B6/P5P is a pseudo phenolic

Title: The effects of pyridoxal-5-phosphate on sulfotransferase activity: actions on tyrosyl protein sulfotransferase and phenol sulfotransferase. Authors: Rosemary H Waring, Robert M Harris and Victoria L Griffiths, School of Biosciences, University of Birmingham, Birmingham. B15 2TT. UK Introduction Sulfotransferase enzymes use PAPS (3’-phospho-adenosive-5’-phosphosulfate) to transfer sulfate residues onto a wide variety of substrates. TPST substrates require sulfation for efficient function while sulfation by SULT 1A1 greatly alters substrate properties, usually decreasing their activity. a) Tyrosylprotein sulfotransferase (TPST) Substrates - tyrosine residues on gastrin, cholecystokinin, mucin proteins Methods TPST activity was measured (using gastrin as substrate) with radiolabelled 35S-PAPS as sulfate donor; assays were incubated at 37°C for 40 minutes. All assays were performed in quadruplicate). Enzyme sources were a) platelet pellets prepared by centrifugation from time-expired platelet packs from the Birmingham Blood Transfusion Service and b) human colon adenocarcinoma HT-29 cells which synthesise mucin proteins and are thought to be the best model for the human g.i. tract. These were grown in McCoys 5A medium supplemented with 10% fetal bovine serum and glutamine/penicillin/streptomycin at 37°C till confluent, then harvested and centrifuged to give a cell membrane pellet. This was resuspended in phosphate buffered saline, then sonicated before assay. Cells were also grown after confluence for 24 hours with varying concentrations of P5P. P5P was also added directly to the platelet assay (0-2.5μM) MgCl2 was added at varying concentrations (0-5μM) before the start of the TPST assay. Results TPST activity was present in both human platelets and HT-29 cells. This enzyme activity was inhibited in the presence of P5P (Fig. 1). Direct effects of MgCl2 on the assay are shown in Fig. 2. As can be seen, MgCl2 concentration had no significant effects on the TPST activity of HT-29 cells but activated TPST activity in platelets (there are different isoforms of the enzyme). The concentration of 0.4μM P5P was then chosen as showing clear reduction of TPST activity in initial experiments (See Fig. 1). Human platelets were treated directly with this concentration while the HT-29 cells were incubated with it for 24h. Varying amounts of MgCl2 were then added to the assays (see Fig. 3). As can be seen, levels of MgCl2 at 0.5μM or greater removed the inhibition caused by 0.4μM P5P. Enzyme assays and Western blotting with specific anti-TPST antibodies showed that P5P did not affect the expression of TPST. All results were carried out in quadruplicate and are expressed as means ± SD. (SD <= 5.3%). b) SULT1A1 (Phenolsulphotransferase) Substrates – Phenols, catecholamines, flavonoids, steroids. Methods SULT1A1 activity was measured using 4-nitrophenol as a substrate (this also picks up any of the SULT1A2 isoform, although this is only present to a small extent in platelet preparations). Again radiolabelled 35S-PAPS was used in the assay as a sulfate donor. The enzyme sources were cytosols from platelets and HT-29 cells, prepared as before and assayed under standard conditions. Results SULT1A1 activity was present in both human platelets and HT-29 cells. This activity was inhibited by P5P (Fig. 4). However, this inhibition was only significant in the platelets; the HT-29 cell SULT1A1 seemed relatively unaffected. Treatment with MgCl2 (1.0μM) on cells exposed to 1.0 μM P5P restored the activity in platelets (Fig. 6) but had no significant effects in the HT-29 cells, which in any case were not greatly affected by the P5P (Fig. 4) SULT1A1 activity in platelets was almost unaffected by MgCl2 although SULT1A1 activity in HT-29 cells decreased slightly with increasing Mg (Fig. 5). Our previous studies have shown that the isoenzymes in these tissues have different activities and slightly different properties. Incubation of HT-29 cells with P5P had no effect on enzyme expression, as seen by Western blotting and enzyme activity. All results were carried out in quadruplicate and are expressed as means ±SD. (SD <= 5.4%). Conclusions 1. Platelet and HT-29 cells show TPST activity which is inhibited by P5P, though only the platelet isoform is greatly affected. This inhibition is reversed by MgCl2 in roughly equimolar amounts. 2. Platelet and HT-29 cells show SULT1A1 activity which is inhibited by P5P, although only the platelet isoform is greatly affected. Again this inhibition is reversed by MgCl2 in roughly equimolar amounts. 3. Neither TPST nor SULT1A1 expression is altered by P5P, which only affects the enzyme activity directly. 4. From the literature, P5P has a pseudo-phenolic structure which is believed to interact with those phenol sulfotransferases for which phenolic rings are a substrate. However, addition of Mg2+ may form a complex which no longer interacts with the enzyme. From the therapeutic point of view, Mg2+ ions should be supplied in at least a 2:1 ratio with P5P to reverse any inhibition and activate those sulfotransferases which respond to increased magnesium levels particularly the platelet enzymes.