Sato M, Hayashi A, Ito H, Tojo M, Arima M
No To Shinkei. 1984 Nov;36(11):1063-8.
Copper concentration, intracellular copper distribution, and inducibility of metallothionein-like metal-binding protein (MLP) by copper or cadmium addition to culture medium were compared among three types of skin fibroblasts derived from patients with Menkes' disease and Wilson's disease, both exhibiting genetic defects of copper metabolism, and from normal subjects (control). Skin fibroblasts were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics in 5% CO2 at 37 degrees C. Cells were harvested with rubber-policeman, washed twice with phosphate-buffered saline, pH 7.2, suspended in deionized water, and homogenized. The homogenate from each cell type was used to determine the concentration of copper by atomic absorption spectrophotometry employing graphite-rod atomizer after lyophilization, ashing in HNO3, and coprecipitation with zirconium. Intracellular copper concentration was elevated in Menkes' cells (420 ng Cu/mg of protein) and Wilson's cells (217 ng Cu/mg of protein) than in control cells (90.0 ng Cu/mg of protein), although one of four Wilson's strains showed normal copper level (70.5 ng Cu/mg of protein). Cytosol copper concentration was 5.8-fold higher in Menkes' cells but only 1.3-fold in Wilson's cells than in control cells, and cytosol copper accounted for only 35% of total intracellular copper in Wilson's cells as compared with 68% and 52% in Menkes' and control cells, respectively. These suggest that accumulated copper in each cell type is differently distributed within cells; in Menkes' cells exclusively into cytosol, but in Wilson's cells into particulates rather than cytosol. Elution profiles from Sephadex G-75 columns indicated that most of copper had bound to MLP in Menkes' cells, though no Cu-MLP was detectable in Wilson's or control cells under these experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
比较了来自患有门克斯病和威尔逊病（二者均表现出铜代谢遗传缺陷）患者以及正常受试者（对照）的三种皮肤成纤维细胞中铜浓度、细胞内铜分布以及通过向培养基中添加铜或镉诱导金属硫蛋白样金属结合蛋白（MLP）的情况。皮肤成纤维细胞在补充有10%胎牛血清和抗生素的杜尔贝科改良伊格尔培养基中于37℃、5%二氧化碳条件下培养。用橡胶刮棒收集细胞，用pH 7.2的磷酸盐缓冲盐水洗涤两次，悬浮于去离子水中并匀浆。每种细胞类型的匀浆经冻干、在硝酸中灰化并与锆共沉淀后，用石墨棒原子化器通过原子吸收分光光度法测定铜浓度。门克斯病细胞（420 ng铜/毫克蛋白质）和威尔逊病细胞（217 ng铜/毫克蛋白质）中的细胞内铜浓度高于对照细胞（90.0 ng铜/毫克蛋白质），尽管四个威尔逊病菌株中的一个显示铜水平正常（70.5 ng铜/毫克蛋白质）。门克斯病细胞中的胞质铜浓度比对照细胞高5.8倍，但威尔逊病细胞中仅高1.3倍，并且威尔逊病细胞中的胞质铜仅占细胞内总铜的35%，而门克斯病细胞和对照细胞中分别为68%和52%。这些表明每种细胞类型中积累的铜在细胞内的分布不同；在门克斯病细胞中仅进入胞质，而在威尔逊病细胞中进入颗粒而非胞质。来自葡聚糖G - 75柱的洗脱图谱表明，在门克斯病细胞中大部分铜与MLP结合，尽管在这些实验条件下在威尔逊病细胞或对照细胞中未检测到铜 - MLP。（摘要截断于250字）
