CALCIUM REGULATION SYSTEMS IN SEA URCHIN EGGS

在受精和受精过程中发生多个细胞内钙瞬变 海胆卵的早期发育。三磷酸肌醇(IP3)具有 已被证明是传递精子-卵子相互作用的第二信使 在地面上动员内部的CA+2商店 受精。负责后来的信使分子(S) 瞬变，无论是不是IP3，都还没有确定。在视野中 其他信使分子可能存在的可能性 开发了一种使用鸡蛋匀浆的无细胞系统来筛选 潜在的钙释放激活剂的数量。有了这个化验系统，我们 鉴定出一种新的具有钙动员活性的NAD代谢物 和IP3一样强大。结构确定确定了 代谢产物是环化的ADP-核糖，我们提出了环化ADP-核糖的概念。 核糖(CADPR)作为它的描述性通用名称。Cadpr不仅仅是 在体外是活跃的，但在显微注射时也会引起皮质反应 放进鸡蛋里。负责合成cADPR的酶似乎 普遍存在，因为它不仅存在于海胆卵提取液中，而且 在哺乳动物组织提取物中也是如此。 我建议在下一步研究cadpr的生理作用。 通过体内时间进程的测量和比较获得授权期 从受精到第一次卵裂的cADPR和IP3的产生 时间进程与细胞内钙离子变化的相关性。一个IP 3- 受体阻滞剂肝素将被显微注射到受精卵中 确定是否有任何Ca+2瞬变不是由IP3介导的。我们会 通过纯化负责的酶来表征cADPR系统 合成并降解它，就会产生抗体来对抗它们。 Ca+2储存在对cADPR AS敏感的蛋匀浆中 以及对IP3和NADP敏感的那些将被分开 密度梯度离心法及其标记酶鉴定 分布和受体结合分析。这些研究将是 扩展到其他小区系统，以确定cADPR是否为普通秒 与IP3类似的细胞内钙离子动员信使。

Multiple intracellular Ca+2 transients occur during fertilization and early development of sea urchin eggs. Inositol trisphosphate (IP3) has been shown to be the second messenger transducing sperm-egg interactions at the surface to the mobilization of internal ca+2 stores during fertilization. The messenger molecule(s) responsible for later transients, whether it is IP3 or not, has not been identified. In view of the possibility that other messenger molecules may exist we have developed a cell free system using egg homogenates for screening a large number of potential Ca+2 release activators. With this assay system, we have identified a novel metabolite of NAD with Ca+2 mobilizing activity as potent as IP3. Structural determination established that the metabolite is a cyclized ADP - ribose and we have proposed cyclic ADP- ribose (cADPR) as a descriptive common name for it. cADPR is not only active in vitro but can also induce cortical reaction when microinjected into the egg. The enzyme responsible for synthesizing cADPR appears to be ubiquitous since it is found not only in sea urchin egg extracts but also in mammalian tissue extracts. I propose to investigate the physiological role of cADPR in the next grant period by measuring and comparing the time course of in vivo production of cADPR and IP3 from fertilization to first cleavage and correlating the time course with intracellular Ca+2 changes. An IP 3 - receptor blocker heparin, will be microinjected into fertilized eggs to determine if any of the Ca+2 transients are not mediated by IP3. We will characterize the cADPR system by purifying the enzymes responsible for synthesizing and degrading it and antibodies will be raised against them. The Ca+2 stores in the egg homogenates which are sensitive to cADPR as well as those that are sensitive to IP3 and NADP will be separated by density gradient centrifugation and characterized by marker enzyme distributions and receptor binding assays. These studies will be extended to other cell systems to determine if cADPR is a general second messenger for intracellular Ca+2 mobilization similar to IP3.