ULTRASTRUCTURAL-LOCALIZATION OF NUCLEIC-ACIDS BY THE USE OF ENZYME-GOLD COMPLEXES
ULTRASTRUCTURAL-LOCALIZATION OF NUCLEIC-ACIDS BY THE USE OF ENZYME-GOLD COMPLEXES
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DOI：
10.1177/29.4.6265546
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发表时间：
1981-01-01
影响因子：
3.2
通讯作者：
BENDAYAN, M
中科院分区：
文献类型：
--
作者：
BENDAYAN, M
本文报道了一种利用酶-金络合物对底物进行超微结构定位的细胞化学技术。用Au颗粒标记RNase A和DNase I [来自牛胰腺]。将获得的RNase-Au和DNase-Au复合物应用于戊二醛固定和Epon包埋组织的薄切片[大鼠和小鼠胰腺和肝脏]。不同的细胞室标记这些酶-金络合物。使用RNase-Au复合物，粗面内质网出现Au颗粒装饰。Au标记也出现在细胞核上，尤其是核仁上;线粒体标记较弱。使用DNA酶-Au复合物，将Au颗粒浓缩在细胞核和线粒体的常染色质上。异染色质和核仁显示出较弱的标记。对于这两种酶-Au复合物，高尔基体区，分泌颗粒和细胞外空间出现免费的标签。在那些对照条件下，底物被添加到酶-金复合物中，观察到标记的主要减少。对标签进行了定量评价，证实了定性观察结果和在对照条件下发生的标签显著减少。酶-底物相互作用的特异性与Au颗粒的大小和电子密度以及组织的良好超微结构保存的组合导致具有高分辨率的非常特异性的标记。这些结果表明，通过酶-Au络合物在EM水平上检测底物的可能性。
A cytochemical technique for the ultrastructural localization of substrates using enzyme-Au complexes is reported. RNase A and DNase I [ from bovine pancreas] were labeled with Au particles. The RNase-Au and DNase-Au complexes obtained were applied on thin sections [rat and mouse pancreas and liver] of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-Au complexes. Using the RNase-Au complex the rough endoplasmic reticulum appeared decorated with Au particles. The Au marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-Au complex, Au particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-Au complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-Au complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed, it confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the Au particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-Au complexes at the EM level.