Wiemer E A, Brul S, Just W W, Van Driel R, Brouwer-Kelder E, Van Den Berg M, Weijers P J, Schutgens R B, Van Den Bosch H, Schram A
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
Eur J Cell Biol. 1989 Dec;50(2):407-17.
The presence and intracellular localization of peroxisomal integral membrane proteins (PMP) were investigated in liver and cultured skin fibroblasts from control subjects and patients with the Zellweger syndrome and related disorders in which peroxisomes are virtually absent. Immunoblotting experiments showed that 22, 36 and 69 kDa PMPs were present and were confined to the membranous fraction both in the control liver and in the livers from the Zellweger patients. The 22 and 36 kDa PMPs were present in significantly lower amounts in the patients' livers than in the control liver. A reduced amount of the 69 kDa PMP was found in liver from one Zellweger but not in liver from another. The subcellular localization in fibroblasts of catalase and the 69 kDa PMP was studied by indirect immunofluorescence. A characteristic punctate fluorescence was seen in control cells incubated with either anti-(catalase) or with anti-(69 kDa PMP). Incubation of mutant cells with anti-(catalase) resulted in a diffuse fluorescence, whereas with anti-(69 kDa PMP) fluorescent particles were visualized which, in some cell lines, were larger and fewer in number than in control cells. Cryosections of control and mutant cells were examined by electron microscopy using immunogold labeling. Control cells contained small structures consisting of a single membrane enclosing a homogeneous matrix; the membranes reacted with anti-(69 kDa PMP) and the matrix with anti-(catalase). The mutant cell lines contained spherical or ellipsoidal structures whose membranes reacted with anti-(69 kDa PMP); no labeling was observed with anti-(catalase). We conclude that peroxisomal ghosts, the membranes of which contain the 69 kDa PMP, are present in peroxisome-deficient cell lines from all complementation groups studied so far.
研究了过氧化物酶体整合膜蛋白（PMP）在对照受试者以及患有 Zellweger 综合征和相关疾病（其中几乎不存在过氧化物酶体）的患者的肝脏和培养的皮肤成纤维细胞中的存在情况及其细胞内定位。免疫印迹实验表明，22 kDa、36 kDa 和 69 kDa 的 PMP 在对照肝脏和 Zellweger 患者的肝脏中均存在且局限于膜部分。患者肝脏中 22 kDa 和 36 kDa 的 PMP 含量明显低于对照肝脏。在一名 Zellweger 患者的肝脏中发现 69 kDa 的 PMP 含量减少，但在另一名患者的肝脏中未发现。通过间接免疫荧光研究了过氧化氢酶和 69 kDa PMP 在成纤维细胞中的亚细胞定位。在用抗（过氧化氢酶）或抗（69 kDa PMP）孵育的对照细胞中可见特征性的点状荧光。用抗（过氧化氢酶）孵育突变细胞导致荧光弥漫，而用抗（69 kDa PMP）孵育时可见荧光颗粒，在某些细胞系中，这些颗粒比对照细胞中的更大且数量更少。使用免疫金标记通过电子显微镜检查对照细胞和突变细胞的冷冻切片。对照细胞含有由单个膜包围均匀基质组成的小结构；膜与抗（69 kDa PMP）反应，基质与抗（过氧化氢酶）反应。突变细胞系含有球形或椭圆形结构，其膜与抗（69 kDa PMP）反应；用抗（过氧化氢酶）未观察到标记。我们得出结论，迄今为止研究的所有互补组的过氧化物酶体缺陷细胞系中都存在过氧化物酶体空壳，其膜含有 69 kDa 的 PMP。
