A modified Ecteola cellulose assay for M and P phenol sulfotransferase

The development and applications of a modified Ecteola cellulose, ion exchange assay for phenol sulfotransferase (EC 2.8.2.1, PST) are described. Mixtures containing dopamine or phenol and 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) were incubated with 100,000 g supernatant solution from human frontal cortex and applied to 0.5 X 2 cm columns of Ecteola cellulose. Dopamine sulfate was eluted with 3 ml of distilled water, while phenyl sulfate, inorganic sulfate and unreacted PAPS were eluted with successive step gradients of 5, 20 and 200 mM NH4HCO3. The solution volume for phenyl sulfate was 11 ml, while those for inorganic sulfate and PAPS were both 6 ml. The new assay method yielded apparent Km values for dopamine, 3-methoxytyramine, tyramine and norepinephrine similar to those obtained by other methods. Comparison of the activities of various amine substrates at a concentration of 20 microM showed that dopamine was the preferred substrate, followed in decreasing order of relative activity by 3-methoxytyramine, norepinephrine, tyramine and octopamine. When mixed substrate inhibition of dopamine sulfation by phenol was examined, phenol was found to effectively inhibit dopamine sulfation over a range of 0.1 to 10 mM. The procedure described in this paper offers a number of significant advantages over currently available assays: these include a rapid, simple product isolation procedure and a complete, discrete separation of the radiolabeled products and reactants. This property allows the detailed study of the flux of radiolabel through the enzymatic system and also makes alternative substrate inhibition studies possible.