High-molecular-weight human epidermal transglutaminase.
High-molecular-weight human epidermal transglutaminase.
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高分子量人表皮转谷氨酰胺酶。
DOI：
10.1111/1523-1747.ep12275357
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Goldsmith,LA
中科院分区：
文献类型：
--
作者：
Negi,M;Colbert,MC;Goldsmith,LA
在含有 EDTA 和苯甲磺酰氟的 Tris-HCl 中提取人角质层，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离，转印到硝酸纤维素纸上，并与兔抗人表皮转谷氨酰胺酶（ETG）抗体反应。通过多步过氧化物酶程序检测蛋白质结合的抗体。当使用抗-ETG时，分子量为50,000 (50kDa)和72,000道尔顿(72kDa)的蛋白质被染色，而当使用单独的第二抗体或来自未免疫动物的血清时则不会被染色。当ETG用胰蛋白酶或有机溶剂处理时，50kDa ETC条带的迁移率没有改变，但72kDa条带完全消失。结合 72kDa 蛋白的抗体，当从印迹中洗脱时，与 50kDa 和 72kDa 蛋白均发生反应；类似地，与 50kDa 蛋白质结合的抗体，当从印迹中洗脱时，会与 50kDa 和 72kDa 蛋白质发生反应。在钙和二硫苏糖醇存在下于 56°C 加热或用胰蛋白酶处理后，部分纯化的 72kDa ETG 活性增加（对照水平的 3 至 16 倍）。这些研究与之前关于 ETG 激活的研究相结合，表明 ETG 存在两种形式。不同的形式可能在调节酶活性方面发挥作用。
Human stratum corneum was extracted in Tris-HCl containing EDTA and phenylmethylsulfonyl fluoride, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transblotted to nitrocellulose papers and reacted with rabbit antihuman epidermal transglutaminase (ETG) antibody. Protein-bound antibody was detected with a multistep peroxidase procedure. Proteins with a molecular weight of 50,000 (50kDa) and 72,000 daltons (72kDa) were stained when anti-ETG Was used and not when second antibody alone or sera from nonimmunized animals were used. When ETG was treated with trypsin or organic solvents, there was no alteration in the mobility of the 50kDa ETC band, but there was complete disappearance of the 72kDa band. Antibody that bound 72kDa protein, when eluted from the blot, reacted with both 50kDa and 72kDa proteins; similarly, antibody that bound to the 50kDa protein, when eluted from the blot, reacted with both the 50kDa and 72kDa proteins. Partially purified 72kDa ETG activity was increased (3 to 16 times control levels) after heating at 56°C in the presence of calcium and dithiothreitol or by treatment with trypsin. These studies, in conjunction with the previous studies of ETG activation, are consistent with there being two forms of ETG. The different forms may play a role in regulating enzyme activity.