Khoo B Y, Sit K H, Wong K P
Department of Biochemistry, National University of Singapore.
Clin Chim Acta. 1990 Dec 24;194(2-3):219-28. doi: 10.1016/0009-8981(90)90136-g.
The phenolsulfotransferase (PST) activity in human liver, platelets and blood was measured under saturating concentrations of the conjugating agent, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Conventional PST assays employ PAP35S at suboptimal concentrations. In addition, the sulfate conjugate formed, namely N-acetyldopamine-sulfate (NADA-sulfate) was quantified directly by high-pressure liquid chromatography cum electrochemical detection (HPLC-ECD). NADA, the biogenic amine acceptor used in this study appeared from kinetic data to be a substrate of both the P and M forms of PST when used in micromolar concentration. Two apparent Km values of 4.2 mumol/l and 22.6 mumol/l were observed. In contrast, only one apparent Km value was evident when the assay was carried out in the presence of 2,6-dichloro-4-nitrophenol (DCNP), a selective inhibitor of the P form of PST or after heat treatment under specified conditions which inactivates the M form of PST. Thus measurement of PST activity with NADA as the acceptor substrate permits the determination of total PST activity and a parallel assay with the inclusion of DCNP would distinguish the two variants of PST, both of which appear to be present in all human tissues.
在共轭剂3'-磷酸腺苷-5'-磷酸硫酸酯（PAPS）饱和浓度下，测定了人肝脏、血小板和血液中的酚磺基转移酶（PST）活性。传统的PST检测采用的是亚最佳浓度的PAP35S。此外，通过高压液相色谱结合电化学检测（HPLC-ECD）直接对形成的硫酸酯共轭物，即N-乙酰多巴胺-硫酸盐（NADA-硫酸盐）进行定量。本研究中使用的生物胺受体NADA，从动力学数据来看，当以微摩尔浓度使用时，似乎是PST的P型和M型的底物。观察到两个表观Km值，分别为4.2 μmol/l和22.6 μmol/l。相比之下，当在2,6-二氯-4-硝基苯酚（DCNP，PST的P型选择性抑制剂）存在下进行检测，或在特定条件下热处理使PST的M型失活后进行检测时，仅出现一个表观Km值。因此，以NADA作为受体底物测定PST活性可确定总PST活性，同时加入DCNP进行平行检测可区分PST的两种变体，这两种变体似乎存在于所有人体组织中。
