de Curtis I, Fumagalli G, Borgese N
J Cell Biol. 1986 May;102(5):1813-25. doi: 10.1083/jcb.102.5.1813.
Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.
通过在适度低渗介质中短暂超声处理，从射精公牛精子中分离出质膜，释放的质膜通过差速离心进行部分纯化。与起始超声处理的精子相比，所得组分的碱性磷酸酶和5'核苷酸酶活性分别富集了8倍和15倍。通过在连续线性蔗糖梯度上进行平衡密度离心，将该总质膜组分分离为两个不同的组分。在密度为1.117和1.148 g/ml处形成了两个光散射物质峰。密度较大的峰包含质膜组分的大部分蛋白质，而几乎所有的伴刀豆球蛋白A结合活性都在较轻的峰中发现。通过SDS-PAGE分析时，这两条带具有明显不同的多肽组成。用兔制备针对每个组分的主要整合膜糖蛋白的多克隆抗体（轻峰中为92,000，重峰中为98,000）。通过间接免疫荧光显微镜在完整精子表面检测到这两种抗原。仅在头部顶体和顶体后前部区域的质膜上检测到92-kD蛋白（存在于较轻的条带中）。存在于较重条带中的98-kD抗原定位于头部顶体后区域的表面、尾部的主段以及头部和尾部之间的连接段。免疫电子显微镜证实了92-kD多肽在头部前部表面的唯一定位。这些数据表明，在蔗糖梯度上分离的两个组分源自精子细胞质膜的不同区域。
