Grbac-Ivankovic S, Antonijczuk K, Varghese A H, Plowman M C, Antonijczuk A, Korza G, Ozols J, Sunderman F W
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington.
Mol Reprod Dev. 1994 Jul;38(3):256-63. doi: 10.1002/mrd.1080380305.
An Ni(2+)-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2 beta, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2 beta agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metal-blot competition assay, the abilities of metal ions to compete with 63Ni2+ for binding to pNiXb were ranked: Zn2+ approximately Cu2+ approximately Co2+ > Cd2+ approximately Mn2+ > Sn2+. This study shows that Xenopus lipovitellin 2 beta is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo.
从成熟非洲爪蟾卵母细胞以及处于N/F阶段42的受精胚胎中分离并鉴定出一种镍离子结合蛋白（pNiXb，31kD）。卵母细胞或胚胎经聚丙烯酰胺凝胶电泳（PAGE）分级分离、电转印至硝酸纤维素膜上，并用63Ni2+进行检测，通过放射自显影法检测到pNiXb。pNiXb是一种位于胚胎肠道的卵黄蛋白，通过硫酸铵沉淀、脱脂、凝胶过滤色谱法和高效液相色谱分析从卵黄小板中纯化得到。在这些步骤中，pNiXb与卵黄脂磷蛋白2共纯化。纯化后的pNiXb的N端序列与从非洲爪蟾卵黄蛋白原A2前体基因的DNA序列推导出来的非洲爪蟾卵黄脂磷蛋白2β的N端序列完全匹配。由于pNiXb和卵黄脂磷蛋白2β在N端序列、氨基酸组成和表观分子量方面一致，它们似乎是相同的。基于金属印迹竞争试验，金属离子与63Ni2+竞争结合pNiXb的能力排序为：Zn2+≈Cu2+≈Co2+＞Cd2+≈Mn2+＞Sn2+。本研究表明，非洲爪蟾卵黄脂磷蛋白2β在体外是一种金属结合蛋白，并增加了其在体内可能具有类似功能的可能性。
