Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa.
Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa.
复制标题
与牛精子顶体外膜相关的大分子复合物的分离和表征。
DOI：
10.1095/biolreprod33.3.761
复制
发表时间：
1985
影响因子：
3.6
通讯作者：
Noland,TD
中科院分区：
文献类型：
--
作者：
Olson,GE;Winfrey,VP;Garbers,DL;Noland,TD
哺乳动物精子的顶体膜分为不同结构和功能的区域。顶体和主节的顶体外膜是参与顶体反应膜融合事件的唯一结构域，但该功能的分子基础尚未解决。在之前对牛精子的研究中，我们注意到顶体外膜的一个独特的结构特征是其管腔表面上有一层电子致密材料的粘附层（ES Surface，Branton 等，1975）。在这项研究中，我们报告了这种材料的分离，并描述了其结构和生化特征。用1% Triton X-100提取附睾精子尾部，溶解细胞质和膜成分；洗涤剂处理溶解了顶体外膜，但不溶解其粘附的电子致密复合物。均质化从精子中释放出这种复合物，然后通过 Percoll 密度梯度离心将其分解为均质部分。该级分的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳揭示了一系列多肽，包括 290 kDa、280 kDa、260 kDa、115 kDa、81 kDa、58 kDa 和 46 kDa 的成分以及 34-12 kDa 范围内的一系列相关成分。该复合物具有蛋白激酶活性，能够以不依赖于 cAMP 的方式磷酸化特定的内源性多肽。此外，34-12 kDa 家族的几种多肽特异性结合 125 I-钙调蛋白。孤立复合体的一个一致的结构反应是其边缘缠绕成螺旋结构。我们推测这种膜相关组装在顶体反应的膜融合事件中发挥功能性作用。
The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X–100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34–12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34–12 kDa family specifically bind125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.