PURIFICATION OF MATURATION-PROMOTING FACTOR, AN INTRACELLULAR REGULATOR OF EARLY MITOTIC EVENTS
PURIFICATION OF MATURATION-PROMOTING FACTOR, AN INTRACELLULAR REGULATOR OF EARLY MITOTIC EVENTS
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DOI：
10.1073/pnas.85.9.3009
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发表时间：
1988-05-01
影响因子：
11.1
通讯作者：
MALLER, JL
中科院分区：
文献类型：
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作者：
LOHKA, MJ;HAYES, MK;MALLER, JL
成熟促进因子注入非洲爪蟾卵母细胞后可引起生殖泡破裂，并在无细胞系统中诱导中期分裂。无细胞测定被用来监测成熟促进因子在其纯化从未受精的非洲爪蟾卵。硫酸铵沉淀和六个色谱程序导致制备纯化> 3000倍，当注射到环己酰亚胺处理的卵母细胞中时，可以在2小时内诱导萌发囊泡破裂。45 kDa和32 kDa的蛋白质与两种测定中活性最高的组分相关。这些组分含有能够磷酸化内源性45-kDa蛋白的蛋白激酶活性，以及组蛋白H1、磷酸酶抑制剂1和酪蛋白。这里描述的高度纯化的制剂应有助于鉴定促成熟因子的作用机制，并阐明蛋白激酶在中期诱导中的作用。
Maturation-promoting factor causes germinal vesicle breakdown when injected into Xenopus oocytes and can induce metaphase in a cell-free system. The cell-free assay was used to monitor maturation-promoting factor during its purification from unfertilized Xenopus eggs. Ammonium sulfate precipitation and six chromatographic procedures resulted in a preparation purified > 3000-fold that could induce germinal vesicle breakdown within 2 hr when injected into cycloheximide-treated oocytes. Proteins of 45 kDa and 32 kDa were correlated with fractions of highest activity in both assays. These fractions contained a protein kinase activity able to phosphorylate the endogenous 45-kDa protein, as well as histone H1, phosphatase inhibitor 1, and casein. The highly purified preparations described here should help to identify the mechanism of action of maturation-promoting factor and to elucidate the role of protein kinases in the induction of metaphase.