Fluorometric assay for alcohol sulfotransferase

Chen, Wei Ti; Liu, Ming Chih; Yang, Yuh‐Shyong

doi:10.1016/j.ab.2004.12.016

Cited by 27 publications

(20 citation statements)

References 26 publications

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“…A linear response was obtained when 1.49 to 14.9 nM (0.1-1 g) SULT2A1 was added in the standard assay condition. In the reaction condition, excess amount of K65ER68G of rat SULT1A1 was added to catalyze the regeneration of PAPS so that the production of PAP catalyzed by SULT2A1 could not accumulate (Chen et al, 2005). The reaction condition for SULT1A3 standard assay was similar to that for SULT2A1 assay except that the reaction mixture contained 1.5 to 7.3 nM (0.1 to 0.5 g) SULT1A3 and 30 M dopamine at 37°C to replace SULT2A1 and DHEA or ADT, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Identification and Characterization of Two Amino Acids Critical for the Substrate Inhibition of Human Dehydroepiandrosterone Sulfotransferase (SULT2A1)

Hsieh

Liu

et al. 2007

Mol Pharmacol

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Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K i value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/ Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.

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Section: Methodsmentioning

confidence: 99%

“…The activities of wild-type and mutant SULT2A1 were determined according to the change of fluorescence based on a coupled-enzyme assay method (Chen et al, 2005). The fluorescence of 4-methylumbelliferone at 460 nm was measured upon excitation at 355 nm.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Two Amino Acids Critical for the Substrate Inhibition of Human Dehydroepiandrosterone Sulfotransferase (SULT2A1)

Hsieh

Liu

et al. 2007

Mol Pharmacol

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confidence: 99%

“…Many strains of Escherichia coli, the main source of urinary tract infections, contain the enzyme ␤-glucuronidase, which hydrolyzes EtG. Given that UGT and SULT activity also occur with some bacteria (19,20 ), we examined whether human pathogens may enable postcollection synthesis of EtG and EtS from ethanol in urine.Fresh human urine specimens (anonymous surplus volumes) with confirmed growth of common pathogenic bacteria (E. coli, n ϭ 36; Klebsiella pneumoniae, n ϭ 6; Enterobacter cloacae, n ϭ 6), as identified by culture on standard solid media, were used (study approved by the local ethics committee). The samples had been submitted for routine diagnostic testing in the Department of Clinical Microbiology, Karolinska University Hospital, and were stored refrigerated until use.…”

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Postcollection Synthesis of Ethyl Glucuronide by Bacteria in Urine May Cause False Identification of Alcohol Consumption

Helander¹,

2007

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Background: Ethyl glucuronide (EtG) is a minor ethanol metabolite used as a specific marker to document recent alcohol consumption; confirm abstinence in treatment programs, workplaces, and schools; and provide legal proof of drinking. This study examined if bacterial pathogens in urine may enable postsampling synthesis of EtG and ethyl sulfate (EtS) from ethanol, leading to clinical false-positive results. Methods: Urine specimens with confirmed growth of Escherichia coli, Klebsiella pneumoniae, or Enterobacter cloacae were stored at room temperature in the presence of ethanol. Ethanol was either added to the samples or generated by inoculation with the fermenting yeast species Candida albicans and glucose as substrate. EtG and EtS were measured by LC-MS. Results: High concentrations of EtG (24-h range 0.5-17.6 mg/L) were produced during storage in 35% of E. coliinfected urines containing ethanol. In some specimens that were initially EtG positive because of recent alcohol consumption, EtG was also sensitive to degradation by bacterial hydrolysis. In contrast, EtS was completely stable under these conditions. Conclusions: The presence of EtG in urine is not a unique indicator of recent drinking, but might originate from postcollection synthesis if specimens are infected with E. coli and contain ethanol. Given the associated risks for false identification of alcohol consumption and false-negative EtG results due to bacterial degradation, we recommend that measurement of EtG be combined with EtS, or in the future possibly replaced by EtS. © 2007 American Association for Clinical ChemistryEarly recognition of problem drinking or relapse is important to ensure adequate alcohol treatment strategies (1 ). This goal has been hampered by a lack of sufficiently sensitive and specific diagnostic methods. The reliability of self-reporting is limited by denial and underreporting (2 ). The time frame for identifying alcohol use by ethanol testing is usually limited to Ͻ12 h, because of rapid metabolism and excretion (3 ). Research has therefore focused on developing alcohol biomarkers with a longer detection window (4 ).A new laboratory marker for detecting recent alcohol consumption is ethyl glucuronide (EtG) (5 ). EtG and ethyl sulfate (EtS) (6 ) are minor ethanol metabolites formed by uridine diphosphate-glucuronosyltransferase (UGT) and sulfotransferase (SULT), respectively, and excreted in urine for a longer time than ethanol (7-10 ). Positive EtG and/or EtS test results thus provide a strong indication that the person has recently consumed alcohol, even when ethanol is no longer detectable (9 ). LC-MS methods are available for EtG and EtS detection (6, 10 ), as is an enzyme immunoassay for EtG (DRI ® EtG, Microgenics). EtG has been recommended for forensic application (11-13 ) and is used for documentation of abstinence in treatment programs, for alcohol testing in the workplace and schools, and as legal proof of drinking (known as "the 80-h alcohol test"). However, the high diagnostic sensitivity of the EtG test has ...

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“…Moreover, other nucleotides can perform this function [51]. The SULT-catalyzed reaction using the alternate activated sulfate source is a convenient method for determining the activities of a variety of SULTs [92][93][94] and determining PAP in biological samples [95].…”

Section: Sulfation With Alternate Activated Sulfate Donormentioning

confidence: 99%

Mechanism of sulfotransferase pharmacogenetics in altered xenobiotic metabolism

Chen

Wang

Hou

et al. 2015

Expert Opinion on Drug Metabolism & Toxicology

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A wealth of information is available in the literature for understanding the detailed mechanisms underlying xenobiotic sulfation. We reviewed information regarding the sequence, structure and reaction mechanism of SULTs and explained how SULT activities altered. In addition to revealing the SULT kinetics, the mRNA expression of specific SULTs in tissues that revealed their distribution in tissues also affects overall SULT activities. Understanding of the structure-function relationship and the reaction mechanism of SULTs is valuable for understanding, preventing and treating diseases.

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Fluorometric assay for alcohol sulfotransferase

Cited by 27 publications

References 26 publications

Identification and Characterization of Two Amino Acids Critical for the Substrate Inhibition of Human Dehydroepiandrosterone Sulfotransferase (SULT2A1)

Identification and Characterization of Two Amino Acids Critical for the Substrate Inhibition of Human Dehydroepiandrosterone Sulfotransferase (SULT2A1)

Postcollection Synthesis of Ethyl Glucuronide by Bacteria in Urine May Cause False Identification of Alcohol Consumption

Mechanism of sulfotransferase pharmacogenetics in altered xenobiotic metabolism

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