Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry.
Localization of a proteolytic enzyme within the efferent and deferent duct epithelial cells of the turkey (Meleagris gallopavo) using immunohistochemistry.
复制标题
使用免疫组织化学法对火鸡（Meleagris gallopavo）传出管和输精管上皮细胞内的蛋白水解酶进行定位。
DOI：
10.1095/biolreprod67.1.276
复制
发表时间：
2002
影响因子：
3.6
通讯作者：
Thurston,RJ
中科院分区：
文献类型：
--
作者：
Holsberger,DR;Rice,CD;Thurston,RJ
火鸡精浆含有一种与精子顶体蛋白不同的丝氨酸蛋白酶。然而，这种酶的起源和生物学作用尚不清楚。我们的实验目的是确定男性生殖道内这种蛋白酶的细胞来源。使用苯甲脒-Sepharose 6B 色谱法从精浆中分离出酶。使用合成底物 Nα-苯甲酰基-dl-精氨酸对硝基苯胺来检测含有该酶的级分。亲和层析技术使酰胺酶活性提高了 150 倍。通过 SDS-PAGE 分析蛋白酶，显示两条相对分子质量分别为 37 000 和 61 000 的蛋白条带。通过酪蛋白消化证明，在较小的条带内检测到了蛋白水解活性。对纯化蛋白质的进一步分析表明，较小的蛋白质条带被糖基化。为了确定蛋白酶的细胞来源，针对纯化的蛋白酶开发了一组小鼠单克隆抗体，并用于免疫组织化学。将肝脏、睾丸、附睾区域和输精管的冷冻组织切片固定在 4% (w/v) 多聚甲醛中，用 0.2% (v/v)（辛基苯氧基）聚乙氧基乙醇透化，然后进行常规免疫组织化学操作。单克隆抗体不与肝脏或睾丸的组织切片或血浆蛋白结合。传出管和输精管的远端部分均呈免疫反应性。我们得出的结论是，火鸡精浆中发现的蛋白酶集中于远端传出管和输精管上皮细胞。
Turkey seminal plasma contains a serine protease found to be distinct from the spermatozoal acrosin. However, the origin and biological roles of this enzyme are unknown. Our experimental objective was to identify the cellular source of this protease within the male reproductive tract. The enzyme was isolated from seminal plasma using benzamidine-Sepharose 6B chromatography. A synthetic substrate, Nα-benzoyl-dl-argininep-nitroanilide, was used to detect fractions containing the enzyme. The affinity chromatography technique yielded a 150-fold increase in amidase activity. Analysis of the protease by SDS-PAGE revealed two protein bands with relative molecular masses of 37 000 and 61 000. Proteolytic activity was detected within the smaller band as evidenced by casein digestion. Further analysis of the purified protein revealed that the smaller protein band was glycosylated. To determine the cellular source of the protease, a panel of mouse monoclonal antibodies was then developed against the purified protease, and used in immunohistochemistry. Frozen tissue sections from the liver, testis, epididymal region, and deferent duct were fixed in 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) (octylphenoxy)polyethoxyethanol followed by routine immunohistochemistry procedures. Monoclonal antibodies did not bind to tissue sections from either the liver or testis, or to blood plasma proteins. Both the distal portion of the efferent duct and the deferent duct were immunoreactive. We concluded that the protease found in turkey seminal plasma is concentrated to the distal efferent duct and the deferent duct epithelial cells.