CHARACTERIZATION OF THE ACROSIN ACTIVE SITE

顶体蛋白（E.C. 3.4.21.10）是一种精子蛋白酶， 前体（前顶体酶）被发现在更大的浓度比所有 精子蛋白水解酶系统的其他成分。 的 顶体蛋白酶原系统非常重要，因为精子必须 具有酶活性的顶体酶以便能够受精 卵。 这项拨款提案的重点是亲和标记，纯化， 序列，并合成顶体酶的活性位点区域。 的 第一个具体的目标将利用公猪精子集中在 部分纯化，并将顶体酶原自动活化为顶体酶 在活性位点亲和标记试剂的存在下。 的 将所得标记的顶体酶进一步纯化，并进行 酶水解 肽将被分离，标记的 鉴定区域，并确定其氨基酸序列。 第二个具体目标的目标是， 顶体酶活性位点区域。 第三个具体目标 重点介绍了活性位点区域序列的应用。 这些 序列将相互比较，并与其他胰蛋白酶进行比较， 比如酶 这两个物种的活性位点区域将是 合成了 一个兔子系统将被用来开发高度 纯化的针对合成肽的多克隆抗体。 将进行实验以确定抗体是否是 能够抑制蛋白水解活性，并观察它们是否会抑制 受精 后者将包括无透明带仓鼠 卵试验以及猪体外受精系统。 这 将澄清抗体作为男性的潜在作用， 避孕药 这些抗体也将用于开发 顶体酶的酶样免疫吸附测定，和 荧光标记的抗兔IgG将用于定位 兔IgG-顶体酶复合物，以跟踪 精子获能过程中的顶体酶和顶体 反应 该项目的独特优势是：1）它允许一个强烈的 积极的临床医生有机会追求一个严肃的学术 2）它将应用新技术，使人们能够 确定顶体酶分子的活性位点序列，3）它 将提供一个基础，在此基础上调查的潜在用途， 具有男性避孕作用的活性位点序列 剂

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.