Seo H C, Ettinger M J
Department of Biochemistry, State University of New York, Buffalo 14214.
J Biol Chem. 1993 Jan 15;268(2):1160-5.
The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cytosols, and some of its properties were determined. When 64Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal 64Cu binding was detected in both the fraction which contained the tetramer plus dimer (approximately 26% less) and the fraction containing the monomer of CuBP (approximately 37% less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q, chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at approximately 20 min after starting the final water elution step, the brindled mouse protein eluted by approximately 5 min. This seemed to be due to abnormal self-association in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Superose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consistent with a significant role for CuBP in intracellular copper metabolism, and an abnormal structure of CuBP may be the basic defect in the brindled mice and, by inference, Menkes disease.
花斑小鼠是一种门克斯病的动物模型，门克斯病是一种致命的、X连锁的铜代谢疾病。从花斑小鼠的肝细胞溶胶中纯化出一种自我缔合的50 kDa铜结合蛋白（CuBP），并测定了它的一些性质。当用Superose对64Cu标记的全肝细胞溶胶进行分级分离时，在含有四聚体加二聚体的级分（约少26%）和含有CuBP单体的级分（约少37%）中，检测到的64Cu结合量在统计学上显著低于正常水平。使用与从正常小鼠纯化该蛋白相同的方法，通过连续的Mono Q柱、螯合Superose柱和苯基Superose柱从花斑小鼠的肝细胞溶胶中纯化CuBP。然而，来自花斑小鼠的CuBP在纯化过程中有些不稳定。此外，来自花斑小鼠的CuBP从苯基Superose柱上的洗脱异常。因此，正常小鼠的蛋白在最终水洗脱步骤开始后约20分钟洗脱，而花斑小鼠蛋白在约5分钟时洗脱。这似乎是由于柱缓冲液中异常的自我缔合。与使用全细胞溶胶的结果一致，从花斑小鼠纯化的CuBP在Superose的四聚体和单体级分中均显示铜结合减少。此外，在相同条件下，来自花斑小鼠的CuBP似乎比正常小鼠的四聚体相对较少，二聚体相对较多。这些结果与CuBP在细胞内铜代谢中的重要作用一致，CuBP的异常结构可能是花斑小鼠以及由此推断的门克斯病的基本缺陷。
