【作者】 郭伟伟；

【导师】 倪晓华；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 双特异性磷酸酶是酪氨酸蛋白磷酸酶家族中的一个新兴家族，能够使磷酸化的酪氨酸残基和丝／苏氨酸残基去磷酸化。它们在有丝分裂、信号传导、胞外刺激和细胞周期等通路中发挥重要作用。 我们从金黄色葡萄球菌基因文库中克隆了两条新的双特异性磷酸酶，命名为低分子量的双特异性磷酸酶sLMWDSP （low molecular weight dual specificity phosphatase，Staphylococcus aureus）和蛋白磷酸酶sPP2C（protein phosphatase 2C，Staphylococcus aureus）。sLMWDSP具有462个碱基，编码的蛋白有154个氨基酸且具有一个双特异性磷酸酶的催化结构域。蛋白产物的分子量为18 kDa，等电点为4.49。sPP2C具有741个碱基，编码的蛋白有247个氨基酸且具有一个磷酸蛋白磷酸酶2C的催化结构域。蛋白产物的分子量为26.1 kDa，等电点为4.95。在E．coli．Rossetta中表达蛋白sLMWDSP、sPP2C。高纯度的sLMWDSP、sPP2C用亲和层析的方法纯化得到。 sLMWDSP和sPP2C的磷酸酶活性用对硝基苯磷酸pNPP （p-nitrophenyl phosphate，pNPP）进行检测，发现sLMWDSP的最适pH是6.7，最适温度是35℃，且不依赖于金属离子；而sPP2C的最适pH是11.0，最适温度是37℃，且该酶的活性必须依赖二价的金属离子，在二价金属离子Mn²⁺存在下活性最高。磷酸酶的通用抑制剂岗田酸、EDTA对sLMWDSP几乎没有抑制作用，但钒酸钠能明显地抑制该酶的活性。sPP2C能被抑制剂EDTA、钒酸钠、焦磷酸钠、磷酸钠完全抑制。酶学研究表明：sLMWDSP和sPP2C对pSer／Thr、pTyr的寡肽均有去磷酸化作用，说明sLMWDSP和sPP2C是两个新的双特异性磷酸酶。更多还原

【Abstract】 Dual-specificity protein phosphatases （DSPs） constitute a new family of protein tyrosine phosphatases （PTPs） characterized by the ability to dephosphorylate both phospho-tyrosyl residues and phospho-seryl/threonyl residues. They play key roles in the regulation of mitosis, signal transduction, extracellular stimulation and the cell cycle.Two novel dual specificity protein phosphatases named sLMWDSP （low molecular weight dual specificity phosphatase, Staphylococcus aureus） and sPP2C （protein phosphatase 2C, Staphylococcus aureus） were cloned from Staphylococcus aureus gene library. sLMWDSP contained 462 bp. The protein contained 154 amino acids and a dual specifity phosphatase catalytic domain. The molecular weight was 18 kDa, and pI was 4.49. sPP2C contained 741 bp. The protein contained 247 amino acids and a protein phosphatases 2C catalytic domain. The molecular weight was 26.1 kDa, and pI was 4.95. sLMWDSP and sPP2C were expressed in E.coli. Rossetta and purified by affinity chromatography.sLMWDSP and sPP2C show phosphatase activity towards pNPP which is a common synthetic protein phosphatase substrate. sLMWDSP shows phosphatase activity with a maximum value at pH 6.7 and 35 ℃ and is independent of metal ions; sPP2C is optimum at about pH 11.0 and 37 ℃ and Mn²⁺ and its phosphatase activity must be dependent of and is enhanced by bivalent metal ions. sLMWDSP can not be inhibited by okadaic acid and EDTA but obviously inhibited by vanadate sodium. However, sPP2C can be inhibited by different inhibitors such as EDTA, vanadate sodium, sodium pyrophosphate and sodium phosphate. Enzyme property research reveals that both of sLMWDSP and sPP2C show phosphatase activity towards oligopeptides containing pSer/Thr and pTyr, indicating that sLMWDSP and sPP2C are two novel protein phosphatases with dual substrate specificity.更多还原