The PH-20 protein in cynomolgus macaque spermatozoa: Identification of two different forms exhibiting hyaluronidase activity
The PH-20 protein in cynomolgus macaque spermatozoa: Identification of two different forms exhibiting hyaluronidase activity
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DOI：
10.1006/dbio.1996.0102
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发表时间：
1996-04-10
影响因子：
2.7
通讯作者：
Overstreet, JW
中科院分区：
文献类型：
--
作者：
Cherr, GN;Meyers, SA;Overstreet, JW
在这些实验中，我们的特点是在猕猴精子的双功能精子蛋白PH-20，并研究其透明质酸酶活性。在顶体反应（AR）之前对完整精子进行了评价，并对顶体胞吐过程中释放的可溶性PH-20进行了研究。顶体完整精子提取物的SDS-PAGE的Western印迹显示64 kDa形式的PH-20被兔中产生的针对纯化的重组食蟹猴精子PH-20的多克隆抗体（R-10）识别。在AR期间释放的被R-10抗体识别的可溶性组分包括64-kDa形式和53-kDa形式的PH-20。一个ELISA样的程序，用于确定PH-20透明质酸酶活性表明，顶体完整的精子表现出两个峰值的透明质酸酶活性接近pH 4和大于或等于pH 7。顶体完整精子提取物中的大多数酶活性发生在中性pH值下，而在AR释放的可溶性透明质酸酶活性主要是酸活性。利用透明质酸底物凝胶电泳研究了PH-20在不同pH最适条件下的酶活性，结果表明，64-kDa多肽具有较宽的活性范围，其中大部分活性在中性pH（pH 7）下。精子提取物中的53-kDa多肽仅在酸性pH（pH 4）下有活性，芹菜素可抑制这两种酶的活性。AR期间释放的可溶性PH-20透明质酸酶活性主要为酸活性53-kDa形式。用R-10 IgG的Fab片段对PH-20进行的精细结构定位表明，PH-20不仅与精子膜有关，而且与分散的顶体内容物有关。这些数据表明，PH-20（64 kDa）的中性活性形式存在于血浆和顶体内膜上，并在AR时产生可溶性酸活性形式。可溶形式的PH-20的产生可能是由于顶体酶的作用，所述顶体酶可能包括蛋白酶、糖苷酶和磷脂酶。(C)出版社：Academic Press，Inc.
In these experiments, we have characterized the bifunctional sperm protein PH-20 in macaque sperm and studied its hyaluronidase activity. Intact sperm were evaluated before the acrosome reaction (AR), and a soluble form of PH-20 released during acrosomal exocytosis was also investigated. Western blots of SDS-PAGE of acrosome-intact sperm extracts revealed a 64-kDa form of PH-20 was recognized by a polyclonal antibody (R-10) raised in rabbits against purified, recombinant cynomolgus macaque sperm PH-20. The soluble components released during the AR which were recognized by the R-10 antibody included both the 64-kDa form and a 53-kDa form of PH-20. An ELISA-like procedure for determining PH-20 hyaluronidase activity indicated that acrosome-intact sperm exhibited two peaks of hyaluronidase activity near pH 4 and greater than or equal to pH 7. The majority of enzyme activity in acrosome-intact sperm extracts occurred at neutral pH, while the soluble hyaluronidase activity released at the AR was predominantly acid-active. Hyaluronidase activity of PH-20 at different pH optima was investigated using hyaluronic acid substrate gel electrophoresis, and results indicated that the 64-kDa polypeptide had a broad range, with the majority of activity at neutral pH (pH 7). The 53-kDa polypeptide in sperm extracts only exhibited activity at acid pH (pH 4), The hyaluronidase activities of both enzymes could be inhibited by apigenin. The soluble PH-20 hyaluronidase activity released during the AR was primarily of the acid-active 53-kDa form. fine structural localization of PH-20 using Fab fragments of R-10 IgG demonstrated that PH-20 was associated not only with sperm membranes, but also with the dispersing acrosomal contents. These data suggest that the more neutral-active form of PH-20 (64 kDa) is present on the plasma and inner acrosomal membranes and gives rise to the soluble acid-active form at the time of the AR. The generation of the soluble form of PH-20 may result from the action of acrosomal enzymes, which could include proteases, glycosidases, and phospholipases. (C) 1996 Academic Press, Inc.