Desnoyers L, Thérien I, Manjunath P
Department of Medicine, University of Montreal, Québec, Canada.
Mol Reprod Dev. 1994 Apr;37(4):425-35. doi: 10.1002/mrd.1080370409.
Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.
最近，我们证明了来自牛精浆的主要蛋白质BSP-A1、-A2、-A3和-30 kDa（统称为BSP蛋白）与胆碱磷脂特异性相互作用。这些蛋白质在射精后覆盖精子表面，据信在获能过程中发生的膜修饰中起重要作用。在本研究中，我们测定了BSP蛋白的等电点（pI）并分析了其分子异质性。使用氯胺T对牛精浆（CBSP）中的总蛋白和纯化的BSP蛋白进行碘化。样品经还原、变性，通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）分离，然后通过放射自显影进行可视化。对CBSP蛋白的分析表明，存在迁移在pH值3.5-7.8范围内、分子量（M(r)）在6至100 kDa之间的多肽。当通过2D-PAGE分析纯化的碘化蛋白时，发现了每种BSP蛋白的同工型。发现BSP-A1的M(r)为16.5 kDa，pI范围为4.7-5.0；BSP-A2为16 kDa，pI为4.9-5.2；BSP-A3为15 kDa，pI为4.8-5.2，BSP-30 kDa为28 kDa，pI为3.9-4.6。使用特异性抗体进行蛋白质印迹后对BSP蛋白进行免疫定位也得到了类似结果。用神经氨酸酶处理纯化的碘化BSP蛋白会使BSP-30 kDa的pI增加到4.8-5.0，并使其M(r)降低到25 kDa，但未观察到BSP-A1、-A2和-A3有变化。用硫酸酯酶或酸性磷酸酶处理BSP蛋白，其M(r)和pI均未改变。此外，当在2D-PAGE中分离CBSP蛋白并用丹磺酰肼对凝胶进行糖蛋白染色时，BSP蛋白是牛精浆中发现的主要糖蛋白之一。总之，BSP蛋白呈酸性且有几种同工型。此外，BSP-30 kDa的异质性主要归因于其唾液酸含量。
