Yamashita M, Fukada S, Yoshikuni M, Bulet P, Hirai T, Yamaguchi A, Lou Y H, Zhao Z, Nagahama Y
Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Japan.
Dev Biol. 1992 Jan;149(1):8-15. doi: 10.1016/0012-1606(92)90259-j.
Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.
成熟促进因子（MPF）活性首次在鱼类卵母细胞中得到证实。我们使用四个色谱柱（Q-Sepharose Fast-Flow、p13suc1-亲和琼脂糖、Mono S和Superose 12）从碾碎的、自然产卵的鲤鱼卵母细胞的100,000g上清液中纯化MPF。最终制剂的纯化倍数超过1000倍，回收率约为1%。在Superose 12上，MPF以单一峰洗脱，表观分子量为100 kDa。Superose 12后活性组分的SDS-PAGE分析显示存在33、34、46和48 kDa的四种蛋白质。一种针对cdc2激酶PSTAIR序列的单克隆抗体识别33 kDa和34 kDa的蛋白质，而46 kDa和48 kDa的蛋白质是其内源底物。46 kDa和48 kDa的蛋白质被一种针对大肠杆菌产生的金鱼细胞周期蛋白B的单克隆抗体识别，但不被抗细胞周期蛋白A抗体识别。当卵母细胞在32P存在下成熟时，34 kDa的蛋白质出现标记，但33 kDa的蛋白质没有。34 kDa的蛋白质对应于MPF活性，而33 kDa的蛋白质则不对应。这些发现表明鲤鱼MPF是cdc2激酶和细胞周期蛋白B的复合物，并且进一步表明活性MPF包含cdc2激酶的磷酸化形式。
