Qian Y, Tiffany-Castiglioni E, Harris E D
Department of Biochemistry, Texas A&M University, College Station 77843, USA.
Am J Physiol. 1996 Jul;271(1 Pt 1):C378-84. doi: 10.1152/ajpcell.1996.271.1.C378.
To define the function of the Cu-transporting ATPase in Menkes disease, Menkes and normal fibroblasts were incubated with 67Cu before and after brief exposure to -SH reagents, p-chloromercuribenzoate (PCMB) and dithiothreitol (DTT). Accumulation and retention were compared among these cells, BeWo cells, and rat C6 glioma cells similarly treated. The Michaelis constant for influx of 67Cu into normal and Menkes fibroblasts was practically the same (0.21 +/- 0.07 vs. 0.24 +/- 0.06 microM). The PCMB treatment stimulated 67Cu accumulation in C6 cells, inhibited accumulation in normal and Menkes fibroblasts, and did not affect BeWo cells. DTT stimulated 67Cu uptake in all cells but BeWo cells. DTT treatment after PCMB further enhanced 67Cu accumulation in normal fibroblasts and C6 cells but had no enhancing effect on Menkes fibroblasts or BeWo cells. Menkes fibroblasts and BeWo cells released 67Cu at rates considerably slower than normal fibroblasts (0.06 and 0.09 vs. 0.22%/min, respectively). The PCMB blocked 67Cu release from normal fibroblasts but did not affect Menkes fibroblasts or BeWo cells. Reverse transcription-polymerase chain reaction analysis of total RNA from BeWo cells failed to show a predicted 943-base pair fragment representing a partial transcript of the Menkes factor. The fragment was present in extracts from normal fibroblasts. We conclude that the mechanism underlying Cu homeostasis varies among different cell types. As exemplified by BeWo and Menkes cells, failure to efflux Cu ions may be linked with the failure to express a functional Cu-transporting ATPase, namely, the Menkes protein.
为了确定铜转运ATP酶在门克斯病中的作用，在短暂暴露于巯基试剂、对氯汞苯甲酸（PCMB）和二硫苏糖醇（DTT）之前和之后，将门克斯病和正常成纤维细胞与67Cu一起孵育。比较了这些细胞、BeWo细胞和经类似处理的大鼠C6胶质瘤细胞中的积累和保留情况。67Cu流入正常和成纤维细胞的米氏常数实际上是相同的（0.21±0.07对0.24±0.06微摩尔）。PCMB处理刺激了C6细胞中67Cu的积累，抑制了正常和成纤维细胞中的积累，并且对BeWo细胞没有影响。DTT刺激了除BeWo细胞外的所有细胞对67Cu的摄取。PCMB处理后再用DTT进一步增强了正常成纤维细胞和C6细胞中67Cu的积累，但对门克斯病成纤维细胞或BeWo细胞没有增强作用。门克斯病成纤维细胞和BeWo细胞释放67Cu的速率明显低于正常成纤维细胞（分别为0.06和0.09对0.22%/分钟）。PCMB阻止了正常成纤维细胞中67Cu的释放，但对门克斯病成纤维细胞或BeWo细胞没有影响。对BeWo细胞总RNA进行的逆转录-聚合酶链反应分析未能显示出代表门克斯因子部分转录本的预测943碱基对片段。该片段存在于正常成纤维细胞的提取物中。我们得出结论，不同细胞类型中铜稳态的潜在机制各不相同。以BeWo细胞和门克斯病细胞为例，未能排出铜离子可能与未能表达功能性铜转运ATP酶，即门克斯蛋白有关。
