REGULATION OF SPERM PROACROSIN CONVERSION TO ACROSIN

这项更新提案的主要目标是充分 表征顶体酶原-顶体酶系统， 可以获得有关其管理的信息。 两 将利用生物化学和生理学方法。 的 生物化学表征将首先集中在确定 猪顶体蛋白原的氨基酸序列，并在定义 顶体酶活性位点的拓扑结构。 后一项研究 将通过使用亲和标记试剂来完成， 确定哪些特定的氨基酸参与顶体酶的 酶活性 第二个目标是描述 猪睾丸和附睾精子中发现的顶体酶原的前体。 这样做是为了彻底了解新生的 前顶体酶，使系统可以定量和调节 他的转变被阐明了。 这些研究将涉及 前体纯化和表征以及体外 猪睾丸顶体酶原mRNA的翻译。 生理 表征将集中在顶体蛋白原的调节上- 精子顶体酶系统。 这将通过 确定了特定抑制剂和 顶体酶原和顶体酶的抗体对精子的能力 穿透卵母细胞 这些结果将通过 动力学酶分析和受影响的特定形式将 通过高灵敏度的明胶-SDS-PAGE酶谱仪鉴定。 出于可能的临床和避孕目的， 该系统将利用人类精子，然后分析它们的插入情况， 没有透明带的仓鼠卵 更深入的方法将侧重于 充分表征的猪精子顶体蛋白原-顶体蛋白酶系统， 公猪精子进行体外获能的能力， 猪卵母细胞体外受精 本研究将 导致对分子事件的更完整的理解 受精所需的，并最终可能导致可能的 避孕或生育的控制手段 增强的目的。 此外，更深入的理解 这种蛋白酶系统的调节机制会产生 增加对其他可能的管制的了解 蛋白水解酶系统控制着许多重要的生物 流程.

The primary goal of this renewal proposal is to sufficiently characterize the proacrosin-acrosin system so that definitive information regarding its regulation can be obtained. Both biochemical and physiological approaches will be utilized. The biochemical characterization will first focus on the determination of the amino acid sequence of boar proacrosin and in defining the topography of the active site of acrosin. These latter studies will be accomplished by using affinity labeling reagents to determine which specific amino acids are involved in acrosin's enzymatic activity. The second aim will be to characterize the precursors to proacrosin found in testis and epididymal boar sperm. This will be done to obtain a thorough understanding of nascent proacrosin so that the system can be quantitated and the regulation of its conversion elucidated. These studies will involve the precursor purification and characterization as well as the in vitro translation of boar testicular proacrosin mRNA. The physiological characterization will focus on the regulation of the proacrosin- acrosin system in live sperm. This will be accomplished by determined the effectiveness that specific inhibitors and antibodies to proacrosin and acrosin have on the sperm's ability to penetrate oocytes. These results will be quantitated by a kinetic enzyme analysis and specific forms that are affected will be identified by a highly sensitive gelatin-SDS-PAGE zymograph. For possible clinical and contraceptive purposes one of the test systems will utilize human sperm and then analyze their penetration of zona-free hamster eggs. A more in depth approach will focus on the well-characterized boar sperm proacrosin-acrosin system and the boar sperm's ability to undergo in vitro capacitation and in vitro fertilization of zona intact porcine ova. This study will result in a more complete understanding of the molecular events required for fertilization and could ultimately lead to possible means of controlling it for either contraceptive or fertility enhancement purposes. In addition, a more thorough comprehension of the regulatory mechanisms of this proteinase system will produce increased understanding of the possible regulation of other proteolytic enzyme systems which control many important biological processes.