Zhu X, Zeng Y, Lehrman M A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1992 May 5;267(13):8895-902.
A cDNA clone isolated from Chinese hamster ovary cells conferred elevated GlcNAc-1-P-transferase (GPT) activity and resistance to tunicamycin in transfected cells (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). It had been assumed that this cDNA, termed TRG for tunicamycin resistance gene, encoded GPT enzyme. However, other functions were not ruled out. Thus, by one of several mechanisms, the TRG protein could have instead functioned by activation of the transfected host's endogenous GPT enzyme. To analyze the biochemical function of the TRG protein, hamster TRG cDNA was stably expressed at high levels in Chinese hamster ovary cells. In addition, several antipeptide polyclonal antibodies directed against the predicted TRG protein were obtained. With these tools in hand, experiments were performed to test the hypothesis that the TRG encodes GPT enzyme, as well as to rule out other possible functions for the TRG protein. These experiments included examination of the effects of solubilization of membranes on TRG-dependent GPT activity, the apparent binding of tunicamycin to the TRG protein, and the immunoadsorption of GPT activity with TRG protein-specific antibodies. From these results, we conclude that the hamster TRG most likely encodes GPT enzyme.
从中国仓鼠卵巢细胞中分离出的一个cDNA克隆，可使转染细胞中的N-乙酰葡糖胺-1-磷酸转移酶（GPT）活性升高，并赋予细胞对衣霉素的抗性（朱，X.，和莱尔曼，M. A.（1990年）《生物化学杂志》265，14250 - 14255）。人们曾认为这个被称为衣霉素抗性基因（TRG）的cDNA编码GPT酶。然而，其他功能并未被排除。因此，通过几种机制中的一种，TRG蛋白可能反而通过激活转染宿主的内源性GPT酶发挥作用。为了分析TRG蛋白的生化功能，仓鼠TRG cDNA在中国仓鼠卵巢细胞中高水平稳定表达。此外，还获得了几种针对预测的TRG蛋白的抗肽多克隆抗体。有了这些工具，就进行了实验来检验TRG编码GPT酶的假设，并排除TRG蛋白的其他可能功能。这些实验包括检测膜溶解对TRG依赖性GPT活性的影响、衣霉素与TRG蛋白的明显结合，以及用TRG蛋白特异性抗体对GPT活性进行免疫吸附。从这些结果中，我们得出结论，仓鼠TRG很可能编码GPT酶。
