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。制备并纯化了抗聚adp核糖单克隆抗体，用于亲和层析。在实验过程中，我们注意到提取的聚adp核糖化蛋白没有与亲和柱结合，这表明还有其他与聚adp核糖结合的蛋白抑制了聚adp核糖化蛋白与亲和柱的结合。为了解决这些问题，我们以人肾细胞系（293T）为细胞裂解液，体外合成了聚adp -核糖基化蛋白，并考察了提取聚adp -核糖基化蛋白的理想条件。结果表明，提取缓冲液中SDS含量为0.1%，提取效果较好。对于缺乏聚（adp -核糖）糖水解酶且在体内积累了聚adp -核糖化蛋白的果蝇突变体，当缓冲液中SDS含量在0.5%及以上时，提取效率较好。然而，polyadp -核糖基化的蛋白不与抗体柱结合也变得很明显，可能SDS确实抑制了这种结合。在初步实验中，我们通过加入非离子型洗涤剂，成功地消除了游离SDS。因此，我们现在正试图纯化聚adp核糖化蛋白。

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.