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白芷细胞外21kD钙调素结合蛋白的生理功能初探 |
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毛国
红 汤文强 孙大业*
摘要：利用Sephadex G-100及CM-Sepharose层析法，从白芷悬浮培养细胞的胞外盐提液中纯化了21kD钙调素结合蛋白（21kD CaMBP），测其等电点的pH为8.9，紫外薄层扫描显示其纯度达94%以上。以此蛋白为抗原，用免疫小鼠腹水法制备了特异性抗体。由纯化的21kD CaMBP及其特异性抗体研究其对白芷悬浮培养细胞增殖的影响，结果表明：加入外源纯化的21kD CaMBP抑制细胞增殖，半抑制浓度约8μg/ml;而加入21kD CaMBP特异性抗体却促进细胞增殖效应。推测胞外内源存在的21kD CaMBP在生理条件下可能具有参与调节胞外活化CaM信号分子的浓度，从而调节胞外CaM的生物学功能。
关键词：白芷, 胞外钙调素结合蛋白, 纯化，抗体制备, 细胞增殖
学科分类号：Q945
Preliminary Study on the Physiological Function of Extracellular 21 kD Calmodulin-binding Protein from Angelica dahurica
MAO Guo-Hong, TANG Wen-Qiang and SUN Da-Ye
(Laboratory of Molecular Cell Biology,Hebei Normal University, Shijiazhuang 050016)
Abstract：The 21kD calmodulin-binding protein（CaMBP）was purified from extracellular extracts of suspension-cultured cells of Angelica dahurica(Figs.1,2). There was only one band either in SDS-PAGE or in IEF (Figs. 1,3). Its isoelectric point is pH 8.9 (Fig.3). The purity of the 21kD CaMBP obtained was shown to be over 94% by gel scanning. The antibody against 21kD CaMBP was prepared by immunizing rat against ascites. Western blotting showed that the antibody was specific to 21kD CaMBP (Fig.4).The addition of purified 21kD CaMBP to the cultured medium of suspension-cultured cells of Angelica dahurica inhibited cell proliferation (Fig.5), whereas its antibody accelerated cell proliferation (Fig.6).Although the regulation of extracellular CaM activity might be complicated, the above results suggest that 21kD CaMBP may act as a factor regulating the activation and deactivation of extracellular CaM by binding to or dissociating from it.
Key words: Angelica dahurica, extracellular calmodulin-binding protein, purification, antibody preparation, cell proliferation
钙调素（CaM）作为胞内钙信使的多功能受体蛋白，在细胞内信号转导途径中发挥重要作用。自Biro等（1984）首次证实了植物细胞外存在CaM以后，我室用多种实验方法，在多种植物实验体系中证实了植物细胞外CaM存在的普遍性（孙大业等1995），且证实了胞外CaM具有促进细胞增殖（Sun等1994）、原生质体分裂和原生质体壁再生（Sun等1995）以及启动和促进花粉萌[1] [2] 下一页
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