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

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.

Effect of phosphatase inhibition of in vitro dopamine sulfation and 3'-phosphoadenosine-5'-phosphosulfate catabolism in human brain.

The effects of inhibition of phosphatase activity in 100,000 g supernatant solution from human frontal cortex on dopamine (DA) conjugation were examined using the phosphatase substrate p-nitrophenyl phosphate (pNPO4). The increases in DA sulfation seen in the presence of pNPO4 suggested that inhibition of phosphatase activity in high speed supernatant solutions of brain may substantially alter the pattern of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) metabolism and subsequently the rate of DA sulfation. Accordingly, the effects of the pyrophosphate analog phosphonoacetic acid (PAA) on the extent of DA sulfation and PAPS metabolism were examined in 100,000 g supernatant solution from human frontal cortex. At concentrations up to 10 mM, PAA markedly reduced PAPS hydrolysis to inorganic sulfate and 3'-phosphoadenosine-5'-phosphate (PAP) and significantly extended the linear time period for the sulfation of DA. These findings suggest that the phosphatase enzymes that degrade PAPS to produce the end product inhibitor, PAP, and possibly other break-down products of PAP, play an important role in determining the observed levels of phenol sulfotransferase activity in tissue from human brain in vitro.

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

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.

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.