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大鼠中巯基丙酮酸硫转移酶的组织和亚细胞分布:共聚焦激光荧光和免疫电子显微镜研究结合生化分析

Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis.

作者信息

Nagahara N, Ito T, Kitamura H, Nishino T

机构信息

Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.

出版信息

Histochem Cell Biol. 1998 Sep;110(3):243-50. doi: 10.1007/s004180050286.

Abstract

In our previous study, we found that mercaptopyruvate sulfurtransferase (MST) was evolutionarily related to mitochondrial rhodanese. To elucidate the difference between MST and rhodanese, the tissue, cellular, and subcellular distribution of rat MST was determined biochemically and immunohistochemically by using anti-MST antibody raised in rabbit. In an immunohistochemical study, tetramethyl rhodamine isothiocyanate-conjugated phalloidin against F-actin and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin as a secondary antibody to the anti-MST antibody were used for double fluorescent staining. They were detected by confocal laser fluorescence microscopy. In the immunoelectron microscopic study of hepatocyte and renal tubular epithelium, a postembedding immunogold method was used. Biochemical studies including western blot analyses of various tissues and subcellular fractions of the liver were also performed. MST was widely distributed in rat tissues but the cellular distribution was found to be different in each tissue. MST was predominantly localized in proximal tubular epithelium in the kidney, pericentral hepatocytes in the liver, cardiac cells in the heart, and neuroglial cells in the brain. This immunocytochemical study also found that MST was localized in both mitochondria and cytoplasm.

摘要

在我们之前的研究中,我们发现巯基丙酮酸硫转移酶(MST)在进化上与线粒体硫氰酸酶相关。为了阐明MST和硫氰酸酶之间的差异,我们使用兔抗MST抗体,通过生化和免疫组织化学方法测定了大鼠MST的组织、细胞和亚细胞分布。在免疫组织化学研究中,异硫氰酸四甲基罗丹明标记的鬼笔环肽用于标记F-肌动蛋白,异硫氰酸荧光素标记的山羊抗兔免疫球蛋白作为抗MST抗体的二抗用于双重荧光染色。通过共聚焦激光荧光显微镜对其进行检测。在肝细胞和肾小管上皮细胞的免疫电子显微镜研究中,采用了包埋后免疫金法。还进行了包括对肝脏各种组织和亚细胞组分进行蛋白质免疫印迹分析在内的生化研究。MST广泛分布于大鼠组织中,但在每个组织中的细胞分布有所不同。MST主要定位于肾脏近端肾小管上皮细胞、肝脏中央周围肝细胞、心脏心肌细胞和大脑神经胶质细胞中。这项免疫细胞化学研究还发现,MST定位于线粒体和细胞质中。

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