Negi M, Colbert M C, Goldsmith L A
J Invest Dermatol. 1985 Jul;85(1):75-8. doi: 10.1111/1523-1747.ep12275357.
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 ETG 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 degrees 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.
人角质层在含有乙二胺四乙酸（EDTA）和苯甲基磺酰氟的三羟甲基氨基甲烷盐酸盐（Tris-HCl）中提取，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上分离，转印至硝酸纤维素纸上，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与蛋白质结合的抗体。当使用抗ETG抗体时，分子量为50,000（50kDa）和72,000道尔顿（72kDa）的蛋白质被染色，而单独使用二抗或未免疫动物的血清时则未被染色。当ETG用胰蛋白酶或有机溶剂处理时，50kDa的ETG条带迁移率没有改变，但72kDa的条带完全消失。从印迹上洗脱的与72kDa蛋白质结合的抗体，与50kDa和72kDa的蛋白质都发生反应；同样，从印迹上洗脱的与50kDa蛋白质结合的抗体，也与50kDa和72kDa的蛋白质都发生反应。在钙和二硫苏糖醇存在下于56℃加热或用胰蛋白酶处理后，部分纯化的72kDa ETG活性增加（为对照水平的3至16倍）。这些研究与之前关于ETG激活的研究一起，表明存在两种形式的ETG。不同形式可能在调节酶活性中起作用。
