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人脑来源的一种具有苯酚硫酸化作用的苯酚硫酸转移酶的纯化及动力学特性研究

Purification and kinetic characterization of a phenol-sulfating form of phenol sulfotransferase from human brain.

作者信息

Whittemore R M, Pearce L B, Roth J A

出版信息

Arch Biochem Biophys. 1986 Sep;249(2):464-71. doi: 10.1016/0003-9861(86)90023-8.

Abstract

The identification of three forms of phenol sulfotransferase (PST) in human brain and the subsequent purification and kinetic characterization of a phenol-sulfating form of the enzyme are described. Two forms of PST which were capable of conjugating phenol and a third form which sulfated dopamine were resolved from one another using DEAE-cellulose chromatography. One of the phenol-sulfating forms (P1-PST) was subsequently purified on Affi-Gel blue and Sephacryl S-200, giving a final purification of almost 390-fold, with an overall yield of approximately 5%. The purified enzyme was sensitive to NaCl and showed an optimum for phenol conjugation at pH 8.5. Kinetic analysis demonstrated that sulfation by P1-PST proceeds via a sequential ordered, bi-substrate reaction mechanism, where 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is the leading substrate. The true Km and Kia values for PAPS were both 0.35 microM, while the true Km value for phenol was 2.8 microM.

摘要

本文描述了在人脑中鉴定出三种形式的酚磺基转移酶(PST),以及随后对该酶的一种酚硫酸化形式进行纯化和动力学表征的过程。使用DEAE - 纤维素色谱法将两种能够结合酚的PST形式与第三种硫酸化多巴胺的形式彼此分离。随后,其中一种酚硫酸化形式(P1 - PST)在Affi - Gel蓝和Sephacryl S - 200上进行纯化,最终纯化倍数接近390倍,总产率约为5%。纯化后的酶对NaCl敏感,在pH 8.5时对酚结合表现出最佳活性。动力学分析表明,P1 - PST的硫酸化反应通过顺序有序的双底物反应机制进行,其中3'-磷酸腺苷-5'-磷酸硫酸酯(PAPS)是主要底物。PAPS的真实Km和Kia值均为0.35 microM,而酚的真实Km值为2.8 microM。

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