Phenol Sulfotransferase Research Articles

Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation.

The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4'-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase Ⅱ metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings.

Evaluation of Phenol Sulfotransferase (SULT1A1) Ligand Binding Order

Cytosolic sulfotransferases (SULTs) modify and regulate many endogenous and xenobiotic compounds. There are some disagreements regarding the binding pattern of their nucleotide (PAPS, PAP) and acceptor site ligands. Here we present an investigation of the homodimeric bovine phenol sulfotransferase (bSULT1A1) using several methods not dependent on catalytic turnover. Binding order was not determinable from intrinsic protein fluorescence changes in response to PAP(S), 7-hydroxycoumarin (7-HC) or pentachlorophenol (PCP). Equilibrium dialysis showed no binding of 7-HC to apo-bSULT1A1 up to 100 µM ligand, but inclusion of PAP induced biphasic binding of 7-HC. Isothermal titration calorimetry revealed exothermic half-sites binding of PAP to the dimeric protein. No binding of 7-HC to apo-SULT1A1 was detected, but SULT1A1:PAP also bound 7-HC at 0.5 per subunit. Differential scanning fluorimetry failed to show thermal stabilization of protein by excess 7-HC or PCP, whereas inclusion of PAP increased T_m dramatically. Susceptibility of bSULT1A1 to cleavage by chymotrypsin was not affected by excess 7-HC, but enzyme with PAP ± 7-HC resisted proteolysis. Excess 7-HC did not change protein cysteine reactivity with DTNB, whereas PAP impeded the fast phase of reaction attributable to C168. These observations indicate an ordered binding scheme for bSULT1A1. Testing other SULT ligand binding patterns with these methods is suggested.

Fluorometric coupled enzyme assay for N-sulfotransferase activity of N-deacetylase/N-sulfotransferase (NDST).

N-Deacetylase/N-sulfotransferases (NDSTs) are critical enzymes in heparan sulfate (HS) biosynthesis. Radioactive labeling assays are the preferred methods to determine the N-sulfotransferase activity of NDST. In this study, we developed a fluorometric coupled enzyme assay that is suitable for the study of enzyme kinetics and inhibitory properties of drug candidates derived from a large-scale in silico screening targeting the sulfotransferase moiety of NDST1. The assay measures recombinant mouse NDST1 (mNDST1) sulfotransferase activity by employing its natural substrate adenosine 3'-phophoadenosine-5'-phosphosulfate (PAPS), a bacterial analog of desulphated human HS, Escherichia coli K5 capsular polysaccharide (K5), the fluorogenic substrate 4-methylumbelliferylsulfate and a double mutant of rat phenol sulfotransferase SULT1A1 K56ER68G. Enzyme kinetic analysis of mNDST1 performed with the coupled assay under steady state conditions at pH6.8 and 37°C revealed Km (K5) 34.8μM, Km (PAPS) 10.7μM, Vmax (K5) 0.53±0.13nmol/min/μg enzyme, Vmax (PAPS) 0.69±0.05nmol/min/μg enzyme and the specific enzyme activity of 394pmol/min/μg enzyme. The pH optimum of mNDST1 is pH8.2. Our data indicate that mNDST1 is specific for K5 substrate. Finally, we showed that the mNDST1 coupled assay can be utilized to assess potential enzyme inhibitors for drug development.

Phenotyping SULT in Man: a Simple Metric Using Paracetamol as Probe

Introduction Sulfotransferase enzymes (SULT) catalyze sulfoconjugation of drugs, as well as endogenous mediators and environmental xenobiotics. To address the scarce evidence on sulfonation activity from clinical research, we developed a clinical metabolic phenotyping method using paracetamol as a probe substrate. Sulfonation accounts for up to 35% of the total paracetamol metabolism in humans and is mediated mainly by the activity of SULT1A1 and SULT1A3 (phenol sulfotransferases), in liver and small intestine respectively. Our aim was to estimate sulfonation capability of phenolic compounds and study its intraindividual variability in man. Population and Methods: This study was approved by the Portuguese National Ethics Committee. We recruited a population of 36 healthy adults (12 men and 24 women, 12 on oral contraceptives). Subjects received 1 g of oral paracetamol on three different occasions. Paracetamol and its metabolites (paracetamol sulfate, paracetamol glucuronate, paracetamol cysteine-S-conjugate and paracetamol mercapturate) were measured in plasma and spot urine samples using liquid chromatography-high resolution mass spectrometry. Paracetamol sulfonation index (PSI) was used to express SULT activity. PSI was calculated for urine (uPSI) and for plasma (pPSI) samples. We tested differences between groups using ANOVA and we used Pearson's correlation coefficient. Results The frequency distribution histograms of pPSI and uPSI were positively skewed in the studied population (Figure 1). The mean pPSI and uPSI were 0.43 (range 0.34 to 0.60) and 0.40 (range 0.31 to 0.55) respectively (Figure 2). PSI showed low intraindividual variability (mean coefficient of variation was 10% for pPSI and 13% for uPSI). There was a good correlation between values in plasma and spot urine samples (Pearson r=0.79). Urinary PSI was independent of factors not related to SULT activity, such as urine pH or eGFR. Gender and oral contraceptive intake had no impact on PSI. Discussion Our SULT phenotyping method is a simple non-invasive procedure requiring urine spot samples, using the safe and convenient drug paracetamol as a probe substrate, and with low intraindividual coefficient of variation. Although it will not give us mechanistic information, it will provide us an empirical measure of an individual's sulfonator status. To the best of our knowledge, our method provides the first in vivo empirical measure of an individual's phenol sulfonation capability and of its intraindividual variability.

Decreased phenol sulfotransferase activities associated with hyperserotonemia in autism spectrum disorders

Hyperserotonemia is the most replicated biochemical abnormality associated with autism spectrum disorders (ASD). However, previous studies of serotonin synthesis, catabolism, and transport have not elucidated the mechanisms underlying this hyperserotonemia. Here we investigated serotonin sulfation by phenol sulfotransferases (PST) in blood samples from 97 individuals with ASD and their first-degree relatives (138 parents and 56 siblings), compared with 106 controls. We report a deficient activity of both PST isoforms (M and P) in platelets from individuals with ASD (35% and 78% of patients, respectively), confirmed in autoptic tissues (9 pineal gland samples from individuals with ASD—an important source of serotonin). Platelet PST-M deficiency was strongly associated with hyperserotonemia in individuals with ASD. We then explore genetic or pharmacologic modulation of PST activities in mice: variations of PST activities were associated with marked variations of blood serotonin, demonstrating the influence of the sulfation pathway on serotonemia. We also conducted in 1645 individuals an extensive study of SULT1A genes, encoding PST and mapping at highly polymorphic 16p11.2 locus, which did not reveal an association between copy number or single nucleotide variations and PST activity, blood serotonin or the risk of ASD. In contrast, our broader assessment of sulfation metabolism in ASD showed impairments of other sulfation-related markers, including inorganic sulfate, heparan-sulfate, and heparin sulfate-sulfotransferase. Our study proposes for the first time a compelling mechanism for hyperserotonemia, in a context of global impairment of sulfation metabolism in ASD.

An activity-based fluorescent sensor for the detection of the phenol sulfotransferase SULT1A1 in living cells.

Human phenol sulfotransferases mediate the transfer of a sulfuryl moiety from the activated sulfate donor PAPS to hydroxy-containing substrates, altering substrate solubility and charge to affect phase II metabolism and cell signaling. Here, we present the development, computational modeling, in vitro enzymology, and biological application of STS-3, an activity-based fluorescent sensor for the SULT1A1 isoform.

Silicon Nanowire Field-Effect Transistor as Biosensing Platforms for Post-Translational Modification.

Protein tyrosine sulfation (PTS), a vital post-translational modification, facilitates protein–protein interactions and regulates many physiological and pathological responses. Monitoring PTS has been difficult owing to the instability of sulfated proteins and the lack of a suitable method for detecting the protein sulfate ester. In this study, we combined an in situ PTS system with a high-sensitivity polysilicon nanowire field-effect transistor (pSNWFET)-based sensor to directly monitor PTS formation. A peptide containing the tyrosine sulfation site of P-selectin glycoprotein ligand (PSGL)-1 was immobilized onto the surface of the pSNWFET by using 3-aminopropyltriethoxysilane and glutaraldehyde as linker molecules. A coupled enzyme sulfation system consisting of tyrosylprotein sulfotransferase and phenol sulfotransferase was used to catalyze PTS of the immobilized PSGL-1 peptide. Enzyme-catalyzed sulfation of the immobilized peptide was readily observed through the shift of the drain current–gate voltage curves of the pSNWFET before and after PTS. We expect that this approach can be developed as a next generation biochip for biomedical research and industries.

Evaluation of a conserved tryptophanyl residue in donor substrate binding and catalysis by a phenol sulfotransferase (SULT1A1)

Structural determinations of members of the sulfotransferase (SULT) family suggest a direct interaction between a conserved tryptophanyl side chain and bound 3′-phosphoadenosine-5′-phosphate (PAP). We have prepared and purified mutants of the bovine SULT1A1, a very conserved homolog to the human SULT1A1, in which tryptophanyl-53 was sequentially trimmed to tyrosine, leucine, and alanine. Differential scanning fluorimetry indicated structural stabilities of the mutant proteins comparable to the wild type SULT1A1; however, less thermal stabilizations by PAP plus pentachlorophenol were observed with the mutants, suggesting weakened ligand binding. Protein fluorescence of the wild type enzyme decreased 6.5% upon binding PAP, whereas no changes occurred with the mutant enzymes. This reveals that W53, or its positional counterpart, has been the source of emission intensity changes used in previous investigations of other SULTs. Fluorescence resonance energy transfer from excited tryptophans to bound 7-hydroxycoumarin, as induced by PAP, indicated weakened binding of ligands to the mutant SULTs. This was also encountered and quantified in initial rate kinetic analyses. Ablation of the PAPS adenine-to-W53 ring interaction, shown by the W53A mutant enzyme, resulted in a 6.4-fold increase in KPAPS and a 92% decrease in kcat/KPAPS. Measured KPAPS values reveal the W53 indole ring contribution to PAPS binding to be 1.1 kcal/mol (4.6 kJ/mol). These results verify the structurally-inferred role for the π−π stacking interaction between PAP(S) and the conserved tryptophanyl residue in SULT1A1 and other members of the SULT family.

Abstract 606: Circulating proteins associated with ovarian cancer risk and survival are enriched for immune-related pathways

Abstract Most women with epithelial ovarian cancer (EOC) are diagnosed after the disease has metastasized and five-year survival rates in this group continue to remain very poor. Circulating proteins may illuminate EOC etiology and have the potential to serve as biomarkers for early detection and diagnosis. We performed a two-sample Mendelian randomization (MR) study with circulating protein levels as exposures and susceptibility to EOC and its histological subtypes as outcomes. We used previously identified quantitative trait loci (QTLs) robustly associated with plasma levels of 1,478 proteins as genetic instruments and corresponding summary genetic association statistics for these instruments derived from 22,406 EOC cases and 40,941 controls. Among proteins that had only a single QTL variant available as a genetic instrument, we identified 12 proteins whose circulating levels were associated with overall EOC risk at a False Discovery Rate (FDR) of < 0.1. For nine of the 12 proteins, the association was driven by variants near ABO, which is both a genome-wide significant high grade serous histotype risk locus and a hub QTL for several proteins. Three of these 12 proteins and their QTLs were, however, not linked to the ABO locus and included the products of the NOTCH signaling genes MFAP2 and DLK1. Among proteins that had multiple QTL variants available for the creation of more robust multi-variant genetic instruments, we identified 33 protein level-EOC risk associations at FDR < 0.1. Once again, we observed that the majority of associations (19/33) were driven by variants at the ABO locus. Nine of the 14 remaining associations were for overall and high grade serous EOC risk and included predominantly immune system proteins: ARTS1 (HLA class I), CPNE1 (involved in innate immunity), Macrophage mannose receptor (HLA class I), DQA2 (HLA class II), RGAP1 (innate immunity), Cathepsin H (innate immunity), and FCG2B (the low affinity receptor for the Fc region of immunoglobulin gamma). We observed one mucinous histotype-specific association (complement C4) and two clear cell histotype-specific associations (KI2S2 and PSG4). KI2S2 is a Killer cell immunoglobulin-like receptor expressed by natural killer cells and PSG4 is a member of the carcinoembryonic antigen gene family that also includes the gene encoding the tumor marker for colorectal cancer. Finally, we also performed two-sample MR analyses using survival as the outcome and identified two proteins whose plasma levels were associated with overall EOC survival at FDR < 0.1: SULT1E, a phenol sulfotransferase that conjugates sulfates to estrogens, and MA2B2. Thus, we identified circulating proteins that are candidate biomarkers for EOC risk and survival. They represent promising starting points for follow-up studies and the measurement of plasma levels of these proteins in EOC cohorts is required to validate these associations. Citation Format: Daniel P. Considine. Circulating proteins associated with ovarian cancer risk and survival are enriched for immune-related pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 606.

Allosteres to regulate neurotransmitter sulfonation

Catecholamine neurotransmitter levels in the synapses of the brain shape human disposition-cognitive flexibility, aggression, depression, and reward seeking-and manipulating these levels is a major objective of the pharmaceutical industry. Certain neurotransmitters are extensively sulfonated and inactivated by human sulfotransferase 1A3 (SULT1A3). To our knowledge, sulfonation as a therapeutic means of regulating transmitter activity has not been explored. Here, we describe the discovery of a SULT1A3 allosteric site that can be used to inhibit the enzyme. The structure of the new site is determined using spin-label-triangulation NMR. The site forms a cleft at the edge of a conserved ∼30-residue active-site cap that must open and close during the catalytic cycle. Allosteres anchor into the site via π-stacking interactions with two residues that sandwich the planar core of the allostere and inhibit the enzyme through cap-stabilizing interactions with substituents attached to the core. Changes in cap free energy were calculated ab initio as a function of core substituents and used to design and synthesize a series of inhibitors intended to progressively stabilize the cap and slow turnover. The inhibitors bound tightly (34 nm to 7.4 μm) and exhibited progressive inhibition. The cap-stabilizing effects of the inhibitors were experimentally determined and agreed remarkably well with the theoretical predictions. These studies establish a reliable heuristic for the design of SULT1A3 allosteric inhibitors and demonstrate that the free-energy changes of a small, dynamic loop that is critical for SULT substrate selection and turnover can be calculated accurately.

Preparation of Tyrosylprotein Sulfotransferases for In Vitro One-Pot Enzymatic Synthesis of Sulfated Proteins/Peptides.

Protein tyrosine sulfation (PTS), catalyzed by membrane-anchored tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications of secretory and transmembrane proteins. PTS, a key modulator of extracellular protein–protein interactions, accounts for various important biological activities, namely, virus entry, inflammation, coagulation, and sterility. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying PTS in biology. A sulfated protein was prepared using a TPST-coupled protein sulfation system that involves the generation of the active sulfate 3′-phosphoadenosine-5′-phosphosulfate (PAPS) through either PAPS synthetase (PAPSS) or phenol sulfotransferase. The preparation of sulfated proteins was confirmed through radiometric or immunochemical assays. In this study, enzymatically active Drosophila melanogaster TPST (DmTPST) and human TPSTs (hTPST1 and hTPST2) were expressed in Escherichia coli BL21(DE3) host cells and purified to homogeneity in high yield. Our results revealed that recombinant DmTPST was particularly useful considering its catalytic efficiency and ease of preparation in large quantities. This study provides tools for high-efficiency, one-step synthesis of sulfated proteins and peptides that are useful for further deciphering the mechanisms, functions, and future applications of PTS.

Targeting the expression of glutathione- and sulfate-dependent detoxification enzymes in HepG2 cells by oxygen in minimal and amino acid enriched medium

Cancer cells exhibit specific metabolism allowing them to survive and proliferate in various oxygen conditions and nutrients' availability. Hepatocytes are highly active metabolically and thus very sensitive to hypoxia. The purpose of the study was to investigate the effect of oxygen on the expression of phase II detoxification enzymes in hepatocellular carcinoma cells (HepG2) cultured in minimal and rich media (with nonessential amino acids and GSH). The cells were cultured at 1% hypoxia, 10% tissue normoxia, and 21% atmospheric normoxia. The total cell count was determined by trypan blue exclusion dye and the expression on mRNA level by RT-PCR. The result indicated that the expression of glutathione-dependent enzymes (GSTA, M, P, and GPX2) was sensitive to oxygen and medium type. At 1% hypoxia the enzyme expression (with the exception of GSTA) was higher in minimal compared to rich medium, whereas at 10% normoxia it was higher in the rich medium. The expression was oxygen-dependent in both types of medium. Among phenol sulfotransferase SULT1A1 was not sensitive to studied factors, whereas the expression of SULT1A3 was depended on oxygen only in minimal medium.It can be concluded that in HepG2 cells, the detoxification by conjugation with glutathione and, to a lower extent with sulfate, may be affected by hypoxia and/or limited nutrients' availability. Besides, because the data obtained at 10% oxygen significantly differ from those at 21%, the comparative studies on hypoxia should be performed in relation to 10% but not 21% oxygen.

Polymorphisms in genes of respiratory control and sudden infant death syndrome.

Sudden infant death syndrome (SIDS) is a multifactorial syndrome and assumingly, among other mechanisms, a deficit in respiratory control leads to a failure of arousal and autoresuscitation when the child is challenged by a stressful homeostatic event, e.g., hypoxia. We hypothesize that genetic polymorphisms involved in respiratory control mediated in the medulla oblongata contribute to SIDS. Therefore, a total of 366 SIDS cases and 421 controls were genotyped for 48 SNPs in 41 candidate genes. Genotyping was performed using Fluidigm nanofluidic technology. Results were obtained for 356 SIDS and 406 controls and 38 SNPs. After correction for multiple testing, one SNP retained a nominally significant association with seasonal SIDS: rs1801030 in the phenol sulfotransferase 1A1 gene (subgroup: death occurring during summer). A borderline association could be also observed for rs563649 in the opioid receptor μ1 gene in a recessive model (subgroup: death occurring during autumn). As a conclusion, although these data suggest two SNPs to be associated with different subgroups of SIDS cases, none of them can fully explain the SIDS condition, consistent with its multifactorial etiology. Given the great complexity of respiratory control and our initial findings reported here, we believe it is worthwhile to further investigate genes involved in the respiratory system.

Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation.

BackgroundAstrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation.MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35–55 immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes.ResultsThe spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes.ConclusionsWe described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0348-y) contains supplementary material, which is available to authorized users.

Characterization of bovine phenol sulfotransferases: evidence of a major role for SULT1B1 in the liver

1. Cattle are an important component of the human food chain. Drugs used either legally or illegally in cattle may therefore enter the food chain and it is thus important to understand pathways of drug metabolism in this species, including sulfation catalyzed by the sulfotransferases (SULTs).2. In this study, we have analyzed the sulfation of 4-nitrophenol and other compounds in male and female bovine liver and characterized recombinant bovine SULT isoforms 1A1 and 1B1 expressed in Escherichia coli.3. We found that, in contrast to most other mammalian species, the major phenol sulfotransferase SULT1A1 is not expressed in bovine liver. Rather SULT1B1 seems to be a major form in both male and female bovine liver.4. We also identified kinetic differences between bovine and human SULT1A1 and, using the human SULT1A1 crystal structure, identified two amino acid positions in the active site of bovine SULT1A1 (Ile89Val and Phe247Val) that may be responsible for these differences.

Apoptosis inducing anthraquinone rhein and emodin differentially suppress human dehydroepiandrosterone sulfotransferase (hSULT2A1) and phenol sulfotransferases (hSULT1A1) in Hep-G2 and Caco-2 cells

The anti-cancer and apoptosis-inducing drugs rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) and emodin (3methyl-1, 6, 8-trihydro-xyanthrax-quinone) are clinically very important. They modulate cell cycle via tumor suppressor gene, immuno-receptors and ligand activated nuclear receptors. Our recent observation suggests for the first time that 10 days of treatment of either drug with various concentrations (0.01 to 100 M) differentially suppressed the sulfotransferases (SULTs) activities and protein expressions in human hepatocellular carcinoma (Hep G2) and intestinal carcinoma (Caco-2) cell lines. SULTs are phase II drug metabolizing enzymes which catalyze the sulfuryl group transfer to hydroxyl containing endobiotics and xenobiotics. In the present investigation, dehydroepiandrosterone SULT (hSULT1A1) was markedly suppressed by these drugs in human cells. This is the first time report which demonstrates that rhein and emodin may regulate human SULTs. Our finding has important physiological and clinical implications. It will help in the understanding of the SULTs regulations by clinically important drugs and xenobiotics. In future, these drugs may be used in a better defined manner, taking into account its SULTs suppression effects with possible physiological consequences.

Substances Used in Wine Clarification as Potential Allergens

Potential allergens also include proteins and substances used in the clarification of wine: fish gelatin or isinglass, protein from chicken eggs (ovalbumin), milk (casein) products, rubber arabicum, lysozyme, pectinase, cellulose, glucosidase, urease, and aroma enzymes (1). Furthermore, moulds—especially Botrytis cinerea, which is responsible for noble rot in grapes—as well as insect proteins (for example, wasp venom or bee venom), which may have contaminated the mash during the pressing process, play an allergenic part that should not be underestimated (1, 2). Triggers for allergic intolerance reactions such as headaches or migraine after ingestion of red wine may also be the phenolic flavonoids that are present in the skin of the grapes (anthocyanins and catechins), because they inhibit catechol-O-methyltransferase in the intestinal wall as well as the phenol sulfotransferase. Other non-organic ingredients, such as the ethanol metabolite acetaldehyde and acetic acid, also play a part in wine intoleranceoften in the shape of urticaria (1). The intolerance to alcohol in general that was observed by the authors is supported by a representative survey in adults in Copenhagen (4142/6000=70.7% response rate), which showed a prevalence of hypersensitivity reactions after alcohol consumption (especially red wine) of 8% (3, 4). In Bavaria, for example, all superior quality wines have to be examined by the Bavarian Health and Food Safety Agency, in order to ensure that the legally defined upper limits of sulfur dioxide are adhered to. In order to protect consumers from intolerance reactions to sulfites, this type of state controlled wine examination in addition to the official testing number on the label would also be desirable for other German states.

Fluorescence assay for protein post-translational tyrosine sulfation

We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein-protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3'-phosphate 5'-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3',5'-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.

A nano switch mechanism for the redox-responsive sulfotransferase

Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase (rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59–70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase (hSULT1A1) and human alcohol sulfotransferase (hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.

An equation to model fluorescent probe displacement by non‐fluorescent competitive ligands

Measurement of ligand binding to a receptor to evaluate Kd values is fundamental. If binding is spectrally silent, then a probe with dynamic optical properties can be employed. Such is the case in our studies of nucleotides binding to phenol sulfotransferase, SULT1A1, one such enzyme that requires 3’‐phosphoadenosine‐ 5’‐phosphosulfate (PAPS). Whereas binding of PAP(S) and other nucleotides is spectrally invisible, 3’‐trinitrophenylated‐adenylates (e.g. TNP‐AMP) form a fluorescent SULT:TNP‐AXP complex. These TNP probes have seen considerable applications for kinases and other ATP‐binding proteins. However, many published analyses of probe displacement by biological nucleotides are confounded by assumptions and questionable analyses. We have derived an equation that predicts binding constants by adequately modeling competition fluorimetric titrations, and have applied it to both published works and to our SULT analyses. The procedure requires titration with the probe and determination of its Kd value, followed by competitive titration with another ligand. Parameters required or derived are: total receptor, probe, and ligand concentrations; Kd (probe), Kd (ligand), Fmax, and observed F values. We see agreement between Kd values obtained by this method and by kinetic and isothermal titration methods, for the binding of PAP to recombinant bovine SULT1A1.