CHARACTERIZATION OF THE ACROSIN ACTIVE SITE

顶体酶(E.C.3.4.21.10)是一种精子蛋白酶和顶体酶。 前体(顶体酶原)的浓度比所有 精子蛋白水解酶系统的其他成分。这个 顶体酶原系统非常重要，因为精子必须 具有酶活性的顶体酶，以便能够受精 奥瓦。这项拨款提案的重点是亲和标签，纯化， 测序，并合成顶体酶活性部位区域。这个 第一个特定目标将利用公猪精子集中在 原顶体酶的部分纯化和自身激活为顶体酶 在活性部位亲和标记剂存在的情况下。这个 合成的标记顶体酶将被进一步纯化，并受到 酶解法。多肽将被分离，标记的 鉴定了这些区域，并测定了它们的氨基酸序列。 第二个具体目标的目标将是描述 人体内活性部位区域的顶体蛋白。第三个具体目标 重点介绍了活性部位区域序列的应用。这些 序列将相互比较，并与其他胰酶进行比较- 就像酶一样。这两个物种的活跃部位区域将是 合成的。将使用兔子系统来高度发展 合成多肽的纯化多克隆抗体。 将进行实验以确定抗体是否 能够抑制蛋白分解活性，并观察它们是否会抑制 受精。后者将包括去透明带仓鼠 卵子试验以及猪的体外受精系统。这 将阐明抗体作为男性的潜在作用 避孕药。抗体也将被用来开发 一种类似酶的免疫吸附测定顶体酶，以及一种 荧光标记的抗兔免疫球蛋白将用于定位 兔免疫球蛋白-顶体酶复合体的位置发生变化 精子获能过程中的顶体酶和顶体 反应。 这个项目的独特优点是：1)它允许一个强有力的 激励临床医生有机会追求严肃的学术 职业生涯，2)它将应用新技术，使人们能够 鉴定顶体酶分子的活性部位序列，3)它 将提供一个基础，用于调查 包括作为男性避孕药的作用的活性部位序列 探员。

Acrosin (E.C. 3.4.21.10) is a sperm protease and the acrosin precursor (proacrosin) is found in greater concentrations than all other components of the sperm proteolytic enzyme system. The proacrosinacrosin system is of major importance since sperm must have enzymatically active acrosin in order to be able to fertilize ova. The focus of this grant proposal is to affinity label, purify, sequence, and synthesize the active site region of acrosin. The first specific aim will utilize boar sperm to concentrate on the partial purification, and auto-activation of proacrosin to acrosin in the presence of an active site affinity labeling reagent. The resultant labeled acrosin will be further purified, and subjected to enzymatic hydrolysis. The peptides will be separated, the labeled regions identified, and their amino-acid sequence determined. The goal of the second specific aim will be to characterize the acrosin active site region in the human. The third specific aim focuses on applications of the active site region sequences. These sequences will be compared to each other and to other trypsin- like enzymes. The active site regions of both species will be synthesized. A rabbit system will be used to develop highly purified polyclonal antibodies to the synthesized peptides. Experiments will be performed to determine if the antibodies are able to inhibit proteolytic activity, and to see if they will inhibit fertilization. The latter will include both the zona-free hamster egg test as well as a porcine in vitro fertilization system. This would clarify the potential role of the antibodies as a male contraceptive agent. The antibodies will also be used to develop an enzyme-liked immunosorbent assay for acrosin, and a fluorescent labeled anti-rabbit IgG will be used to localize the rabbit IgG-acrosin complex to follow changes in the location of acrosin in the sperm during capacitation and the acrosome reaction. The unique strengths of this project are: 1) It allows a strongly motivated clinician the opportunity to pursue a serious academic career, 2) It will apply new technology which will enable one to identify the active site sequence of the acrosin molecule, 3) It will provide a basis on which to investigate potential uses of the active site sequence including a role as a male contraceptive agent.