LYSOSOMAL PROTEASES OF SPERMATOZOA

精子的溶酶体蛋白酶

基本信息

批准号：
2199292
负责人：
JOHN K MC DONALD
金额：
$ 10.82万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-30 至 1995-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2199292
关键词：
acrosin acrosome cystine endopeptidases enzyme inhibitors enzyme mechanism enzyme substrate fluorescence spectrometry gel filtration chromatography guinea pigs high performance liquid chromatography ion exchange chromatography lysosomes protein purification sperm spermatogenesis

项目摘要

The long term objectives of this application are intended to establish the biochemical properties and functional significance of new lysosomal-type protease (endopeptidases and exopeptidases) recently detected in guinea pig epididymal spermatozoa. One of the endopeptidases resembles lysosomal cathepsin L, but is totally masked in epididymal sperm. A second cysteine proteinase (which may be responsible for unmasking "cathepsin L") has also been detected. This endopeptidase shares some properties with cathepsin S -- a poorly studied lysosomal cysteine proteinase thus far found only in lymphatic tissues. These two cathepsins are uniquely and selectively concentrated (to the exclusion of detectable levels of cathepsins B and H) in guinea pig sperm. The principal exopeptidase to be investigated resembles dipeptidyl peptidase II (DPP II), a lysosomal enzyme belonging to the serine catalytic class that is present at extraordinary levels in epididymal sperm. Efforts will be made to purify these proteases from freshly collected (guinea pig) epididymal sperm homogenates for the purpose of establishing their molecular properties, substrate specificity, reaction kinetics, inhibitor sensitivities, and immunological cross reactivity with their lysosomal counterparts. Purification methods will be employed that exploit differential solubilities, molecular size, charge, isoelectric points, hydrophobicity, and special affinities. In view of the limited supply of tissue available for this purification work, high performance adaptations of traditional methodologies will be utilized. These (FPLC) procedures, which are currently being employed in this laboratory, include ion (anion and cation) exchange chromatography, molecular exclusion chromatography, chromatofocusing, and hydrophobic interaction chromatography. Specific fluorometric assays together with azo-dye and fluorescence enzyme histochemical procedures will be employed (or developed) as needed to assay and to localize these "new proteases" during spermatogenesis and sexual development. The possible functional significant of latent cathespin L will be assessed in relation to capacitation, the acrosome reaction, and proacrosin activation. Insights gained from the proposed studies will contribute to a basic understanding of the role of acrosomal proteases in reproduction and facilitate a more rational approach to problems of contraception, unexplained, infertility, and in vitro fertilization.

本应用程序的长期目标旨在建立新溶酶体类型的生化特性和功能意义蛋白酶（内肽酶和外肽酶）最近在豚鼠中检测到附子精子。内肽酶之一类似于溶酶体组织蛋白酶L，但在附睾精子中被完全掩盖。第二个半胱氨酸蛋白酶（可能负责揭露“组织蛋白酶L”）也已有被检测到。该内肽酶与组织蛋白酶有一些特性 - 到目前为止，研究良好的溶酶体半胱氨酸蛋白酶仅在淋巴组织。这两个组织蛋白蛋白是独特的，有选择性的集中（排除可检测水平的组织蛋白酶B和H）在豚鼠精子中。要研究的主要外肽酶类似于二肽基肽酶II（DPP II），一种属于的溶酶体酶丝氨酸催化类以非凡的水平存在附子精子。将努力从中净化这些蛋白酶新鲜收集的（豚鼠）附睾精子匀浆为此目的建立其分子特性，底物特异性，反应动力学，抑制剂敏感性和免疫交叉反应性与他们的溶酶体对应物。将采用纯化方法利用差异溶解度，分子大小，电荷，等电点，疏水性和特殊亲和力。鉴于有限可用于此净化工作的组织供应，高性能将利用传统方法的适应。这些（FPLC）目前正在本实验室使用的程序包括离子（阴离子和阳离子）交换色谱，分子排除色谱，色谱和疏水相互作用色谱法。特定的荧光测定与偶氮-DYE和将采用荧光酶组织化学程序（或根据需要开发的），以分析和本地化这些“新蛋白酶” 精子发生和性发展。可能的功能意义潜在的ctastespin l将根据电容进行评估 ACROSOM反应和前乳糖素激活。从中获得的见解拟议的研究将有助于基本了解在繁殖方面的Arosomal蛋白酶，并促进了更合理的方法避孕问题，无法解释，不育和体外受精。