Manjunath P, Sairam M R
Biochem J. 1987 Feb 1;241(3):685-92. doi: 10.1042/bj2410685.
Three major acidic proteins of bovine seminal plasma, BSP-A1, BSP-A2 and BSP-A3, were purified to homogeneity, by employing fast protein liquid chromatography, gel filtration and h.p.l.c. The proteins were purified on the basis of their stimulatory effect on the basal release of gonadotropins by rat anterior-pituitary cells in culture. All three proteins migrated as distinct single bands in the presence or absence of 2-mercaptoethanol in SDS/polyacrylamide-gel electrophoresis. Their Mr values were estimated to be between 15,000 and 16,500 by SDS/polyacrylamide-gel electrophoresis. Similar Mr estimates were obtained when they were subjected to gel filtration on a calibrated column of Sephadex G-75 equilibrated in 0.05 M-acetic acid, pH 3.0. However, BSP-A1 and BSP-A2 were eluted as aggregated molecules (Mr 60,000-120,000) during gel filtration on Sephadex G-200 equilibrated in 0.05 M-NH4HCO3, pH 8.5, or phosphate buffer, pH 7.0, containing 0.15 M-NaCl. In the presence of 8 M-urea both BSP-A1 and BSP-A2 were eluted at positions corresponding to Mr values of 17,000-20,000. BSP-A1 and BSP-A2 had an identical amino acid composition, which differed largely from that of BSP-A3. All three proteins contained aspartic acid as the N-terminal residue, and cysteine was identified as the C-terminal residue. BSP-A1 and BSP-A2 are glycoproteins containing galactosamine, sialic acid and neutral sugars, but BSP-A3 did not contain any covalently attached sugars. Whereas BSP-A2 and BSP-A3 were eluted unadsorbed, BSP-A1 bound to wheat-germ lectin-Sepharose 6MB and could be eluted by the competing sugar N-acetyl-D-glucosamine. Treatment of BSP-A1 and BSP-A2 with trypsin resulted in complete loss of gonadotropin-release activity, but BSP-A3 retained full activity. Antibody raised against BSP-A1 did not cross-react with BSP-A3, or vice versa. All these properties indicated marked structural differences between BSP-A3 and BSP-A1 (or BSP-A2). On the basis of amino acid composition it was concluded that BSP-A1, BSP-A2 and BSP-A3 are the same as the gonadostatins [Esch, Ling, Bohlen, Ying & Guillemin (1983) Biochem. Biophys. Res. Commun. 113, 861-867].
利用快速蛋白质液相色谱、凝胶过滤和高效液相色谱法，将牛精浆中的三种主要酸性蛋白BSP-A1、BSP-A2和BSP-A3纯化至同质。这些蛋白质是根据它们对培养的大鼠垂体前叶细胞促性腺激素基础释放的刺激作用进行纯化的。在SDS/聚丙烯酰胺凝胶电泳中，无论有无2-巯基乙醇，这三种蛋白质均迁移为清晰的单一条带。通过SDS/聚丙烯酰胺凝胶电泳估计它们的Mr值在15,000至16,500之间。当它们在0.05 M乙酸（pH 3.0）平衡的校准Sephadex G-75柱上进行凝胶过滤时，得到了相似的Mr估计值。然而，在0.05 M碳酸氢铵（pH 8.5）或含0.15 M氯化钠的pH 7.0磷酸盐缓冲液平衡的Sephadex G-200上进行凝胶过滤时，BSP-A1和BSP-A2以聚集分子（Mr 60,000 - 120,000）形式被洗脱。在8 M尿素存在下，BSP-A1和BSP-A2均在对应于Mr值17,000 - 20,000的位置被洗脱。BSP-A1和BSP-A2具有相同的氨基酸组成，与BSP-A3的氨基酸组成有很大差异。这三种蛋白质均以天冬氨酸作为N端残基，且已鉴定半胱氨酸为C端残基。BSP-A1和BSP-A2是含有氨基半乳糖、唾液酸和中性糖的糖蛋白，但BSP-A3不含有任何共价连接的糖。BSP-A2和BSP-A3未被吸附而被洗脱，而BSP-A1与麦胚凝集素-Sepharose 6MB结合，并可被竞争性糖N-乙酰-D-葡糖胺洗脱。用胰蛋白酶处理BSP-A1和BSP-A2会导致促性腺激素释放活性完全丧失，但BSP-A3保留全部活性。针对BSP-A1产生的抗体与BSP-A3不发生交叉反应，反之亦然。所有这些特性表明BSP-A3与BSP-A1（或BSP-A2）之间存在明显的结构差异。根据氨基酸组成得出结论，BSP-A1、BSP-A2和BSP-A3与促性腺抑制素相同[埃施、凌、博伦、英和吉耶曼（1983年）《生物化学与生物物理研究通讯》113, 861 - 867]。
