An HPLC-ECD procedure for measuring total phenolsulfotransferase (PST) activity in human liver, platelets and blood.
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.
Khoo
B Y
BY
Department of Biochemistry, National University of Singapore.
Sit
K H
KH
Wong
K P
KP
D015239:Arylsulfotransferase / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004298:Dopamine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013462:Sulfur Radioisotopes
D013462:Sulfur Radioisotopes; C068801:N-acetyldopamine-sulfate; D010724:Phosphoadenosine Phosphosulfate; D015239:Arylsulfotransferase; D004298:Dopamine