A STUDY ON MEMBRANES ISOLATED FROM SEA URCHIN GAMETES

海胆质膜Na~+/H~+交换的激活 配子负责触发精子的运动并启动 受精卵的发育。这项工作的目标是理解 交换过程的机理及导致其发生的分子事件 激活。 本实验室的研究表明精子中的Na+/H+交换系统 鞭毛膜对膜电位很敏感，并被关闭 去极化。这种独特的电压门控机制将被描述为 再远一点。交换的动力学问题将得到解决。22Na+/Na+ 将对汇兑进行衡量。浇注机构的对称性 关于膜电势的极性将被确定。这个 交换的Na+结合部位将被探测并标记为 基团特定的化学修饰技术。最后，鞭毛 交换器将被溶解和重新组合。 参与激活鸡蛋中Na+/H+交换的机制将 用从鸡蛋中提取的质膜制剂进行检查 受精卵。由于蛋白激酶C似乎参与其中，该酶将是 提纯和鉴定。激酶C在细胞内的分布 将通过亚细胞分馏来确定。内源性膜 被激酶C磷酸化的蛋白质将被鉴定出来。的影响 磷酸化对Na+/H+交换活性的影响将在分离的 膜泡。 多聚磷脂酰肌醇(PPI)分解在激活过程中的作用 将在体内进行研究。PPI分解的抑制剂新霉素将 被注射到鸡蛋中，这些鸡蛋将被监测对Ca++的影响 通过受精或激活产生的动员和Na+/H+交换 A23187和佛波醇酯。GTP结合蛋白在细胞周期调控中的可能作用 将对PPI明细进行检查。最后，包被囊泡的内吞作用 被研究为一种可能的从 受精卵的质膜。

Activation of a Na+/H+ exchange at the plasma membrane of sea urchin gametes is responsible for triggering motility in sperm and initiating development in fertilized eggs. The goal of this work is to understand the mechanism of the exchange process and the molecular events leading to its activation. Studies from this laboratory indicate the Na+/H+ exchange system in sperm flagellar membranes is sensitive to membrane potential and is turned off by depolarization. This unique voltage-gating mechanism will be characterized further. The kinetics of the exchange will be resolved. The 22Na+/Na+ exchange will be measured. The symmetry of the gating mechanism with respect to the polarity of the membrane potential will be determined. The Na+-binding site of the exchanger will be probed and labeled by group-specific chemical modification techniques. Finally, the flagellar exchanger will be solubilized and reconstituted. Mechanisms involved in the activation of the Na+/H+ exchange in eggs will be examined using plasma membrane preparations derived from eggs and zygotes. Since protein kinase C appears to be involved, the enzyme will be purified and characterized. The intracellular distribution of kinase C will be determined by subcellular fractionation. The endogenous membrane proteins phosphorylated by kinase C will be identified. The effects of phosphorylation on the Na+/H+ exchange activity will be studied in isolated membrane vesicles. The role of polyphosphoinositides (PPI) breakdown in the activation process will be investigated in vivo. Neomycin, an inhibitor of PPI breakdown will be injected into eggs, and these eggs will be monitored for effects on Ca++ mobilization and Na+/H+ exchange produced by fertilization or activation by A23187 and phorbol esters. The possible role of GTP-binding protein in the PPI breakdown will be examined. Finally, coated-vesicle endocytosis will be investigated as a possible means for retrieving the Na+/H+ exchange from the plasma membrane of the zygotes.