In-vitro sulfation of piceatannol by human liver cytosol and recombinant sulfotransferases.

OBJECTIVES: The aim of this study was to investigate the concentration-dependent sulfation of piceatannol, a dietary polyphenol present in grapes and wine and known for its promising anticancer and anti-inflammatory activity. METHODS: Sulfation of piceatannol was investigated in human liver cytosol as well as using a panel of recombinant sulfotransferase isoforms. Furthermore, the chemical structures of novel sulfates were identified by liquid chromatography/mass spectrometry (LC/MS). KEY FINDINGS: In the presence of 3'-phosphoadenosine-5'-phosphosulfate, three metabolites could be detected whose structures were identified by LC/MS/MS as piceatannol disulfate (M1) and two monosulfates (M2, M3). The kinetics of M1 formation exhibited a pattern of substrate inhibition with a Ki of 21.8 +/- 11.3 microm and a Vmax/Km of 7.63 +/- 1.80 microl/mg protein per min. Formation of M2 and M3 showed sigmoidal kinetics with apparent Km and Vmax values of 27.1 +/- 2.90 microm and 118.4 +/- 4.38 pmol/mg protein per min, respectively, for M2; and 35.7 +/- 2.70 microm and 81.8 +/- 2.77 pmol/mg protein per min, respectively, for M3. Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 was formed equally by SULT1A1*1 and SULT1B1 and to a lesser extent by SULT1A1*2. M2 was preferentially catalysed by SULT1A1*2, 1A3 and 1E1. The formation of M3, however, was mainly catalysed by SULT1A2*1 and SULT1A3. CONCLUSIONS: Our results elucidate the importance of piceatannol sulfation in human liver, which must be taken into account in humans after dietary intake of piceatannol.

Human chondroitin 6-sulfotransferase: cloning, gene structure, and chromosomal localization.

Chondroitin 6-sulfotransferase (C6ST) is the key enzyme in the biosynthesis of chondroitin 6-sulfate, a glycosaminoglycan implicated in chondrogenesis, neoplasia, atherosclerosis, and other processes. C6ST catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to carbon 6 of the N-acetylgalactosamine residues of chondroitin. Based on the previously published avian sequence, we searched the database of expressed sequence tags (dbEST) and obtained partial-length cDNAs that we completed by 5'-RACE using human chondrosarcoma and endothelial-cell RNA as template. Stable transfection of our full-length expression construct into CHO-K1 cells resulted in marked increases in C6ST and keratan sulfate sulfotransferase (KSST) enzymatic activities in cell homogenates. The predicted 411 amino acid sequence of human C6ST contains an N-terminal hydrophobic domain consistent with membrane insertion, four potential sites for N-linked glycosylation, several consensus sequences for protein phosphorylation, and one RGD sequence. The human and chick C6ST cDNA share 51% nucleotide identity, 40% amino acyl identity, and 75% amino acyl conservation. The human C6ST gene structure has been elucidated and exhibits an intron-less coding region, and the gene has been mapped to human chromosome 11 by radiation hybrid panel mapping.

Depletion of hepatic 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and sulfate in rats by xenobiotics that are sulfated.

Sulfation is considered a high-affinity but low-capacity conjugation mechanism that is limited by the availability of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the cosubstrate for sulfation. Salicylamide, phenol and 1-naphthol are all known substrates for the sulfation reaction. This study was conducted to determine whether the xenobiotics that are sulfated when administered to rats will lower hepatic PAPS and its precursor, sulfate. Urinary sulfate excretion was reduced 85% to 95% by these compounds. Hepatic PAPS was reduced 73%, 39%, and 87% by salicylamide, phenol and naphthol, respectively, 2 hr after administration of 2 mmol/kg. These compounds also decreased serum sulfate concentrations by 45% to 86% and lowered hepatic sulfate concentrations. In summary, these studies demonstrate that salicylamide, phenol and 1-naphthol lower hepatic PAPS and sulfate concentrations, as well as serum sulfate concentrations. These findings imply that increased sulfation, as a result of the sulfation of xenobiotics, results in depletion of hepatic PAPS concentrations, possibly because the utilization of PAPS by the sulfotransferases exceeds its generation via sulfate activation. Thus the capacity-limited sulfation of high dosages of xenobiotics appears to be due to the reduced availability of hepatic PAPS, which in turn is limited by the availability of sulfate.

Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti.

In this paper we describe the two-step coupled 35S-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol. These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (KD) of 117 nM, similar to that observed with a synthetic tritiated ligand. The first step of the 35S-labelling involves the synthesis of 3'-phosphoadenosine 5'-phospho[35S]sulphate ([35S]PAPS) from ATP and [35S]sulphate using yeast enzymes. The second step exploits the sulphotransferase activity of the R. meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors. This enzyme was found to be active in E. coli cultured at 18 degrees C but not 37 degrees C. NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent Km values of 56 and 70 microM respectively, and exhibited an apparent Km value for non-sulphated Nod factors of 28 microM. Coupling the two steps of the radiolabelling resulted in an efficiency of 35S incorporation from inorganic sulphate to the Nod factors of approximately 10%. These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors.

Affinity labeling of aryl sulfotransferase IV. Identification of a peptide sequence at the binding site for 3'-phosphoadenosine-5'-phosphosulfate.

2'-O-[(R)-Formyl(adenin-9-yl)-methyl]-(S)-glyceraldehyde 3'-triphosphate (also designated as ATP dialdehyde or ATPDA) was utilized as an affinity label for the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding site of an aryl sulfotransferase. The sulfotransferase employed in these studies was rat hepatic aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase, EC 2.8.2.9), for which a cDNA had been previously cloned and expressed in Escherichia coli and the resulting enzyme purified to homogeneity. ATPDA was a time-dependent irreversible inhibitor of the recombinant AST IV, and this inhibition was prevented by including either PAPS or adenosine 3',5'-diphosphate (PAP) in the incubation of AST IV with ATPDA. Experiments relating covalent binding of [2,8-3H]ATPDA with catalytic activity indicated that 1 nmol of the affinity label was bound per nmol of AST IV subunit. Incubation of [2,8-3H]ATPDA with the enzyme followed by reduction with sodium cyanoborohydride, proteolysis with trypsin, and separation of the resulting peptides by high pressure liquid chromatography yielded two labeled peptide fractions. Automated sequence analysis showed that both modified peptide fractions were derived from the same sequence in AST IV: 63-Leu-Glu-Lys-Cys-Gly-Arg-68. Both the sequencing results and examination of the two peptide fractions by matrix-assisted laser desorption ionization mass spectrometry indicated that the ATPDA affinity label was bound to the hexapeptide at both lysine 65 and cysteine 66. These affinity labeled amino acids are located within a region of sequence in AST IV that shows considerable homology with various sulfotransferases that possess diverse specificities for acceptor substrates, and this may provide insight into PAPS binding in other sulfotransferases.

Measurement of contribution from intracellular cysteine to sulfate in phosphoadenosine phosphosulfate in rat ovarian granulosa cells.

Synthesis of the large dermatan sulfate (DS) proteoglycan by rat ovarian granulosa cells was studied using metabolic radiolabel precursors in culture media with varying concentrations of environmental sulfate (20-800 microM) and cysteine (130 and 650 microM). Experiments using [3H]glucosamine and [35S]sulfate showed that the average size of the DS chains and the rate of DS proteoglycan synthesis were independent of the sulfate and cysteine concentrations in the medium. Experiments with [35S]cysteine were then used to determine the contribution that metabolic conversion of cysteine sulfur to sulfate makes to the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) pool which provides the substrate for sulfoester formation in DS synthesis. When 35S in cysteine is metabolized into [35S]PAPS, the specific activity is reduced from that of the [35S]cysteine pool, by dilution with other sulfur sources such as extracellular sulfate, and this dilution factor directly reflects the contribution of cysteine to the PAPS pool. The decreases of 35S specific activity were measured under various sulfate-depleted and cysteine-supplemented conditions by comparing the specific activity of [35S]sulfate ester in the DS chains with that of [35S]cysteine residues in the core protein of the DS proteoglycan. The contribution of sulfur in cysteine to the intracellular PAPS pool was 0.03% in culture medium with normal sulfate (800 microM). Depleted environmental sulfate (20 microM) and increased cysteine supply (650 microM) only increased the sulfur contribution from cysteine to PAPS up to 0.74 and 1.5%, respectively, even though the DS chains were greatly undersulfated (55 and 82% of the control value). Thus, the source of sulfur in the intracellular pool of PAPS was mainly derived from environmental sulfate, and the contribution from cysteine was minimal in these cells.

Acetaminophen decreases adenosine 3'-phosphate 5'-phosphosulfate and uridine diphosphoglucuronic acid in rat liver.

The purpose of this investigation was to determine the effects of acute administration of acetaminophen on adenosine 3'-phosphate 5'-phosphosulfate (PAPS) and UDP-glucuronic acid concentrations in fed male rats. Acetaminophen produced a dosage-dependent decline in rat hepatic PAPS concentrations which was significant after dosages of 150, 300, or 600 mg/kg, ip. The time course of the decline in PAPS values after 600 mg acetaminophen/kg showed that PAPS concentrations reached a nadir 1 hr after dosing (40% of control values). Serum sulfate concentrations were also decreased by large dosages of acetaminophen (32 and 15% of control 2 hr after 150 and 600 mg/kg, respectively) and a significant, positive correlation between serum sulfate and hepatic PAPS concentrations was noted. In addition, hepatic cysteine and glutathione concentrations were lowered by high dosages of acetaminophen. Hepatic UDP-glucuronic acid concentrations were greatly decreased for 2 hr after a dosage of 600 mg acetaminophen/kg (15, 23, and 42% of control 0.5, 1.0, and 2.0 hr after dosing, respectively) whereas 150 mg/kg produced a less pronounced and more transient decrease. These findings demonstrate that both PAPS and UDP-glucuronic acid concentrations in liver are decreased after administration of acetaminophen and imply that capacity-limited sulfation and glucuronidation of acetaminophen are due to a decrease in co-substrate availability.