PURIFICATION AND CHARACTERIZATION OF A CASEIN-KINASE-II-TYPE ENZYME FROM XENOPUS-LAEVIS OVARY - BIOLOGICAL EFFECTS ON THE MEIOTIC CELL-DIVISION OF FULL-GROWN OOCYTE
PURIFICATION AND CHARACTERIZATION OF A CASEIN-KINASE-II-TYPE ENZYME FROM XENOPUS-LAEVIS OVARY - BIOLOGICAL EFFECTS ON THE MEIOTIC CELL-DIVISION OF FULL-GROWN OOCYTE
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DOI：
10.1111/j.1432-1033.1988.tb13765.x
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发表时间：
1988-01-15
期刊：
影响因子：
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通讯作者：
BELLE, R
中科院分区：
文献类型：
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作者：
MULNERLORILLON, O;MAROT, J;BELLE, R
从非洲爪蟾卵巢中高度纯化出一种Ⅱ型酪蛋白激酶。本文提出了一种新的纯化方法，包括四个层析步骤：酪氨酸-琼脂糖疏水层析、DEAE-Sepharose离子交换层析、肝素-Sepharose亲和层析和Mono Q快速蛋白液相层析。考虑到起始材料中存在的抑制剂掩盖了粗级分中的活性，纯化> 20 000。总收率> 20%。完全生长的非洲爪蟾卵母细胞含有64毫单位/卵母细胞，对应于纳摩尔范围内的细胞内浓度。该酶与哺乳动物酪蛋白激酶II具有以下特征：（a）可比较的亚单位组成（42-kDa双联体，38 kDa和26 kDa），（B）26-kDa亚基的自磷酸化，（c）使用GTP以及ATP作为磷酸供体的能力，（d）不能使用Mn 2+代替Mg 2+来支持活性，（e）酪蛋白的苏氨酸和丝氨酸残基的磷酸化，（f）低剂量肝素的抑制。高度纯化的酶的生物学效应进行了研究后，显微注射到非洲爪蟾完全成熟的卵母细胞。在纳摩尔浓度（约0.001）下，3 nM）的酶抑制孕酮诱导减数分裂细胞分裂，而它促进成熟促进因子水平的减数分裂成熟。这些结果表明，在减数分裂细胞分裂的前期/中期过渡过程中参与的磷酸化级联的激酶的作用，无论是在减数分裂前期逮捕的机制，并在细胞质因子成熟促进因子的活性。当显微注射到卵母细胞超过45 nM，激酶引起复杂的变化，在卵内32 P-标记的蛋白质的配置文件，表明其目标可能是其他激酶/磷酸酶调节蛋白。
A casein kinase of type II has been highly pourified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was > 20 000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was > 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (.apprxeq. 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.