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性， 受精卵的发育。 这项工作的目标是了解 交换过程的机制和导致其 activation. 该实验室的研究表明，精子中的Na+/H+交换系统 鞭毛膜对膜电位敏感， 去极化 这种独特的电压门控机制将被表征 进一步. 交换的动力学将得到解决。 22 Na +/Na+ 交换将被衡量。 门控机构的对称性 将确定相对于膜电位的极性。 的 交换剂的Na+结合位点将被探测并标记为 特殊化学修饰技术。 最后，鞭毛 交换剂将被溶解和重构。 卵子中Na+/H+交换的激活机制将 使用来自鸡蛋的质膜制备物进行检查， 受精卵 由于蛋白激酶C似乎参与其中，因此该酶将 纯化和表征。 激酶C的细胞内分布 将通过亚细胞分级分离来确定。 内源性膜 将鉴定被激酶C磷酸化的蛋白质。 的影响 磷酸化对Na+/H+交换活性的影响将在分离的 膜囊泡 多磷酸肌醇（PPI）在活化过程中的作用 将在体内进行研究。 新霉素是PPI分解的抑制剂， 注射到鸡蛋中，并监测这些鸡蛋对Ca++的影响 受精或活化产生的Na+/H+交换 A23187和佛波酯。 GTP结合蛋白在肿瘤发生中的作用 将研究生产者价格指数的分项数字。 最后，包被囊泡内吞作用将 作为一种可能的手段，用于恢复Na+/H+交换， 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.