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標題: | 吳郭魚肝臟細胞之核型蛋白質磷酸化酪胺酸解磷酸?以c-Src磷酸化的拓樸異構晦-I為受質 c-Src Phosphorylated Topoisomerase I as a Substrate of Nuclear Phosphotyrosyl Protein Phosphatase from the Liver of Tilapia mossambica |
作者: | 劉棠必 |
出版年 : | 1996 |
學位: | 碩士 |
摘要: | 本論文採用水生脊椎動物:Tilapia mossambica為實驗材料,對魚類細胞中影響酪胺酸磷酸化的蛋白質酪胺酸解磷酸?與近來受到熱烈討論並成為化學療法對象的拓樸異構?-I進行活性調控的研究。 純化的步驟共使用1次親合力層析法,2次陰離子交換層析法與1次膠體過濾層析法。純化到的核型PTPase其比活性為5.095unit/mg of protein。分子量為62kDa(由膠體過濾管柱SuperoseTM 12層析法推算)與67kDa與60kDa(由SDS-PAGE推算)。pI值為5.79±0.02。pH 7.0為酵素活性最適作用酸鹼值。Ca2+、Mg2+、Mn2+與VO43-與Zn2+五種金屬離子對純化到之核型蛋白質酪胺酸解磷酸?活性的影響皆為抑制效果其中Mn2+,VO43-與Zn2+對PTPase活性具有很強的的抑制作用。 依據等當於蛋白質酪胺酸解磷酸?純化步驟所得到之拓樸異構?-I的比活性為53.000unit/mg of protein。由SDS-PAGE估算之分子量為66kDa,65kDa,64kDa與58kDa,並且可被anti human topoisomerase I-antibody辨認。 純化得到的拓樸異構?-I在事先未經任何處理的狀態下,經由人類鹼性解磷酸?作用後活性幾乎消失,而核型PTPase對於此一狀態的拓樸異構?-I並無影響活性的作用。但若經c-Src以[γ-32P]-ATP封拓樸異構?-I進行磷酸化後,一方面可在放射性自動顯影中觀察到pl由5.32,5.23,5.11與4.93往酸端移動成為4.32,4.23,4.06與3.96,另方面造成拓樸異構?-I的活性下降,之後再經過核PTPase解磷酸化作用後,拓樸異構?-I的解旋能力回復至原來活性。c-Src與核蛋白質酪胺酸解磷酸?對拓樸異構?-I的作用關係與發生的可能性進一步在本論文中討論。 A phosphotyrosyl protein phosphatase (PTPase) was purified from the hepatic nuclear matrix of Tilapia mossambica with a final specific activity of 5.095 units/mg of protein. 2% CHAPS and 2 M NaCl were applied to extract the matrix bound protein and one affinity column (HiTrap Heparine). two anion exchangers (HiTrap Q and Mono Q HR 5/5) plus one gel filtration column (SuperdexTM 200) were conducted consecutuvely to purify the enzyme. The PTPase had a relative molecular mass of 67 kDa and 60 kDa as estimated by SDS-PAGE and of 62 kDa by gel filtration chromatography. This monomeric and neutral PTPase was with an isoelectric point of 5.79 ± 0.02 and an optimal activity at pH 7.0 in the dephosphorylation of a 32P-labeled synthetic insulin receptor peptide [ -RDI(PY)ETD(PY)(PY)R- ] as substrates. Orthovanadate, Zn2+ and Mn2+ were strong inhibitors for the purified PTPase and had IC50 at the level of μM; Mg2+ and Ca2+ had higher IC50 in mM level. Through comparable purification procedures, topoisomerase I was purified in proteolytic forms with relative molecular mass of 66 kDa. 65 kDa and 59 kDa, and had a final specific activity of 53,000 units/mg of protein. The influence of different topoisomerase I phosphorylation states on the enzyme activity was investigated: incubation of the purified topoisomerase I with human alkaline phosphatase abolished the relaxing activity in a time-dependent pattern but not with PTPase. The activity of topoisomerase I was also inhibited (49% decreased) by recombinant human c-Src-mediated tyrosyl phosphorylation and re-activated by PTPase-treatment. In isotope labeling experiments which employed the purified topoisomerase I, [γ-32P] ATP and recombinant human c-Src, a pI-shifting was observed in Immobiline DryStrip Gel by isoelectric focusing. The interaction between Src, topoisomerase I and PTPase was futher discussed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76230 |
全文授權: | 未授權 |
顯示於系所單位: | 動物學研究所 |
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