Hossain M Z, Murphy L J, Hertzberg E L, Nagy J I
Department of Physiology, University of Manitoba, Winnipeg, Canada.
J Neurochem. 1994 Jun;62(6):2394-403. doi: 10.1046/j.1471-4159.1994.62062394.x.
The gap junction protein connexin43 (Cx43) has been reported to exist as several phosphorylated forms migrating at approximately 43 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as an unphosphorylated 41-kDa form. In brain, Cx43 is expressed predominantly in astrocytes and is also expressed in several other cell types. Whereas the phosphorylated forms of Cx43 predominate in heart, several studies have indicated that high levels of the unphosphorylated form of Cx43 are present in brain. Various experiments in this report indicate that the 41-kDa molecular form in brain is a postmortem dephosphorylation product of phosphorylated Cx43. In rats killed by cranial high-energy microwave irradiation leading to rapid inactivation of brain metabolism, Cx43 in cerebral cortex was present almost exclusively as the 43-kDa phosphorylated form. Rapid dissection of brain followed by heat treatment or inclusion of phosphatase inhibitors during tissue homogenization also largely prevented the conversion of the 43- to the 41-kDa form. The 41-kDa species was generated after alkaline phosphatase digestion of the 43-kDa material obtained by immunoprecipitation from microwave-irradiated brain. Immunolabeling patterns and relative regional levels of Cx43 as seen by immunohistochemical and western blot detection were the same whether or not metabolism to the 41-kDa species was prevented. In developing rat brain, Cx43 levels in frontal cortex and brainstem increased with age, but the degree of dephosphorylation of the 43- to the 41-kDa form was greater at earlier ages in the brainstem. It appears that brain contains a phosphatase that may be involved in modulating the phosphorylation state of Cx43 and thus may regulate intercellular communication via astrocytic gap junctions.
据报道，间隙连接蛋白连接蛋白43（Cx43）以几种磷酸化形式存在，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上迁移至约43 kDa，同时还存在一种未磷酸化的41 kDa形式。在大脑中，Cx43主要在星形胶质细胞中表达，也在其他几种细胞类型中表达。虽然Cx43的磷酸化形式在心脏中占主导，但多项研究表明，大脑中存在高水平的未磷酸化形式的Cx43。本报告中的各种实验表明，大脑中的41 kDa分子形式是磷酸化Cx43的死后去磷酸化产物。在因颅骨高能微波辐射导致脑代谢迅速失活而死亡的大鼠中，大脑皮层中的Cx43几乎完全以43 kDa的磷酸化形式存在。在组织匀浆过程中对大脑进行快速解剖并随后进行热处理或加入磷酸酶抑制剂，也在很大程度上阻止了43 kDa形式向41 kDa形式的转化。通过对从微波辐射大脑中免疫沉淀得到的43 kDa物质进行碱性磷酸酶消化后产生了41 kDa的物质。无论是否阻止向41 kDa物质的代谢，通过免疫组织化学和蛋白质印迹检测所观察到的Cx43的免疫标记模式和相对区域水平都是相同的。在发育中的大鼠大脑中，额叶皮层和脑干中的Cx43水平随年龄增加，但在脑干中，43 kDa形式向41 kDa形式的去磷酸化程度在较早年龄时更大。看来大脑中含有一种磷酸酶，可能参与调节Cx43的磷酸化状态，从而可能通过星形胶质细胞间隙连接调节细胞间通讯。
