CALCIUM REGULATION SYSTEMS IN SEA URCHIN EGGS

细胞内钙离子的动员在许多细胞中起着重要的作用 流程。人们普遍认为，三磷酸肌醇是一种 转导表面受体激活的第二信使 体内Ca+2的动员。肌醇三磷酸可能不是 最近的几项研究表明，只有Ca+2信使。我们有 在海胆卵中发现了一种钙动员系统，它完全是 不依赖于肌醇三磷酸途径。这个系统是 被NAD+的代谢产物激活，我们称之为环状ADP-核糖(CADPR)。 我们已有的证据表明，cADPR可能是一种内源性调节因子 钙离子诱导细胞内钙离子释放的机制。详细描述了 Cadpr系统应提供有用的信息，帮助理解 细胞内钙离子动员的基本机制。 有人建议进行研究，以阐明cadpr的生理作用。 首先，通过确定cADPR是否增加了钙离子敏感性 完整鸡蛋和匀浆中的钙诱导-钙释放系统， 第二，测量从受精到受精的卵子中内源cADPR的水平 第三，开发特定的抑制剂来阻断cADPR 系统，并评估由此产生的细胞功能变化。我们会 设计方法来纯化ADP的可溶性形式和膜结合形式- 核糖环化酶，cADPR合成酶，并产生特异的 针对它们的抗体。CADPR的结构和可溶性ADP-核糖基 将用X射线结晶学方法测定海兔卵巢中的环化酶。 CADPR将被化学修饰，以产生激动剂、拮抗剂和 代谢物的免疫原。CADPR的降解酶是 特色化的。我们将开发放射性标记的方法 CADPR的微粒体受体。一般适用的程序 我们会在细胞内引入cadpr，以方便进行评估。 Cadpr系统在细胞中有多普遍。我们将描述 鸡蛋匀浆中还有另一个独立的钙释放系统，即 被NADP的一种衍生物激活。

Mobilization of intracellular Ca+2 plays an important role in many cellular processes. It is generally accepted that inositol trisphosphate is a second messenger for transducing surface receptor activation to mobilization of internal Ca+2. That inositol trisphosphate may not be the only Ca+2 messenger as suggested by several recent studies. We have identified a Ca+2 mobilization system in sea urchin eggs which is totally independent of the inositol trisphosphate pathway. This system is activated by a metabolite of NAD+ we named cyclic ADP-ribose (cADPR). Evidence we have suggests that cADPR may be an endogenous regulator of the Ca+2-induced-Ca+2 release mechanism in cells. Detailed characterization of the cADPR system should provide useful information toward understanding the basic mechanisms involved in mobilization of cellular Ca+2. Research is proposed to elucidate the physiological role of the cADPR system by, first, determining if cADPR increases the Ca+2 sensitivity of the Ca+2-induced-Ca+2 release system in intact eggs and homogenates, second, measuring the endogenous cADPR levels in eggs from fertilization to cleavage and, third, developing specific inhibitors to block the cADPR system and assessing the resultant changes in cell functions. We will devise methods to purify both the soluble and membrane bound forms of ADP- ribosyl cyclase, the cADPR synthesizing enzyme, and generate specific antibodies against them. Structures of cADPR and the soluble ADP-ribosyl cyclase from Aplysia ovotestis will be determined by X-ray crystallography. cADPR will be chemically modified to produce agonists, antagonists and immunogens of the metabolite. The degradation enzyme of cADPR will be characterized. We will develop methods to radioactively label the microsomal receptor for cADPR. Generally applicable procedures for introducing cADPR into cells will be devised to facilitate the assessment of how widespread the cADPR system among cells is. We will characterize yet another independent Ca+2 release system in egg homogenates that is activated by a derivative of NADP.