Bendayan M, Shore G C
J Histochem Cytochem. 1982 Feb;30(2):139-47. doi: 10.1177/30.2.7061817.
Two mitochondrial proteins, carbamyl phosphate synthetase (CPS) and a structural membrane protein, OMM-35, were specifically localized in the hepatocyte mitochondrial matrix and inner and outer membranes, respectively, using the protein A-gold technique. Three embedding media, Epon, glycol methacrylate (GMA), and Lowicryl K4M, were tested for their ability to provide good ultrastructural preservation of mitochondrial membranes, while at the same time retaining protein antigenicity in embedded liver. Epon embedding proved to be relatively unsuitable, since mitochondrial membranes were poorly preserved. GMA and Lowicryl however gave excellent ultrastructural preservation and retained protein antigenicity sufficiently well to enable the localization of the structural membrane protein. Both qualitative and quantitative immunocytochemical demonstration of CPS have ascertained its localization to the rat hepatocyte mitochondrial matrix. The enzyme was undetectable, however, in the mitochondria of liver endothelial cells. OMM-35 was specifically located in the mitochondrial membranes and the quantitative evaluation confirms the biochemical data that OMM-35 is clearly enriched in the outer mitochondrial membrane. OMM-35 was detected in mitochondria of both hepatocytes and endothelial cells. The labeling of a relatively minor structural membrane protein such as OMM-35 gives and indication of the high sensitivity of the protein A-gold immunocytochemical technique.
采用蛋白A-金技术，两种线粒体蛋白，即氨甲酰磷酸合成酶（CPS）和一种结构膜蛋白OMM-35，分别特异性定位于肝细胞线粒体基质以及内膜和外膜。测试了三种包埋介质，即环氧树脂、甲基丙烯酸乙二醇酯（GMA）和低温包埋剂K4M，它们在提供线粒体膜良好超微结构保存的同时，能否保留包埋肝脏中蛋白质的抗原性。结果表明，环氧树脂包埋相对不合适，因为线粒体膜保存不佳。然而，GMA和低温包埋剂能提供出色的超微结构保存，并充分保留蛋白质抗原性，从而能够定位结构膜蛋白。对CPS进行的定性和定量免疫细胞化学证明确定了其在大鼠肝细胞线粒体基质中的定位。然而，在肝内皮细胞的线粒体中未检测到该酶。OMM-35特异性定位于线粒体膜，定量评估证实了生化数据，即OMM-3明显富集于线粒体外膜。在肝细胞和内皮细胞的线粒体中均检测到了OMM-35。对相对较小的结构膜蛋白如OMM-35进行标记，表明蛋白A-金免疫细胞化学技术具有高灵敏度。
