Studies of three major proteases associated with guinea pig sperm acrosomes.
Studies of three major proteases associated with guinea pig sperm acrosomes.
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与豚鼠精子顶体相关的三种主要蛋白酶的研究。
DOI：
10.1002/jez.1402440212
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Gerton,GL
中科院分区：
文献类型：
--
作者：
Arboleda,CE;Gerton,GL
应用免疫学和电泳技术对豚鼠精子中的主要蛋白酶进行了研究。在含有0.1%明胶的凝胶中，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后检测到三种主要蛋白酶。相对于还原蛋白标准品，这些酶的分子量为47，000、34，000和32，000，相对于未还原标准品，这些酶的分子量为58，500、40，000和37，500。所有三种蛋白酶活性均存在于精子的酸性提取物、精子的洗涤剂提取物以及在与Ca 2 +-离子载体A23187诱导顶体反应后释放的精子的可溶性顶体组分中。通过间接免疫荧光法测定，纯化的猪顶体酶抗体与豚鼠精子的顶体发生特异性交叉反应。荧光减少与精子失去顶体有关。免疫印迹分析表明，该抗体与47，000 Mr蛋白酶反应，但不与32，000和34，000 Mr蛋白酶反应。所有三种蛋白酶在pH 7.0-8.5区域中最大活性，并被顶体酶活性的经典抑制剂抑制。在3小时孵育期间，MgCl 2（10 mM）抑制32，000和34，000 Mr蛋白酶的活性，而47，000 Mr蛋白酶受到刺激。尽管这些蛋白酶具有将其归类为胰蛋白酶样蛋白酶的共同特性，但基于与纯化的猪顶体酶的抗体的交叉反应，仅47，000 Mr蛋白酶可以明确归类为顶体酶原-顶体酶家族的成员。
The major proteases associated with guinea pig sperm were investigated by using immunological and electrophoretic techniques. Three major proteases were detected following sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in gels containing 0.1% gelatin. These enzymes had molecular weights of 47,000, 34,000, and 32,000 relative to reduced protein standards and 58,500, 40,000, and 37,500 relative to unreduced standards. All three protease activities were present in acid extracts of sperm, detergent extracts of sperm, and the soluble acrosomal components of sperm released following induction of the acrosome reaction with the Ca2+‐ionophore A23187. As determined by indirect immunofluorescence, an antibody to purified boar acrosin specifically cross‐reacted with the acrosomes of guinea pig sperm. Decreased fluorescence was associated with sperm that had lost their acrosomes. Immunoblot analysis demonstrated that this antibody reacted with the 47,000 Mrprotease but not the 32,000 and 34,000 Mrproteases. All three proteases were maximally active in the pH 7.0–8.5 region and were inhibited by classical inhibitors of acrosin activity. During a 3‐hour incubation period, MgCl2(10 mM) inhibited the activities of the 32,000 and 34,000 Mrproteases while the 47,000 Mrprotease was stimulated. Although these proteases shared properties that would classify them as trypsin‐like proteases, only the 47,000 Mrprotease could be definitely classified as a member of the proacrosin‐acrosin family based upon cross‐reaction with an antibody to purified boar acrosin.
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DOI：
--
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