Lajemi M, Demignot S, Borge L, Thenet-Gauci S, Adolphe M
Laboratoire de Pharmacologie Cellulaire de l'Ecole Pratique des Hautes Etudes, Centre de Recherches Biomédicales des Cordeliers, Paris, France.
Histochem J. 1997 Aug;29(8):593-606. doi: 10.1023/a:1026484227851.
The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase in living cells was studied. Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce cytotoxicity when used at 0.5 mM in culture media and diffused throughout the cell. After appropriate fixation using methanol, Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase. Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into proteins strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co-distributed with Fluoresceincadaverine-labelled structures, which resembled a lattice. Fluoresceincadaverine-labelled proteins detected by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled lattice. This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active form in this compartment. This opportunity to perform morphological and biochemical analyses in the search for transglutaminase substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth.
研究了使用荧光尸胺作为伯胺供体来检测活细胞中活性转谷氨酰胺酶的内源性底物。发现荧光尸胺适用于标记培养中的细胞，因为当在培养基中以0.5 mM使用时它不会诱导细胞毒性，并且能扩散到整个细胞中。在使用甲醇进行适当固定后，通过直接荧光显微镜观察荧光尸胺标记的细胞，从而可以看到活性转谷氨酰胺酶的底物。同时检测转谷氨酰胺酶和掺入蛋白质中的荧光尸胺强烈表明，胞质转谷氨酰胺酶在这些活细胞中是无活性的。然而，转谷氨酰胺酶与类似晶格的荧光尸胺标记结构共分布。使用抗荧光素抗体通过蛋白质印迹法检测到的荧光尸胺标记蛋白质表明，在活细胞中，主要的转谷氨酰胺酶底物迁移的表观分子量为220 kDa，纤连蛋白也是如此。发现纤连蛋白与荧光尸胺标记的晶格共分布。这证实了这些晶格结构是细胞外的，因此，转谷氨酰胺酶在这个区室中处于活性形式。在活细胞中寻找转谷氨酰胺酶底物时进行形态学和生化分析的这个机会，应该有助于确定转谷氨酰胺酶在特定细胞类型以及普遍细胞事件（如细胞凋亡或细胞生长）中的特定功能。
