Alkaline butanol extraction of bile salt and steroid sulfate esters: application to the assay of sulfotransferases

A butan-1-ol solvent-extraction procedure has been evaluated for the assay of 3'-phosphoadenosine-5'-phosphosulfate:sulfotransferase activity with various bile salt and steroid substrates. Although butanol extracted the sulfate esters of steroids and bile salts from aqueous solution at neutral pH, extraction at basic pH gave optimum recovery which was independent of protein in the sample. Greater than 99.9% of unreacted 3'-phosphoadenosine-5'-phospho[35S]sulfate remained in the aqueous phase. The data for sulfotransferase activities obtained with this solvent-extraction assay were not significantly different from those obtained with a standard thin-layer chromatography method. Solvent extraction has enabled multiple, rapid assays of several steroid and bile salt sulfotransferases during chromatographic purification of these enzymes from tissue fractions.     

A radioassay of enzymes catalyzing the glucosylation and sulfation steps of glucosinolate biosynthesis in Brassica species

A new method for assaying the enzymes uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:desulfoglucosinolate sulfotransferase has been designed. The assay system is based on the separation of nonionic [14C]desulfobenzylglucosinolate from anionic [14C]UDPglucose and anionic [14C]benzylglucosinolate, respectively, by differential adsorption to DEAE-ion-exchange disks. The procedure eliminates elaborate chromatographic techniques. The method was used to measure both enzymes in several Brassica spp. In addition, sulfotransferase activity was monitored during partial purification from seedlings of Brassica napus (cv Westar).     

A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV.

We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from the desulfated 3'-phosphoadenosine-5'-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure beta-form during purification, and utilized for the regeneration. The activity of betaAST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. betaAST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotransferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and other tissues for specific sulfotransferase enzyme activity and substrate concentration.     

Paper disk assay for glycosaminoglycan sulfotransferases

A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the [35S]sulfate-labeled products are precipitated and then separated from a sulfate donor ([35S]PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.     

Enzymatic assay for flavonoid sulfotransferase

A novel enzyme assay for flavonoid sulfotransferase is described. It makes use of tetrabutylammonium dihydrogen phosphate which forms a pair of ions with the flavonoid sulfate esters formed. This renders the sulfate ester soluble in organic solvents such as ethyl acetate, whereas the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, remains in the aqueous reaction mixture. The procedure is simple, rapid, and reproducible. It eliminates the need for chromatographic separation of the reaction products, except when their identification is required, and is suitable for use in the purification and kinetic studies of sulfotransferases.     

Inhibitors and dipeptide substrates for a microsomal tyrosylsulfotransferase from rat brain

The sulfotransferase associated with a microsomal fraction from rat brain was previously shown to transfer sulfate groups from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to peptides derived from the cholecystokinin (CCK) molecule. Three tyrosine-containing dipeptide derivatives, i.e., Cbz-Glu-Tyr, Cbz-Gly-Tyr and Ac-Phe-Tyr are shown here to accept the [35S] sulfate group from [35S] PAPS under the action of this sulfotransferase. The sulfotransferase activity evaluated with either any of these dipeptide derivatives or CCK-8 as acceptors is similarly inhibited by a series of compounds, i.e., lipophilic polycyclic compounds like fluphenazine, tyrosine derivatives like Boc-O-benzyl-tyrosine and phenolsulfotransferase inhibitors like 4,4-di-isothiocyano 2',2'-disulfonic acid stilbene.     

Demonstration of a novel sulfotransferase in fetal bovine serum, which transfers sulfate to the C6 position of the GalNAc residue in the sequence iduronic acid alpha1-3GalNAc beta1-4iduronic acid in dermatan sulfate.

A novel sulfotransferase activity was discovered in fetal bovine serum using pig skin dermatan sulfate as an acceptor and [35S]3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor. The enzyme was separated from chondroitin:GalNAc 6-O-sulfotransferase by chromatographic techniques. Enzymatic analysis of the reaction products demonstrated that the enzyme transferred sulfate to the C6 position of the GalNAc residue in the sequence -iduronic acid alpha1-3GalNAc beta1-4iduronic acid-. Thus, the enzyme has been identified as a hitherto unreported dermatan sulfate:GalNAc 6-O-sulfotransferase. The finding is in sharp contrast to the current concept that in dermatan sulfate biosynthesis GalNAc 4-O-sulfation is a prerequisite for iduronic acid formation by C5 epimerase.     

Properties and Localization of the Sulfate-Activating System in Rat Brain

The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isotope exchange at equilibrium indicates a steady state ordered kinetic mechanism for human sulfotransferase

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biosignaling molecular biological activities and detoxifies hydroxyl-containing xenobiotics. The universal sulfuryl group donor for SULTcatalyzed sulfation is adenosine 3'-phosphate 5'-phosphosulfate (PAPS). The reaction products are a sulfated product and adenosine 3',5'-diphosphate (PAP). Although the kinetics has been reported since the 1980s,SULT-catalyzed reaction mechanisms remain unclear. Human SULT1A1 catalyzes the sulfation of xenobiotic phenols and has very broad substrate specificity. It has been recognized as one of the most important phase II drug-metabolizing enzymes. Understanding the kinetic mechanism of this isoform is important in understanding drug metabolism and xenobiotic detoxification. In this report, we investigated the SULT1A1-catalyzed phenol sulfation mechanism. The SULT1A1-catalyzed reaction was brought to equilibrium by varying substrate (1-naphthol) and PAPS initial concentrations. Equilibrium constants were determined. Two isotopic exchanges at equilibrium ([14C]1-naphthol <=>[14C]1-naphthyl sulfate and[35S]PAPS<=>[35S]1-naphthyl sulfate) were conducted. First-order kinetics, observed for all the is otopic exchange reactions studied over the entire time scale that was monitored, indicates that the system was truly at equilibrium prior to addition of an isotopic pulse. Complete suppression of the 35S isotopic exchange rate was observed with an increase in the levels of 1-naphthol and 1-naphthyl sulfate in a constant ratio,while no suppression of the 14C exchange rate was observed with an increase in the levels of PAPS and PAP in a constant ratio. Data are consistent with a steady state ordered kinetic mechanism with PAPS and PAP binding to the free enzyme.     

Dermatan is a better substrate for 4-O-sulfation than chondroitin: implications in the generation of 4-O-sulfated, L-iduronate-rich galactosaminoglycans

The biosynthesis of dermatan sulfate is a complex process that involves, inter alia, formation of L-iduronic acid residues by C5-epimerization of D-glucuronic acid residues already incorporated into the growing polymer. It has been shown previously that this reaction is promoted by the presence of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. In the present investigation, the role of sulfation in the biosynthesis of L-iduronic acid-rich galactosaminoglycans was examined more closely by a study of the substrate specificities and kinetic properties of the sulfotransferases involved in dermatan sulfate biosynthesis. Comparison of the acceptor reactivities of oligosaccharides from chondroitin and dermatan, in an in vitro system containing microsomes from cultured human skin fibroblasts and 3'-phosphoadenosine-5'-phosphosulfate, showed that Km values for the dermatan fragments were substantially lower than those for their chondroitin counterparts. Calculation of Vmax values likewise showed that dermatan was the better substrate. Whereas dermatan incorporated [35S]sulfate exclusively at the C4 position of N-acetylgalactosamine residues, approximately equal amounts of radioactivity were found at the C4 and C6 positions in the labelled chondroitin. Under standard assay conditions, the 4-O-sulfation of dermatan proceeded about six times faster than the 4-O-sulfation of chondroitin. On the basis of these results, we propose that L-iduronic acids, formed in the course of the biosynthesis of dermatan sulfates, enhance sulfation of their adjacent N-acetylgalactosamine residues, and will thereby be locked in the L-ido configuration.     

Inhibition of bovine phenol sulfotransferase (bSULT1A1) by CoA thioesters. Evidence for positive cooperativity and inhibition by interaction with both the nucleotide and phenol binding sites

Previous work with the bovine phenol sulfotransferase (bSULT1A1, EC ) demonstrated inhibition by CoA that was competitive with respect to the sulfuryl donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Leach, M., Cameron, E., Fite, N., Stassinopoulos, J., Palmreuter, N., and Beckmann, J. D. (1999) Biochem. Biophys. Res. Commun. 261, 815-819). Here we report that long chain acyl-CoAs are more potent inhibitors of bSULT1A1 and also of human dopamine sulfotransferase (SULT1A3) when compared with unesterified CoA and short chain-length acyl-CoAs. A complex pattern of inhibition was revealed by systematic variation of palmitoyl-CoA, PAPS, and 7-hydroxycoumarin, the acceptor substrate. Convex plots of apparent K(m)/V(max) versus [palmitoyl-CoA] were adequately modeled using an ordered rapid equilibrium scheme with PAPS as the leading substrate and by accounting for the possible binding of two equivalents of inhibitor to the dimeric enzyme. Interestingly, the first K(i) of 2-3 microm was followed by a second K(i) of only 0.01-0.05 microm, suggesting that positive subunit cooperativity enhances binding of long chain acyl-CoAs to this sulfotransferase. Simultaneous interaction of palmitoyl-CoA with both the nucleotide and phenol binding sites is suggested by two experiments. First, the acyl-CoA displaced 7-hydroxycoumarin from the highly fluorescent bSULT1A1.PAP.7-HC complex in a cooperative manner. Second, palmitoyl-CoA prevented the quenching of bSULT1A1 fluorescence observed with pentachlorophenol. Finally, titrations of bSULT1A1-pentachlorophenol complex with palmitoyl-CoA caused the return of protein fluorescence, and the binding of palmitoyl-CoA was highly cooperative (Hill constant of 1.9). Overall, these results suggest a model of sulfotransferase inhibition in which the 3'-phosphoadenosine-5'-diphosphate moiety of CoA docks to the PAPS domain, and the acyl-pantetheine group docks to the hydrophobic phenol binding domain.     

A novel type of aryl sulfotransferase obtained from an anaerobic bacterium of human intestine

A novel type of aryl sulfotransferase is produced by an anaerobic bacterium of human intestine, Eubacterium A-44. Aryl sulfotransferase separated from this bacterium differs from the sulfotransferase which uses 3'-phosphoadenosine 5'-phosphosulfate as a donor. The enzyme catalyzes stoichiometric transfer of a sulfate group from a phenol sulfate ester to other phenolic compounds, with strict specificity. The optimal pH and molecular weight were 8-9 and 315,000, respectively.     

Inhibition of M and P Phenol Sulfotransferase by Analogues of 3''-Phosphoadenosine-5''-Phosphosulfate

Structural analogues of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (3',5'-PAPS) were examined for their ability to inhibit dopamine and phenol sulfation by the M and P forms of phenol sulfotransferase (PST), respectively. The Ki values for each of the adenosine derivatives were calculated from the rate equation for PST. For both M and P PST, the naturally occurring product 3'-phosphoadenosine-5'-phosphate, (3',5'-PAP), was shown to be the most effective inhibitor. The weakest inhibitors of the two sulfotransferases were 5'-adenosine phosphosulfate and the three AMP derivatives, which were less than 1,000 times as effective as 3',5'-PAP. 5'-ATP, 2',5'-PAPS, 2',5'-PAP, and 5'-ADP were similar in their inhibition of M and P PST and were all approximately 100 times less effective than the natural end product. These data reveal that there is a rigid structural requirement for binding of the ribose portion of adenosine to both M and P PST that involves the groups on both the 3' and 5' positions. The effectiveness of binding to the two enzymes may depend on both steric factors as well as the distribution of negative charges on the ribose ring.     

Studies on bovine adrenal estrogen sulfotransferase. III. Facile synthesis of 3'-phospho- and 2'-phosphoadenosine 5'-phosphosulfate.

A practical synthesis of 3'-phosphoadenosine 5'-phosphosulfate (IV) in yields of 68-72% from adenosine 2',3'-cyclic phosphate 5'-phosphate (II) is described. Reaction of II with triethylamine-N-sulfonic acid affords adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (III) which, on treatment with ribonuclease-T2, provides IV. Spleen phosphodiesterase, on the other hand, converts III to 2'-phosphoadenosine 5'-phosphosulfate (V). The biological activity of IV, measured by sulfate transfer to [6,7-3H2]estrone as mediated by bovine adrenal estrone sulfotransferase (3'-phosphoadenylyl-sulfate:estrone 3-sulfotransferase, EC 2.8.2.4), is identical with that obtained with a sample of IV prepared by an established biochemical procedure. By contrast, V exhibits approximately one-third the activity of the natural isomer.     

DUSP13B/TMDP inhibits stress-activated MAPKs and suppresses AP-1-dependent gene expression

Sulfotransferases catalyze the transfer of sulfate group from para-nitrophenyl sulfate (pNPS) or 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto acceptor molecules in the biosynthesis of sulfate esters. Human pathogenic mycobacteria are known to produce numerous sulfated molecules on their cell surface which have been implicated as important mediators in host-pathogen interactions. The open reading frame stf9, a predicted homologue of sulfotransferase in the Mycobacterium avium genomic data, was cloned and over expressed in Escherichia coli. The recombinant STF9 conserved the characteristic PAPS binding motif of sulfotransferase and was purified as a 44?kDa soluble protein which exhibited transfer of sulfate group from pNPS (K (m) 1.34?mM, V (max) 7.56?nmol/min/mg) onto 3'-phosphoadenosine-5'-phosphate (K (m) 0.24?mM, V (max) 10.36?nmol/min/mg). The recombinant STF9 protein was also capable of transferring sulfate group from PAPS onto certain acceptor substrates in E. coli, and showed binding affinity to the PAP-agarose resin, supporting the sulfotransferase activity of the recombinant STF9 protein. This is the first report of molecular evidence for sulfotransferase activity of a protein from M. avium. Mutation of Arg96 to Ala and Glu170 to Ala abolishes sulfotransferase activity, indicating the importance of Arg96 and Glu170 in STF9 activity catalysis.     

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.     

Apparent sulfation of glycosaminoglycans by ascorbic acid 2-[3 5-S] sulfate: an explanation.

The sulfation of glycosaminoglycans by ascorbic acid 2-[35S]sulfate was studied in costal cartilage and chondrocytes in vitro. Negligable (if any) sulfation of glycosaminoglycans was detected with immediately isolated ascorbic acid 2-[35S]sulfate. However, formation of [35S]glycosaminoglycans was readily detected with ascorbic acid 2-[35S]sulfate which had been stored at minus 20 degrees C for several days. The [35S]glycosaminoglycans did not result from the direct transfer of 35S from ascorbic acid 2-sulfate but rather from a decomposition product of ascorbic acid 2-[35S]sulfate. Evidence is presented to show that the sulfation pathway with the decomposition product involves exchange with inorganic sulfate, and strongly suggests that sulfation proceeds via 3'-phosphoadenosine 5'-phosphosulfate. The decomposition product appears similar to inorganic sulfate in several test systems. In view of these observations, it is suggested that previous conclusions implicating as acid 2-sulfate as a biological sulfate donor, based on the use of ascorbic acid 2-[35S]sulfate be re-evaluated.     

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.     

Characterization of a sulfotransferase responsible for the 4-O-sulfation of terminal beta-N-acetyl-D-galactosamine on asparagine-linked oligosaccharides of glycoprotein hormones

The Asn-linked oligosaccharides on the glycoprotein hormones lutropin (LH) and thyrotropin terminate with the sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2 Man alpha-. Using a chemically synthesized trisaccharide GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOCH3 (GGnM-MCO), we have developed a sensitive assay for the sulfotransferase responsible for the 4-O-sulfation of the terminal beta-D-GalNAc. GGnM-MCO is incubated with a bovine pituitary membrane extract and [35S]3'-phosphoadenosine 5'-phosphosulfate ([35S]PAPS). The sulfated product [35S]SGGnM-MCO is separated from [35S]PAPS, PAPS degradation products and endogenous sulfated products by a two-step procedure utilizing an Ecteola cellulose column and a Sep-Pak (C18) cartridge. Characterization of the [35S]SGGnM-MCO produced in the assay indicates that sulfate is incorporated exclusively on the 4-position of GalNAc. Linear incorporation of sulfate into GGnM-MCO can be maintained for greater than 10 h. GGnM-4-sulfotransferase has a pH optimum of 7.2, requires the presence of a reducing agent, and is stimulated by, but does not require, divalent cations. Initial velocity studies indicate an apparent Km (Henri-Michaelis-Menten equilibrium constant) for PAPS of 4 microM and for GGnM-MCO of 9 microM. Incorporation of sulfate into the trisaccharide is stimulated 3-fold by the presence of basic proteins including deglycosylated LH. The stimulation by deglycosylated LH suggests that the protein component of glycoproteins that bear oligosaccharides terminating with GalNAc-GlcNAc-Man- may modulate GGnM-4-sulfotransferase.     

3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. An optimized assay in homogenates of developing brain.

An optimized in vitro assay of 3'-phosphoadenylysulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11, galactosylceramide sulfotransferase, formerly known as galactocerebroside sulfotransferase) activity is presented, that can be used in crude homogenate of brain tissue of various developmental stages. The enzyme activity is determined by measuring the [35S]sulfatides formed by the enzymic transfer of [35S]sulfate from 3'-phosphoadenoside 5'-phospho [35S]sulfate to galactosylceramides. The sulfatide formation at 30 degrees C is linear up to 30 min and up to a protein concentration of 1 mg per 0.5 ml assay volume. The presence of 0.4% Triton X-100 and 50 micrometer exogenous bovine cerebrosides are optimal for enzyme activity. The pH optimum of the reaction is at pH 6.5 using 0.1 M imidazole buffer. The enzyme reaction is stimulated by NaCl, KCl, MgCl2, CaCl2, MnCl2, ATP and inhibited by ADP. The developmental enzyme activity pattern of mouse brain is the same, if derived from homogenates and microsomes, respectively, under our assay conditions.