleton. Similar results were also obtained with a microsomal preparation of rat cerebellum. On the other hand, the supernatant fraction also displayed important InsP3-binding activity (188 +/- 67 fmol/mg of protein; Kd of 10.4 +/- 2.2 nM). InsP3 binding in both fractions was inhibited by heparin and was increased upon pH elevations from 6 to 9. These are two characteristic properties of InsP3 receptors. Solubilized InsP3 receptors displayed a molecular size around 1,000,000, as estimated by gel filtration through Sepharose-4B column. Radiation inactivation analyses of the receptors of bovine adrenal cortex and rat cerebellum revealed unusual inactivation curves, indicating binding domains of Mr 65,000, much smaller than the smallest covalent structure (subunit) of Mr 260,000 estimated by gel electrophoresis. These results suggest that the binding domain of the receptor behaves independently from the rest of the molecule and that a direct hit on the domain is needed for inactivation. Our data show that the binding sites for InsP3 in the adrenal cortex have properties similar to those of sites recently purified from rat cerebellum, and they suggest that InsP3 receptors from both sources might be the same molecular entity.

摘要

肌醇 1,4,5 - 三磷酸(InsP3)是细胞内信使,在多种细胞受到钙离子动员刺激时产生。在肾上腺皮质和许多其他组织中已鉴定出 InsP3 的特异性结合位点。本研究的目的是溶解并进一步表征牛肾上腺皮质的 InsP3 受体。当肾上腺皮质微粒体与 1% 的 Triton X - 100 孵育 45 分钟,然后在 100,000×g 下离心 1 小时时,沉淀部分中回收了大量的 InsP3 结合活性(82±46 fmol/mg 蛋白质;解离常数为 2.7±1.2 nM),这表明可能与细胞骨架相关。用大鼠小脑的微粒体制备物也得到了类似结果。另一方面,上清部分也显示出重要的 InsP3 结合活性(188±67 fmol/mg 蛋白质;解离常数为 10.4±2.2 nM)。两个部分中的 InsP3 结合均受到肝素抑制,并且在 pH 从 6 升高到 9 时增加。这些是 InsP3 受体的两个特征特性。通过 Sepharose - 4B 柱凝胶过滤估计,溶解的 InsP3 受体的分子大小约为 1,000,000。对牛肾上腺皮质和大鼠小脑受体的辐射失活分析显示出不寻常的失活曲线,表明结合域的分子量为 65,000,远小于通过凝胶电泳估计的最小共价结构(亚基)的分子量 260,000。这些结果表明受体的结合域与分子的其余部分独立发挥作用,并且失活需要直接作用于该域。我们的数据表明,肾上腺皮质中 InsP3 的结合位点具有与最近从大鼠小脑中纯化的位点相似的特性,并且表明来自这两个来源的 InsP3 受体可能是相同的分子实体。

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