純化及鑑定Nucleolin之蛋白激?

Abstract

從C3H小老鼠，肝癌細胞株（129P）之核萃取液，純化可結合於AGP?動子中負向調控區域之蛋白質，找出 nucleolin為可能之因數。而nucleolin為核仁之主要蛋白質，其生物功能包括核糖體的生合成，調節pre-rRNA轉錄及幫助核糖體蛋白運回核仁：此與其轉錄後調節相關。因此針對nucleolin的胺基酸序列有許多個可能之磷酯化位置，我們欲純化核萃取液中的蛋白激?，研究nucleolin磷酯化後之生化及其與DNA結合親和力之影響。 首先將核萃取液中nucleolin純化出來，再與具較高磷酯化活性的流出部分進行重組磷酯化試驗。找出的 nualeolin蛋白激?，做進一步純化，並鑑定其對nucleolin磷酯化胺基酸位置為絲胺酸。這種蛋白激?作用之pH值範圍很廣，對熱穩定性不佳，而對其活性促進劑或抑制劑之反應與已知的nuoleolin蛋白激?casein kinaseⅡ相似。這些生化性質可做為純化出此蛋白激?及研究nucleolin功能之參考。

Nucleolin (C23) has been found to be a candidate repressor that binds to a negative cis-element in the AGP promoter region. Nucleolin is one of the major nucleolar proteins in exponentially growing cells and plays a direct role in ribosome assembly, regulating the transcription of pre-ribosomal RNA, and shuttling the ribosomal proteins to the nucleolus. Furthermore, its posttranslational modifications-phosphorylation and proteolysis are related to these functional regulations. Nucleolin has several potential phosphorylation sites in its N-terminal domain. To further understand the regulation of nucleolin phosphorylation, I purify its protein kinase from 129P nuclear extract. Nucleolin was purified from the nuclear extract by Heparin-Sepharose, Q-Sepharose, and gel filtration column chromatography. The kinase was purified by Q-Sepharose, Mono-S, and gel filtration column chromatography. The partially purified kinase can phosphorylate the serine residue(s) of nucleolin. Further characterization of the nucleolin kinase showed that it has a wide pH optimum, poor heat stability and responds to some stimulators and inhibitors of casein kinase Ⅱ