Establishment of a quantitative method for determining cyclic ADP-ribose concentration

最近，NAD^+的代谢产物环ADP-核糖被证明是细胞内Ca^2+动员的第二信使，并在胰腺β细胞分泌胰岛素中起重要作用。因此，需要一种定量测定环ADP-核糖浓度的方法来研究哺乳动物细胞中环ADP-核糖的生理功能。本课题的目的是建立一种灵敏的测定ADP-核糖环化酶的方法，本课题的主要内容如下：（1）从黑田氏圆藻中分离纯化了ADP-核糖环化酶，该酶能将NAD^+转化为ADP-核糖。(2)We使用纯化的Aestasia ADP-核糖基环化酶从NAD^+合成环状ADP-核糖，并通过HPLC纯化环状ADP-核糖。（3）以ADP-核糖环化酶合成的环状ADP-核糖为标准品，建立了一种通过监测哺乳动物细胞（如大鼠小脑）释放Ca^2+来定量测定环状ADP-核糖浓度的方法 ...更多信息 UM或胰岛细胞）微粒体。(4)We发现在微粒体中加入钙调素可敏化并激活ADP核糖介导的Ca^2+释放。(5)By将钙调素加入到无细胞的环ADP-核糖测定体系中，建立了一种灵敏的测定环ADP-核糖浓度的方法。(6)We（7）我们发现在葡萄糖刺激下，胰岛细胞产生环状ADP-核糖。(8)We已经报道的人白细胞抗原CD 38具有ADP-核糖基环化酶和环ADP-核糖水解酶活性。（9）此外，我们发现葡萄糖代谢过程中产生的ATP抑制环ADP-核糖水解酶的活性，导致环ADP-核糖的形成增加。（10）我们产生了在胰岛β细胞中过表达CD 38的转基因小鼠，并获得了CD 38在胰腺β细胞中通过葡萄糖对胰岛素分泌具有调节作用的第一个证据。少

Recently, cyclic ADP-ribose, a metabolite from NAD^+, has been shown to be a second messenger for intracellular Ca^<2+> mobilization and to play an essential role in insulin secretion from pancreatic beta-cells. Thus, a quantitative method for determining cyclic ADP-ribose concentration has been required for studying of physiological functions of cyclic ADP-ribose in mammalian cells. The aim of this project was to establish a sensitive method for determining cyclic ADP-ribose concentration.In this project,(1)We have purified ADP-ribosyl cyclase, which converts NAD^+ to cyclic ADP-ribose, from Aplysia kurodai.(2)We synthesized cyclic ADP-ribose from NAD^+ using the purified Aplysia ADP-ribosyl cyclase and purified cyclic ADP-ribose by HPLC.(3)Using cyclic ADP-ribose synthesized by Aplysia ADP-ribosyl cyclase as a standard, we have developed a quantitative method for determining cyclic ADP-ribose concentration by monitoring the release of Ca^<2+> from mammalian cell (such as rat cerebell … More um or islet cells) microsomes in a cell free system.(4)We found that the addition of calmodulin to microsomes sensitized and activated the cyclic ADP-ribose-mediated Ca^<2+> release.(5)By addition of calmodulin to the cell free cyclic ADP-ribose assay system, we have established a sensitive method for determining cyclic ADP-ribose concentration.(6)We have also developed a method for preparing the cell extract, in which the cyclic ADP-ribose concentration is determined.Using above cyclic ADP-ribose assay system,(7)We found that cyclic ADP-ribose is generated in pancreatic islets by glucose stimulation.(8)We showed that CD38, which has been reported to be a human leukocyte antigen, has both ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities.(9)Moreover, we found that ATP,generated in the process of glucose metabolism, inhibits the cyclic ADP-ribose hydrolase activity, resulting in the increased formation of cyclic ADP-ribose.(10)We produced transgenic mice overexpressing CD38 in islet beta-cells and obtained the first evidance that CD38 has a regulatory role in insulin secretion by glucose in pancreatic beta-cells. Less