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

为了了解PolyADP-核糖化的生理功能，鉴定PolyADP-核糖化的受体蛋白是绝对必要的。我们制备并大量制备了抗多聚ADP-核糖的单抗，并对其进行了纯化，用于亲和层析。在实验过程中，我们注意到提取的多聚ADP核糖化蛋白没有与亲和柱结合，这表明还有其他与多ADP核糖结合的蛋白质抑制了多ADP核糖化蛋白与柱的结合。为了解决这些问题，我们以人肾细胞系(293T)为细胞裂解液，在体外合成了多聚ADP核糖化蛋白，并考察了提取多ADP核糖化蛋白的理想条件。结果表明，提取缓冲液中应含有0.1%的十二烷基硫酸钠，提取效果较好。对于缺乏聚腺苷二磷酸核糖水解酶且体内有聚腺苷二磷酸核糖化蛋白积累的果蝇突变体，当缓冲液中含有0.5%或更多的十二烷基硫酸钠时，提取聚腺苷二磷酸核糖化蛋白的效率较高。然而，也很明显，PolyADP-核糖化蛋白不与抗体柱结合，推测十二烷基硫酸钠确实抑制了结合。在初步实验中，我们通过添加非离子洗涤剂，在消除游离十二烷基硫酸钠方面取得了一些成功。因此，我们现在正试图提纯多聚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.