Moroni-Rawson P, Palmer D N, Jolly R D, Jordan T W
School of Biological Sciences, Victoria University of Wellington, New Zealand.
Am J Med Genet. 1995 Jun 5;57(2):279-84. doi: 10.1002/ajmg.1320570233.
Two-dimensional polyacrylamide gel electrophoresis has been used to search for disease-related protein variation in South Hampshire sheep with ovine ceroid-lipofuscinosis. Several hundred proteins in homogenates and subcellular fractions from livers have been examined, using isoelectric focusing as the first dimension separation, and SDS PAGE in the second dimension. Under these circumstances it was not possible to detect subunit c of the Fo region of ATP synthase, as this protein did not enter the isoelectric focusing gels. However, our studies emphasize the selective nature of misprocessing of subunit c, as we have not been able to detect any other consistent variation between affected and control animals for over 200 mitochondrial fraction proteins. Comparison of the presence or absence, and abundance, of proteins from isolated storage bodies with their counterparts in subcellular fractions from normal liver indicated that storage bodies contained a small subset of mitochondrial proteins, in addition to subunit c, with possible minor contributions from lysosomal, microsomal, and soluble proteins. Analysis of extramitochondrial proteins showed greater than 10-20-fold accumulation of ferritin light chains in microsomes, and partial loss of a putatively lysosomal protein, in ovine ceroid-lipofuscinosis. In addition, senescence marker protein was more abundant in the cytosolic fraction of controls, compared with affected individuals. We are currently investigating the basis and significance of these differences.
二维聚丙烯酰胺凝胶电泳已被用于寻找患有绵羊蜡样脂褐质沉积症的南汉普郡绵羊中与疾病相关的蛋白质变异。使用等电聚焦作为第一维分离方法，十二烷基硫酸钠聚丙烯酰胺凝胶电泳作为第二维分离方法，对肝脏匀浆和亚细胞组分中的数百种蛋白质进行了检测。在这种情况下，无法检测到ATP合酶F₀区域的亚基c，因为该蛋白质未进入等电聚焦凝胶。然而，我们的研究强调了亚基c错误加工的选择性，因为对于超过200种线粒体组分蛋白质，我们未能在患病动物和对照动物之间检测到任何其他一致的变异。将分离的储存体中的蛋白质与正常肝脏亚细胞组分中的对应蛋白质进行有无及丰度比较，结果表明，除了亚基c外，储存体还包含一小部分线粒体蛋白质，可能还有来自溶酶体、微粒体和可溶性蛋白质的少量贡献。对线粒体外蛋白质的分析表明，在绵羊蜡样脂褐质沉积症中，微粒体中铁蛋白轻链积累了10至20倍以上，一种推测为溶酶体的蛋白质部分丢失。此外，与患病个体相比，衰老标记蛋白在对照的细胞溶质组分中更为丰富。我们目前正在研究这些差异的基础和意义。
