Freedman J H, Peisach J
Institute for Structural and Functional Studies, University City Science Center, Philadelphia, PA.
Biochim Biophys Acta. 1989 Aug 18;992(2):145-54. doi: 10.1016/0304-4165(89)90003-2.
The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophoresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, pI values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-reactivity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6-12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.
利用一系列在富含铜的培养基中培养的耐铜肝癌细胞系，研究了对铜毒性作用的抗性机制。对羧酰胺化细胞提取物进行凝胶电泳，结果表明野生型和抗性细胞系中存在一对低分子量的富含半胱氨酸的蛋白质。这些蛋白质被纯化至同质，且约含细胞总铜量的60%。将纯化的肝癌蛋白质的分子量、等电点值和氨基酸组成与 authentic 大鼠肝脏金属硫蛋白进行比较，以及与抗大鼠金属硫蛋白抗体的交叉反应性，证实富含半胱氨酸的肝癌蛋白质是金属硫蛋白。这些肝癌铜金属硫蛋白的细胞浓度与金属抗性水平以及各个细胞系积累的铜量均成正比。此外，从富含铜的培养基中移出6至12个月但仍保持其抗性水平的抗性细胞，其金属硫蛋白浓度仅略有下降。因此，有人提出对金属毒性的抗性水平是由铜金属硫蛋白的浓度介导的。还表明铜金属硫蛋白的稳态水平受金属暴露程度的控制。
