Vitellogenin-Derived Yolk Proteins of White Perch, Morone americana: Purification, Characterization, and Vitellogenin-Receptor Binding1
Vitellogenin-Derived Yolk Proteins of White Perch, Morone americana: Purification, Characterization, and Vitellogenin-Receptor Binding1
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白鲈（Morone americana）的卵黄蛋白原衍生卵黄蛋白：纯化、表征和卵黄蛋白原受体结合1
作者：
N. Hiramatsu;A. Hara;K. Hiramatsu;H. Fukada;G. M. Weber;N. Denslow;C. Sullivan
摘要 本研究的目的是 1) 纯化和表征白鲈 (Morone americana) 的卵黄蛋白原蛋白，2) 开发卵黄蛋白原蛋白的非同位素受体结合测定法，3) 鉴定卵巢卵黄蛋白原受体识别的卵黄蛋白原蛋白结构域。通过选择性沉淀、离子交换色谱和凝胶过滤，从卵黄发生鲈鱼的卵巢中分离出四种源自卵黄蛋白原的卵黄蛋白（YP1、YP2 单体 [YP2m] 和二聚体 [YP2d] 和 YP3）。凝胶过滤后纯化的YP1、YP2m和YP2d的表观分子量分别为310 kDa、17 kDa和27 kDa。 YP3 在 SDS-PAGE 中显示为约 20 kDa 条带，加上一些弥散的较小条带，可以通过用与硝酸铝复合的考马斯亮蓝染色磷蛋白来可视化这些条带。 YP1、YP2s和YP3的免疫学和生化特征分别鉴定为白鲈卵黄蛋白、β'-组分和卵黄高磷蛋白。基于地高辛 (DIG) 标记的卵黄蛋白原示踪剂与固定在 96 孔板中的卵巢膜蛋白的结合，开发了一种新型卵黄蛋白原受体结合测定法。在测定中，来自白鲈的卵黄蛋白和来自鲈鱼和其他硬骨鱼的卵黄蛋白原有效地取代了特异性结合的DIG-卵黄原蛋白，但卵黄高磷蛋白和β'-组分却不能，这首次证明硬骨鱼卵黄原蛋白的脂卵黄蛋白结构域介导其与卵母细胞受体的结合。在这方面，卵黄蛋白原不如卵黄蛋白原有效，这表明卵黄原蛋白的其余卵黄蛋白结构域可能与其卵黄蛋白原结构域相互作用，以促进卵黄蛋白原与其受体的结合。
Abstract The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a ∼20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, β′-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the β′-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.
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