核□酸與酚亞硫酸基轉移酵素結合誘導構型改變之研究

Abstract

亞硫酸基轉移酵素所催化的硫酸化作用在生物體內扮演相當重要的角色。 其中酚亞硫酸基轉移酵素可對含酚化合物進行解毒作用。本論文利用基因 重組過的亞硫酸基轉移酵素進行研究。首先利用陰離子交換層析以及硫銨 沈降法進行純化步驟，得到均質的亞硫酸基轉移酵素。再以聚丙烯醯胺膠 體電泳探討亞硫酸基轉移酵素與核□酸以及硫醇基試劑作用結合後在構型 上的變化。結果顯示除了亞硫酸基轉移酵素本身具有的輔酵素， adenosine 3',5'-bisphossphate（PAP）對於酵素有影響外，其它核□酸 替代物以及硫醇基試劑也同樣能夠造成亞硫酸基轉移酵素在構型上的變化 ，pI值亦 Sulfotransferase catalyzes sulfuryl group transfer from adenosine 3'-phosphate 5'-phosphosulfate(PAPS) to a variety of nucleophilic acceptor in biological system. As a memeber of the enzymes of detoxication, phenol sulfotransferase(PST) detoxifies some phenolic compounds through sulfation. Using recombinant DNA techniques, anionic exchange chromatography and ammonium sulfate precipitation, homogeneous recombinant PST was obtained. Polyacrylamide gel electrophoresis（PAGE） was used to study the conformationAddition of 3',5'-bisphosphate(PAP) into the mixture including PST and 4-nitrophenol (pNP) produced a new pNP spectrum with an absorption maximum at 375 nm. The binding order of nucleotides and phenols that may be important for the inhibition of the phenol sulfotransferase is determined by the change of UV/vis spectra. We also determine stoichiometry of pNP and PAP. A different spectrum was observed when AMP and GMP were used to replace PAP. A model study for pNP in organic solvents to mimic the phenol bind