Villa-Moruzzi E
Department of Biomedicine, University of Pisa, Italy.
Biochem Biophys Res Commun. 1993 Nov 15;196(3):1248-54. doi: 10.1006/bbrc.1993.2386.
The cdc2-activator cdc25C was immunoprecipitated from HeLa cell extracts and assayed as tyrosine phosphatase (PTP) using tyrosine-phosphorylated myelin basic protein. The PTP activity was 12-fold higher in immunocomplexes from mitotic (nocodazole-arrested) than from asynchronous cells. This difference is due to enzyme activation, since the same amount of cdc25C was immunodetected in both conditions. However, mitotic cdc25C had M(r) 59,000, while a 56,000-59,000 doublet was detected in immunocomplexes from asynchronous cells. The PTP activity of mitotic cdc25C was decreased by treatment with Phosphatase-2A catalytic subunit (but not with Phosphatase-1), with re-appearance of the 56,000 polypeptide. cdc25C was also found associated with cdc2-p13-Sepharose complex and its PTP activity was 7-fold higher in samples from mitotic than from asynchronous cells. cdc25C and cdc2 co-migrated during gel filtration and the higher activity of mitotic cdc25C was retained through gel filtration.
从HeLa细胞提取物中免疫沉淀cdc2激活剂cdc25C，并使用酪氨酸磷酸化的髓鞘碱性蛋白作为酪氨酸磷酸酶（PTP）进行检测。有丝分裂期（诺考达唑阻滞）免疫复合物中的PTP活性比非同步细胞中的高12倍。这种差异是由于酶的激活，因为在两种情况下检测到的cdc25C量相同。然而，有丝分裂期的cdc25C分子量为59,000，而非同步细胞免疫复合物中检测到的是56,000 - 59,000的双峰。用磷酸酶2A催化亚基（而非磷酸酶1）处理会降低有丝分裂期cdc25C的PTP活性，同时56,000的多肽会重新出现。还发现cdc25C与cdc2 - p13 - 琼脂糖复合物相关，其PTP活性在有丝分裂期样品中比非同步细胞样品中高7倍。在凝胶过滤过程中cdc25C和cdc2共同迁移，并且有丝分裂期cdc25C的较高活性在凝胶过滤后得以保留。
