DETERMINATION OF UNKNOWN POLY(ADP-RIBOSYL)ATED PROTEINS AND BINDING SITES

为了了解聚ADP核糖基化的生理功能，鉴定聚ADP核糖基化的受体蛋白是绝对必要的。制备了抗聚ADP-核糖的单克隆抗体，并进行了亲和层析纯化。在实验过程中，我们注意到提取的聚ADP-核糖基化蛋白质不与亲和柱结合，这表明存在与聚（ADP-核糖）结合的其他蛋白质，其抑制聚ADP-核糖基化蛋白质与柱的结合。针对这些问题，本研究以人肾细胞系（293 T）为细胞裂解液，体外合成了多聚ADP核糖基化蛋白，并对多聚ADP核糖基化蛋白的理想提取条件进行了考察。结果表明，为了更好地提取，提取缓冲液应含有0.1%的SDS。对于从缺乏聚（ADP-核糖）糖水解酶并且在体内积累了聚ADP-核糖基化蛋白的果蝇突变体中提取聚ADP-核糖基化蛋白，当缓冲液含有0.5%或更高的SDS时，获得了良好的提取效率。然而，聚ADP-核糖基化蛋白不与抗体柱结合也变得明显，推测SDS确实抑制了结合。在初步实验中，我们通过加入非离子洗涤剂，成功地去除了游离SDS。因此，我们现在试图纯化聚ADP核糖基化蛋白。

To understand the physiological function of polyADP-ribosylation, it is absolutely necessary to identify the acceptor proteins of polyADP-ribosylation. We prepared and quantities of monoclonal antibody against poly(ADP-ribose) and purified it for affinity chromatography. During the course of experiments, we noticed that the extracted polyADP-ribosylated proteins did not bind to the affinity column suggesting that there are other proteins binding to poly(ADP-ribose) that inhibit the binding of polyADP-ribosylated proteins to the column. To solve these problems, we used human kidney cell line (293T) as cell lysate and synthesized polyADP-ribosylated proteins in vitro and checked the ideal condition of extraction of polyADP-ribosylated proteins. It turned out that extraction buffer should contain 0.1% SDS for better extraction. For extracting the polyADP-ribosylated proteins from Drosophila mutant that lacks poly(ADP-ribose) glycohydrolase and have accumulated polyADP-ribosylated proteins in vivo, good extraction efficiency was obtained when the buffer contains 0.5% or more SDS. However it became also evident that polyADP-ribosylated proteins do not bind to the antibody column, presumably SDS did inhibit the binding. We had some success in eliminating the free SDS by adding the nonionic detergent in our preliminary experiments. Thus we are now trying to purify the polyADP-ribosylated proteins.