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首页> 外文期刊>American Journal of Physiology >Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase.
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Characterization of Cos-7 cells overexpressing the rat secretory pathway Ca2+-ATPase.

机译：过表达大鼠分泌途径Ca2 + -ATPase的Cos-7细胞的表征。

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On the basis of sequence similarities to the yeast PMR1 and hSPCA gene, the rat alternatively spliced mRNA has been suggested to be a Golgi secretory pathway Ca2+-ATPase (SPCA). Data in this report lend further support for this hypothesis in that sucrose gradient fractionation of rat liver microsomes resulted in SPCA comigrating with the Golgi calcium binding protein CALNUC, which was well resolved from the endoplasmic reticulum marker calreticulin. Also, in PC-12 cells, antibody to SPCA colocalized with an antibody to the Golgi marker alpha-mannosidase II. To study the biological effects of SPCA expression, we performed stable overexpression of SPCA in COS-7 cells. Seven clones were selected for further comparison with COS-7 cells containing an empty expression vector. Overexpression of SPCA resulted in a significant reduction of plasma membrane Ca2+-ATPase, sarco(endo)plasmic reticulum Ca2+-ATPase, and calreticulin expression in these clones. In contrast, the expression of the Golgi calcium-binding protein CALNUC increased significantly. The phosphoenzyme intermediate formed using membranes from clone G11/5 was calcium dependent, significantly more intense than in COS-7 cells, and not affected by La3+ treatment. Calcium uptake by G11/5 microsomes was ATP dependent and significantly greater than in microsomes from parent COS-7 cells. The overexpression of SPCA significantly increased the growth rate of these cells compared with COS-7 cells containing only the empty vector. These data demonstrate that overexpression of the rat SPCA results in significant changes in the expression of calcium transport and storage proteins in COS-7 cells.
机译：基于与酵母PMR1和hSPCA基因的序列相似性，已建议大鼠交替剪接的mRNA是高尔基体分泌途径Ca2 + -ATPase（SPCA）。该报告中的数据进一步支持了这一假说，因为大鼠肝脏微粒体的蔗糖梯度分级分离导致SPCA与高尔基钙结合蛋白CALNUC竞争，这可以从内质网标志物钙网蛋白得到很好的解决。同样，在PC-12细胞中，SPCA抗体与高尔基标记α-甘露糖苷酶II的抗体共定位。为了研究SPCA表达的生物学效应，我们在COS-7细胞中进行了稳定的SPCA过表达。选择七个克隆用于与含有空表达载体的COS-7细胞进行进一步比较。 SPCA的过度表达导致这些克隆中质膜Ca2 + -ATPase，肌膜（内质网）Ca2 + -ATPase和钙网蛋白的表达显着降低。相反，高尔基体钙结合蛋白CALNUC的表达显着增加。使用来自克隆G11 / 5的膜形成的磷酸酶中间体是钙依赖性的，比COS-7细胞中的磷酸酶强度高得多，并且不受La3 +处理的影响。 G11 / 5微粒体对钙的吸收是ATP依赖性的，并且显着高于亲代COS-7细胞的微粒体中的钙吸收。与仅包含空载体的COS-7细胞相比，SPCA的过表达显着提高了这些细胞的生长速率。这些数据表明，大鼠SPCA的过表达导致COS-7细胞中钙转运和贮藏蛋白表达的显着变化。

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