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Vol. 28, Issue 9, 1063-1068, September 2000
Department of Surgery, Central Arkansas Veterans' Health Care System, Little Rock, Arkansas (L.T.F., N.P.L.); Divisions of Molecular Epidemiology (S.O., H.-C.C., F.F.K.), Biometry (R.R.D.), and Biochemical Toxicology (D.R.D.), National Center for Toxicological Research, Jefferson, Arkansas; and University of Arkansas for Medical Sciences, Little Rock, Arkansas (S.A.N., N.P.L., F.F.K.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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A thermostable phenol sulfotransferase, SULT1A1, has been
implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its
putative role in mediating risk for human environmental disease. A
simple endpoint colorimetric assay is described that can be used for
rapid phenotyping of SULT1A1 activity in human populations. The assay
utilizes a microtiter-plate format and relatively small amounts of
platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of
2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of
p-nitrophenyl sulfate to the assay contributes to an
effective PAPS-regenerating system. In contrast to other
sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP)
does not accumulate during the incubation to interfere with enzyme
activity, but instead serves as a cofactor to cause the removal of
sulfate from p-nitrophenyl sulfate to regenerate PAPS.
This reaction concomitantly results in generation of
p-nitrophenol that can be quantified colorimetrically at
405 nm (
= 18,200 M
1) to give an indirect measure of
sulfotransferase activity. Using platelet enzyme preparations from
adult human subjects, sulfation rates of two prototypical thermostable
phenol sulfotransferase substrates (2-naphthol and
p-nitrophenol) and one thermolabile phenol
sulfotransferase substrate (dopamine) were determined using standard
radiochemical protocols. These data were then compared with results
from the colorimetric assay using 2-naphthol as substrate. There was a
good correlation between the phenotyping assay and radiochemical assays
for both 2-naphthol sulfotransferase and p-nitrophenol
sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there
was no correlation between SULT1A1 activity and dopamine
sulfotransferase activity (r = 0.07) in these human
platelet preparations. This inexpensive and rapid method for
phenotyping SULT1A1 activity may help investigators assess a role for
this enzyme in disease susceptibility.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Sulfation
is a mechanism by which a wide variety of hormones, neurotransmitters,
drugs, and xenobiotic compounds are detoxified (Jakoby and Zeigler,
1990
). Alternatively, sulfation can be an important mechanism of
bioactivation, because reactive sulfuric esters of carcinogenic
arylamines and heterocyclic amines have been shown to bind covalently
to DNA (Kadlubar et al., 1976; Kato and Yamazoe, 1987; Abu-Zeid et al.,
1992). There are seven known human cytosolic enzymes involved in
sulfate conjugation, an estrogen sulfotransferase (designated SULT1E1),
three hydroxysteroid sulfotransferases (designated SULT2A1, SULT2B1a,
and SULT2B1b), and three phenol sulfotransferases
(PSTs),3 designated
SULT1A1, SULT1A2, and SULT1A3 [reviewed in Her et al. (1998) and
Sakakibara et al. (1998)]. Expression of other human cytosolic PSTs
(SULT1C1, SULT1B1/2) have also been reported (Fujita et al., 1997;
Sakakibara et al., 1998; Wang et al., 1998; Windmill et al., 1998). The
thermolabile form of PST appears to be expressed in a variety of
tissues, and is important for the conjugation of dopamine and other
monoamines. The two thermostable PSTs, SULT1A1 and SULT1A2, detoxify
numerous phenolic compounds, and are also thought to play the primary
role in sulfotransferase-mediated activation of proximate carcinogens,
such as N-hydroxy-2-acetylaminofluorene, N-hydroxy-4-aminobiphenyl, and
N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Ozawa et al., 1994; Chou et al., 1995a,b). However, of these, only
SULT1A1 is expressed at appreciable levels in human liver (Ozawa et
al., 1998; named ST1A3 in this reference). Anderson et al. (1998) have
further reported the purification of a single major thermostable PST,
presumably SULT1A1, in human platelets. The thermolabile SULT1A3, as
measured by dopamine sulfotransferase activity, is also expressed in
significant amounts in human platelets (Anderson et al., 1998).
However, on a per milligram of protein basis, both SULT1A1 and SULT1A3
are expressed at relatively lower levels in platelets as compared with
human liver. Other sulfotransferase isoforms have not been reported in
human platelets.
Disease mechanisms related to SULT1A1 and SULT1A3 expression have been
studied previously using human peripheral blood. Weinshilboum and
collaborators reported good concordance between thermostable PST
activity in human platelets and jejunal mucosa, liver, and cerebral
cortex; however, there was no such correlation for the thermolabile PST
in platelets and these tissues (Weinshilboum, 1988; Sundaram et al.,
1989; Weinshilboum, 1990). Thus, the coordinate regulation between
SULT1A1 in both liver and blood platelets provides the opportunity for
the utilization of minimally invasive phenotyping methods for
estimating hepatic SULT1A1 levels and hence its role in carcinogen and
drug metabolism.
Pharmacogenetic studies of platelet-derived SULT1A1 and SULT1A3
activities further indicate that the activities of these two isoforms
are regulated by separate genetic mechanisms or polymorphisms (Price et
al., 1988; Raftogianis et al., 1996). With respect to SULT1A1 activity,
this may account, at least in part, for up to a 50-fold individual
variability in phenotype (Raftogianis et al., 1997). A largely genetic
basis for variability in human SULT1A1 activity is also supported by
the recent characterization of multiple variant alleles (Henkel et al.,
1995; Weinshilboum et al., 1997; Ozawa et al., 1998). Less is known
about host-specific and environmental factors that impact on
sulfotransferase expression and possibly contribute to disease
susceptibility. However, numerous studies in animal, human, and cell
models suggest modulation by gender, hormones, disease, developmental
factors, hypoxia, diet, season, and xenobiotic exposure (reviewed by
Coughtrie et al., 1998).
At present, the link between SULT1A1 activity and risk for human disease is relatively unexplored. The standard phenotyping assay involves the use of expensive radioactive substrates and labor-intensive precipitation and centrifugation steps, factors that limit its application to large scale phenotyping sudies. However, in this report, a simple and reproducible endpoint colorimetric assay is described that has been adapted to a 96-well microtiter plate format. The assay was validated by measuring 2-naphthol sulfation rates in preparations of normal human platelets (n = 8), by correlating these results to those obtained using standard radiochemical methods, and by product identification using liquid chromatography-mass spectrometry. Three prototypical SULT substrates have been used to verify that the colorimetric assay is specific for SULT1A1, and not for SULT1A2 or SULT1A3, and were supported by measurements of either SULT mRNA or immunoreactive protein and by the use of specific inhibitors. These validation studies are critical for showing that the colorimetric assay is a useful and inexpensive alternative, particularly for SULT1A1 phenotyping of population-based molecular epidemiological studies.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents.
The following chemicals (and their sources) were obtained commercially:
phosphate-buffered saline (pH 7.4; #1000-3), trisodium citrate, citric
acid, triethanolamine, 2-mercaptoethanol, acetylsalicyclic acid,
2-naphthol, potassium monobasic and dibasic phosphate, magnesium chloride, 5'-phosphoadenosine 3'-phosphosulfate (PAPS),
p-nitrophenylsulfate, parygyline, dopamine, barium
hydroxide, zinc chloride, and barium acetate from the Sigma Chemical
Co. (St. Louis, MO); N,N-dimethylformamide, triethylamine, and 1,3-dicyclohexylcarbodiimide from Aldrich Chemical Co., Inc. (Milwaukee, WI); dextrose from Fisher Scientific (Houston, TX); sucrose from Bio-Rad Laboratories (Hercules, CA); and
[35S]PAPS from New England Nuclear, Inc.
(Boston, MA). Rabbit anti-human SULT1B1/2 (Wang et al., 1998) was
kindly provided by Dr. Charles Falany (University of Alabama at Birmingham).
Human Subjects and Blood Processing. To assess intraindividual variability in platelet PST activities, blood platelets were isolated (on four separate occasions at weekly intervals) from healthy human volunteers, 24 to 65 years old, who were participants in an ongoing colon cancer case control study. For each individual, approximately 24 to 32 ml of blood was collected in four to six Vacutainer tubes ("yellow-top ACD" tubes; Becton-Dickinson, from Fisher Scientific, Houston, TX). The tubes were maintained at room temperature with gentle rocking for up to 24 h before processing. Isolation of the platelets from blood cells was then carried out by differential centrifugation, also at room temperature. Briefly, blood lymphocytes and platelets were collected from the upper interphase of discontinuous Histopaque gradients (polysucrose; Sigma) that were prepared in four to six (15 ml) conical polystyrene centrifuge tubes according to the manufacturer's instructions.
Three citrate/phosphate-buffered saline washes, interspersed with 150g centrifugation steps, removed contaminating lymphocytes from platelets, which were retained in the supernatants. The washes were pooled in 50-ml polystyrene centrifuge tubes, and the platelets were sedimented by centrifugation at 500g. The purified platelet pellets were then resuspended in buffer (10 mM triethanolamine-HCl buffer, 0.25 M sucrose, 5 mM 2-mercaptoethanol, and 67 µM acetylsalicylic acid, pH 7.4) and transferred to standard microcentrifuge tubes. A small aliquot was set aside for determination of platelet yield and purity (Model STKS; Coulter Corp., Irving, TX). By this method, isolated platelets routinely showed negligible contamination with other cell types (
0.08%).
Platelets used in correlation studies were obtained from these and
additional control subjects participating in an ongoing colon cancer
case control study.
Human livers were obtained as surgical samples from the John L. McClellan Memorial Veterans' Hospital and the U.S. Cooperative Tissue
Network. These tissues were excess surgical samples that were
immediately frozen in liquid N2 and stored at
80°C before use. Cytosols were prepared as described by Chou et al.
(1995a,b).
Immunoblotting for SULT1B2 was carried out by the method of Towbin et
al. (1979) using 1:1000-fold dilution of the anti-SULT1B1/2. Detection
of the immunoreactive bands was performed using the ECL
chemiluminescence method from Amersham Pharmacia Biotech
(Arlington Heights, IL).
Determination of SULT1A1, SULT1A2, and SULT1A3 mRNA transcripts in
human platelets was performed according to the method of Ozawa et al.
(1998). Total RNA was isolated from platelets by the acid guanidinium
isothiocyanate method using TriReagent (Molecular Research Center,
Cincinnati, OH) according to the manufacturer's instructions. RNA (10 µg) was reverse-transcribed using the Promega (Madison, WI) Access
reverse transcription-polymerase chain reaction system, which uses
avian myeloblastosis virus reverse transcriptase for first strand cDNA
synthesis. Second strand synthesis and subsequent DNA amplification
were catalyzed by the thermostable Tfl DNA polymerase from
Thermus flavus using the specific primers described in Ozawa et al. (1998).
Preparation of Platelet Samples for PST Assays.
After addition of a sonication solution (0.1 ml/ml) containing 1 M KCl,
10 mM EDTA, and 0.3% 2-mercaptoethanol, purified platelet suspensions
were subjected to three 3-s "bursts" of a sonicator, while on ice.
Supernatants were recovered after a 10-min spin in a refrigerated
microcentrifuge (14,000g, 4°C), removing intact cells and
most subcellular membrane contaminants that contribute to optical
interference. In some experiments, a further ultracentrifugation step
(100,000g for 1 h, 4°C) replaced the microcentrifuge
spin. If time permits, this step is recommended, because it minimizes potential optical interference in some samples. For either platelet supernatant isolation procedure, several aliquots were prepared and
frozen at 80°C for up to 3 months without any detectable effects on
enzyme activities. Samples were subjected to a single freeze-thaw cycle
before assay.
Comparison of SULT1A1 Activity by Colorimetric and Radiochemical
Assays.
In the colorimetric assay, SULT1A1 activity was measured as the release
of p-nitrophenol from a PAPS-regenerating system originally developed by Mulder et al. (1977). The basis for the SULT1A1
phenotyping assay is depicted in Fig. 1A.
Although some overlapping substrate specificity has been reported for
various sulfotransferase isoforms, reported
Km values are approximately two orders of
magnitude lower for reactions with prototypical substrates compared
with nonpreferred substrates (Veronese et al., 1994). For the
phenotyping assays reported here, the prototypical SULT1A1 substrate,
2-naphthol, was used.
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1
M1, which directly correlates with the
concentration of 2-naphthylsulfate formed (Mulder et al., 1977). A
shorter pathlength of 0.63 cm, however, replaced the standard 1-cm
pathlength in final calculations. Results are reported as nmol/min per
mg of protein. Under these conditions, the assays were linear with time
for up to 60 min and directly proportional to protein concentration.
Controls without PAPS and protein were used in each assay set;
time-dependent changes in optical density in these reactions were not
detectable. Protein was measured using a microscale method (Bio-Rad
Coomassie dye reagent) with bovine serum albumin as the reference standard.
This assay was validated by liquid chromatography-mass spectrometry on
a Micromass Platform II single quadrupole mass spectrometer (Manchester, UK), equipped with an electrospray interface with an ion
source temperature of 150°C. Separation was achieved on a Beta Basic
C18 column (2× 150 mm; 3 µm), with isocratic elution (40%
acetonitrile in water); the flow rate was 0.2 ml/min. In a typical
experiment, the colorimetric assay yielded a measure of 23.6 pmol of
2-naphthylsulfate, which was determined by liquid chromatography-mass
spectrometry to be 24.2 pmol.
For the radiochemical SULT assays, measurement techniques were adapted
from the method of Foldes and Meek (1973). The basis for the
radiochemical SULT1A1 assay is depicted in Fig. 1B. Triplicate assay
tubes were prepared (plus and minus substrate). Briefly, reaction
mixtures for 2-naphthol sulfation consisted of 50 mM potassium
phosphate buffer (pH 6.5), 5 mM magnesium chloride, 20 µM
[35S]PAPS, and platelet supernatant, in the
presence or absence of 2-naphthol (0.1 mM). For
p-nitrophenol assays, the reaction mixture consisted of 50 mM potassium phosphate buffer (pH 6.8), 5 mM
MgCl2, 20 µM [35S]PAPS,
platelet supernatant, in the presence or absence of
p-nitrophenol (4 µM). For dopamine assays, the reaction
mixture contained 50 mM potassium phosphate (pH 6.8), 5 mM
MgCl2, 20 µM [35S]PAPS,
1 mM pargyline (monoamine oxidase inhibitor), platelet supernatant,
plus or minus dopamine (10 µM). These mixtures were incubated for 10 min at 37°C and then were terminated by the addition of barium
hydroxide, zinc chloride, and barium acetate to precipitate excess
[35S]PAPS. After centrifugation, each sample
was subjected to a second centrifugation, and the individual
supernatants were finally transferred to vials, where the radioactivity
was measured by liquid scintillation counting. The difference in
radioactivity due to addition of substrate was then determined. The
results were expressed as average reaction rates (pmol/min/mg of
protein) from triplicate determinations, ± S.D.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Substrate Selection and Assay Linearity.
Initial studies were carried out with liver cytosol using a variety of
phenolic substrates at a range of concentrations. Of these, 2-naphthol
exhibited the highest turnover (Fig. 2).
A 2-naphthol substrate concentration of 0.1 mM was optimal for the
colorimetric assay, as higher concentrations resulted in significant
substrate inhibition, a characteristic not uncommon for this and other
sulfotransferase isoforms (Campbell et al., 1987). Although the
colorimetric assay was linear with time for 15 min (Fig. 2) and
proportional to protein concentration up to 0.5 mg/ml using the liver
100,000g supernatant with the microtiter plate format, the
reaction was found to be proportional to platelet protein concentration
and linear with time for at least 45 min using either the
14,000g (up to 0.2 mg/ml protein) or the 100,000g
(up to 0.04 mg/ml protein) platelet supernatant preparations. Protein
values greater than this resulted in p-nitrophenol absorbance values (>0.400) that did not obey Beer's law. Moreover, studies with recombinant SULT1A1 and SULT1A2 (Ozawa et al., 1994) showed that the former had 20-fold higher activity toward 2-naphthol, thus supporting our selection of this substrate for SULT1A1
phenotyping. Moreover, quercetin (10 µM) and
2,6-dichloro-4-nitrophenol (10 µM), which are reported to be
selective for inhibition of SULT1A1, and not SULT1A2 or other SULTs
(Glatt et al., 1999; Raftogianis et al., 1999), inhibited the
2-naphthol colorimetric assay by 60% and 90%, respectively.
Furthermore, the addition of substrates (100 µM) for SULT1A3
(dopamine), SULT1B1/2 (triiodothyronine), and SULT1E1 (estrone) did not
contribute to absorbance at 405 nm in this phenotyping assay. Finally,
an antibody to human SULT1B2 did show cross-reactivity toward platelet
cytosol (Fig. 3A). Similarly, measurement
of SULT1A1, -1A2, and -1A3 mRNA transcripts in platelet RNA revealed
the presence of only SULT1A1 and -1A3 mRNAs (SULT1A3 does not
contribute to the assay); SULT1A2 mRNA was not detected (Fig. 3B).
Thus, SULT1A1 is the only isoform contributing to this assay in
platelet cytosol.
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Substrate Specificity. To validate the use of this phenotype (colorimetric) assay for SULT1A1 activity in platelet fractions, a series of comparisons were made to assess the correlation with standard radiochemical PST assays. As shown in Fig. 4A, a high degree of correlation (r = 0.87) was found between 2-naphthol PST activities measured colorimetrically and by radiochemical detection (n = 8). Note that actual values were much higher in the colorimetric assay (0.2-3.7 nmol/min/mg) than in the corresponding radiochemical assay (15-170 pmol/min/mg) for the same samples.
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Intraindividual Variability. Figure 6 shows the relatively low intraindividual variability when healthy human adult volunteers were phenotyped for SULT1A1 on four occasions, with sampling at weekly intervals. Individuals that had the highest sulfotransferase activities on the initial assay also ranked highest on subsequent assays. Likewise, low 2-naphthol sulfotransferase activity was a good correlate of the slow SULT1A1 phenotype, whether based on single or multiple samples.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A high correlation between 2-naphthol sulfotransferase activities
measured by the colorimetric and radiochemical PST methods was
observed, indicating that both assays measure the same sulfotransferase isoform. Irrespective of the assay technique, there was a good correlation between 2-naphthol and p-nitrophenol
sulfotransferase activities. This was expected, because both 2-naphthol
and p-nitrophenol are considered prototypical substrates for
the SULT1A1 isoform. The low specific activity of the SULT1A2 isoform
toward 2-naphthol and the results of enzyme purification studies in
human platelets (Anderson et al., 1998) essentially rule out any
contribution of SULT1A2 in platelet supernatants to the SULT1A1
phenotyping assay. Similarly, SULT1A3 activities using dopamine as a
substrate showed no correlation with the 2-naphthol phenotyping assay,
thus excluding this isoform as a confounding factor.
Activity values derived from the colorimetric 2-naphthol assay were
consistently higher than the corresponding radiochemical assay. One
possible explanation may be that 3'-phosphoadenosine 5'-phosphate (PAP)
is generated as a reaction byproduct in the traditional radiochemically
based assays but not in the colorimetric assay (cf. Fig. 1, A and B).
PAP is a known potent inhibitor of sulfotransferase enzymes
(Rens-Domiano and Roth, 1987) but is not accumulated in the
colorimetric assay. Therefore, compared with other standard assay
techniques, the colorimetric method may give the better estimate of
SULT1A1 reaction rates.
Within our small sample size, interindividual variability in SULT1A1
activities ranged from 1 to 2.4 nmol/min/mg (n = 8). Other reports show 50-fold or greater variability (Frame et al., 1997;
Raftogianis et al., 1997). Intraindividual and intra-assay variabilities were very low with few identifiable technical problems for effective phenotyping. As with other experimental approaches, these
results support the idea that SULT1A1 expression has a strong genetic
component. It is interesting that certain sulfotransferase isoforms in
humans and animals are modulated by season (Marazziti et al., 1995),
hormones (Runge-Morris, 1998), disease state (el Mouelhi et al., 1987),
diet, and other factors (Burchell and Coughtrie, 1997). To date,
nongenetic factors that modulate SULT1A1 activity have not been
reported. However, using this simple phenotyping assay, nongenetic as
well as genetic factors may be explored readily. For example, the assay
may be useful for screening variant SULT1A1 allele(s) to correlate with
SULT1A1 activity and risk for human disease. It may also be useful for
characterization of age- and disease-associated alterations of this
xenobiotic-metabolizing enzyme.
The assay described in this report is inexpensive and simple to
perform. The microtiter format greatly decreases the assay time per
sample, such that in a few days to weeks, several hundred cytosol
samples may be measured for SULT1A1 activity. For example, using this
phenotyping technique, we were able to complete phenotyping of 367 individual blood samples in 10 working days (Frame et al., 1997).
Although other methods for measurement of PST activities have been
proposed (Arand et al., 1987; Ramaswamy and Jakoby, 1987; Beckmann,
1991; Anderson et al., 1998), these have not been validated for SULT1A1
for use in any human epidemiological study. It is thus anticipated that
this simple phenotyping method for SULT1A1 will improve the
investigation of genetic, environmental, and host-specific factors that
impact on SULT1A1 expression and disease risk.
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Acknowledgments |
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The technical assistance of Tracy L. Gatlin and Candee Teitel is gratefully acknowledged.
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Footnotes |
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Received June 7, 1999; accepted June 1, 2000.
1 Present address: Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX 79416.
2 Present address: Department of Pharmacology, National Institute of Health Sciences, Tokyo 158, Japan.
This work was supported by National Cancer Institute Grants CA58697 and CA55751, by Environmental Protection Agency Grant R825280, and by National Institute on Aging Grant AG15722.
Send reprint requests to: Dr. Fred F. Kadlubar, Division of Molecular Epidemiology, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079. E-mail: fkadlubar{at}nctr.fda.gov
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Abbreviations |
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Abbreviations used are: PST, phenol sulfotransferase; SULT1A1, SULT1A2, isozymes of the thermostable phenol sulfotransferases; SULT1A3, isozyme of the thermolabile phenol sulfotransferase; PAP, 3'-phosphoadenosine 5'-phosphate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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),
triiodothyronine (
), and ascorbic acid (×); and 0.05 mM for
1-naphthol (+) and 2-chlorophenol (
). For active substrates, higher
concentrations were inhibitory. Assay conditions are as described under
Materials and Methods using 0.5 mg/ml cytosol protein.
The data for each substrate shown were all obtained at the same time
using a single, freshly prepared, human cytosol preparation. Replicate
assays comparing 2-naphthol with each of the substrates tested were
carried out before this experiment with different liver cytosols, and
the data shown are essentially identical with these replicates.
