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(Received for publication, January
24, 1995; and in revised form, April 13, 1995) From the
Three aryl sulfotransferases (ASTs) isolated from rat liver
catalyze the sulfuric acid esterification of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). These
three ASTs were separated by high resolution anion exchange
chromatography and were designated Q1, Q2, and Q3. Q1 and Q2 had high N-OH-2AAF sulfonation activity, whereas Q3 showed low
activity. Reversed phase high performance liquid chromatography/mass
spectrometry analysis showed Q1-Q3 to be comprised of 33,945- and
35,675-Da protein subunits. Q1 contained only the 35,675-Da protein
subunit, Q2 contained equal quantities of 33,945- and 35,675-Da
subunits, and Q3 contained only the 33,945-Da subunit. The subunit
compositions of Q1-Q3 were confirmed by immunochemical analysis.
Size exclusion high performance liquid chromatography confirmed that
the active quaternary structure of the three isoenzymes was dimeric.
Analysis of liver cytosols for the relative contributions of
Q1-Q3 to total cytosolic N-OH-2AAF sulfotransferase
activity indicated that Q1, Q2, and Q3 accounted for 44, 46, and 10% of
the activity, respectively. These results demonstrate the existence of
both homodimeric and heterodimeric aryl sulfotransferases and show that
two ASTs, a homodimer of 35,675-Da subunits and a heterodimer of a
33,945- and a 35,675-Da subunit, are primarily responsible for hepatic N-OH-2AAF sulfotransferase activity.
Cytosolic sulfotransferases have been shown to catalyze the
PAPS( Rapid advances in the knowledge of
the molecular structure of sulfotransferases (see (15) , and
references therein) have stimulated efforts to associate the individual
catalytic activities with specific sulfotransferase amino acid
sequences(4) . It has been suggested previously that a
principal source for liver cytosolic N-OH-2AAF
sulfotransferase activity is aryl sulfotransferase IV (AST
IV)(1, 16, 17, 18) . This enzyme was
reported to be a dimeric molecule comprised of two identical subunits
of known amino acid sequence and a subunit mass of 33,906
Da(19, 20) . The primary sequence for the AST IV
subunit is identical to the deduced amino acid sequences reported for
cDNAs of phenol sulfotransferase (PST-I; (21) ) and for
minoxidil sulfotransferase(22) . Recently, two additional
sources of liver cytosolic N-OH-2AAF activity have been
reported, HAST-I and HAST-II(23) . HAST-II was shown to have a
higher enzymatic activity toward N-OH-2AAF than HAST-I and to
be cross-reactive with a polyclonal antibody raised against HAST-I.
Recently, the HAST-I polyclonal antibody was used to isolate an HAST-I
cDNA clone, ST1C1(24) . This cDNA was found to code for a
35,768-Da protein that displayed high N-OH-2AAF
sulfoconjugation activity when transfected and expressed in COS-1 cells. The 36-kDa HAST-I protein was found to have a 51%
amino acid sequence homology with the 34-kDa PST-1/AST IV protein. In order to further evaluate the role of AST IV as an N-OH-2AAF sulfotransferase, studies were conducted to
determine the possible contribution of the 34-kDa and 36-kDa
sulfotransferase subunits to AST IV activity. The use of improved
purification techniques and protein characterization methodologies have
revealed the presence of three sulfotransferase forms. The first form
is a homodimer of two 33.9-kDa subunits, which shows low N-OH-2AAF sulfotransferase activity. A second form is a
homodimer comprised of two 35.7-kDa subunits and has the highest level
of N-OH-2AAF sulfotransferase activity. The third form is a
heterodimer comprised of a 33.9-kDa and a 35.7-kDa protein subunit,
possessing high catalytic activity for N-OH-2AAF sulfonation.
The existence of heterodimeric sulfotransferases constitutes a new,
previously unreported level of sulfotransferase organization.
Figure 1:
Elution profile of AST IV and
characterization of sulfotransferase activity following hydroxylapatite
chromatography. AST IV purification was performed through the
hydroxylapatite purification step as described under
``Experimental Procedures.'' Panel a, eluted
fractions were analyzed for absorbance at 280 nm(- - -) and 2-naphthol
sulfoconjugation activity at pH 5.5 (
Figure 2:
MonoQ FPLC resolution of three
sulfotransferase activity peaks in the HA1 and HA2 sulfotransferase
fractions. Sulfotransferase activity for N-OH-2AAF (
Figure 3:
Size exclusion HPLC analysis of Q1, Q2,
and Q3 molecular masses under non-denaturing conditions. Profiles were
generated using 0.5-ml fractions from size exclusion chromatography of
Q1-Q3 as described under ``Experimental Procedures.''
The first profile (standards) shows the elution positions as
determined by absorbance at 280 nm for bovine serum albumin (66 kDa)
(
To further evaluate the basis
for the Q1, Q2, and Q3 dimer heterogeneity, subunit composition was
assessed by subjecting the dimeric forms to C18 reversed phase
chromatography. Elution profiles showed (Fig.4) that Q1 and Q3
were each comprised of a single protein peak with distinct elution
positions. In contrast, the Q2 profile contained two protein peaks of
equivalent size, which eluted at positions corresponding to the Q1 and
Q3 peaks. Subsequent reversed phase liquid chromatography and mass
spectrometry of protein peaks found in Q1, Q2, and Q3 (Fig.5)
indicated that Q1 contained a single component with a mass of 35,675
± 3 Da and that Q3 was comprised of a single component with a
mass of 33,946 ± 4 Da. Mass spectrometry of the two protein
peaks observed in Q2 were found to have masses identical to the masses
for Q1 and Q3. The mass for the Q3 protein approximated the theoretical
mass of an N-terminally acetylated form of the deduced amino acid
sequence from the PST-1 cDNA(21) , i.e. 33,951 Da. The
mass of the protein comprising Q1 did not closely correspond with any
reported sulfotransferase amino acid sequence. However, since it
displayed high N-OH-2AAF activity, the possibility existed
that it was a modified form of the ST1C1 subunit comprising
HAST-I(24) . N-Acetylation following the removal of
the N-terminal methionyl residue of the ST1C1 subunit would result in a
mass of 35,679 Da.
Figure 4:
C18 reversed phase HPLC analysis of Q1,
Q2, and Q3. Purified Q fractions were analyzed by reversed phase HPLC
as described under ``Experimental Procedures.'' The subunit
elution positions of the Q fractions are shown, as monitored by
absorbance at 215 nm. The first eluting peak, observed in Q1 and Q2,
was labeled A, as shown at the top of the figure. The later
eluting peak, labeled B, was observed in both Q2 and
Q3.
Figure 5:
Mass
spectrometry analysis of the protein subunits comprising the
sulfotransferase isoforms Q1, Q2, and Q3. Molecular mass determination
by mass spectrometry of the subunit peaks A and B, isolated by C18
reversed phase HPLC of Q1, Q2, and Q3, was performed as described
under ``Experimental Procedures.'' A representative mass
spectra, spectral data, and corresponding masses are shown for peak A (a) and B (b).
To further assess the identity of the
sulfotransferase subunits found in Q1, Q2, and Q3, N-terminal amino
acid sequencing was attempted on purified subunit proteins. Both
subunits were found to be N-terminally blocked and thus were subjected
to tryptic digestion followed by chromatographic isolation of peptides
and subsequent analysis of peptides for amino acid sequence. As shown
in Table1, amino acid sequencing of peptides showed that the
subunit comprising Q1 was homologous to ST1C1 (24) and the
subunit for Q3 was homologous to PST-1(21) . These findings
indicate that cytosolic N-OH-2AAF sulfotransferase activity is
a sum of the activities of three different dimer forms in which Q1 is
an ST1C1
Figure 6:
DEAE-HPLC sulfotransferase elution profile
of male cytosol. Male rat liver cytosol (5 ml) was prepared as
described previously, applied to a DEAE-HPLC column, and eluted with a
gradient as described under ``Experimental Procedures.''
Fractions (3 ml/fraction) were collected and analyzed for N-OH-2AAF (
Figure 7:
Immunochemical analysis of
sulfotransferase subunit compositions of fractions from DEAE-HPLC of
male cytosol. The apex fractions from the sulfotransferase peaks
labeled Q1, Q2, and Q3 in Fig.6were evaluated by Western blot
immunohistochemical analysis for the presence of the 33.9-kDa and
35.7-kDa subunits. Panel a demonstrates the ability of the
polyclonal antiserum to be used for detection of the two subunits: lanes1 and 2, 0.1 µg of 33.9-kDa
subunit; lanes 3-5, 0.1 µg of 35.7-kDa subunit; lanes6 and 7, 2 µg of cytosol protein.
Immunochemical staining was performed with polyclonal antibody
pretreated as follows: lanes2 and 4,
preabsorbed with purified 33.9-kDa subunit; lanes5 and 7, preabsorbed with both 33.9-kDa and 35.7-kDa
subunits. In panels b and c, lanes1-3 contained 0.1 µg of Q1, Q2, and Q3,
respectively. The immunochemical staining was performed with
sulfotransferase polyclonal antiserum preabsorbed with 33.9-kDa subunit
in b and 35.7-kDa subunit in c. See
``Experimental Procedures'' for additional
details.
The N-OH-2AAF sulfotransferase activity of rat liver
AST IV has been shown to be comprised of the activities of three
different dimers with distinct structural and functional features. The
three dimers, Q1, Q2, and Q3, were elucidated by subjecting purified
AST IV to high performance liquid chromatography with an anion exchange
resin. Each dimer was shown to have N-OH-2AAF sulfotransferase
activity, and upon SDS-PAGE generated a single 34-kDa band that stained
positively upon Western immunochemical staining with polyclonal
antiserum to AST IV. Denaturation of the individual native AST IV
dimers to monomers by reversed phase HPLC showed that each dimer had a
unique subunit composition, and established reversed phase HPLC as an
important tool for examining heterogeneity of dimer composition.
Subsequent analysis of the subunits by mass spectrometry and amino acid
sequencing provided a basis for the structural identification of the
three dimers. This approach has allowed a definitive determination of
sulfotransferase subunit composition. Q1 was shown to be a homodimer
comprised of a 35,675 ± 3-Da protein subunit, which, based upon
partial amino acid sequence analysis, was found to be homologous to the
ST1C1 sulfotransferase subunit ((24) ; deduced amino acid
sequence molecular mass of 35,768 Da). Q1 showed high N-OH-2AAF sulfotransferase activity and may correspond to the
HAST I sulfotransferase whose composition also included the ST1C1
subunit (24) . The Q3 dimer was shown to be a homodimer
comprised of a protein subunit with a molecular mass of 33,946 ±
4 Da and amino acid sequence homologies that identified it as the PST-1
sulfotransferase subunit ((21) ; deduced amino acid sequence
molecular mass of 33,906 Da). It showed significant but much lower N-OH-2AAF sulfotransferase capacity than the Q1 homodimer and
had also been implicated previously as a source of N-OH-2AAF
sulfotransferase activity(16, 19) . The Q2 dimer was
found to be a heterodimer comprised of one 35.7-kDa subunit (ST1C1) and
one 33.9-kDa subunit (PST-1). It possessed a high N-OH-2AAF
sulfotransferase capacity, which was approximately 50% that of the
ST1C1 homodimer (Q1). Q2 appeared to have an anion exchange
chromatography elution position similar to that of a partially
characterized sulfotransferase termed HAST II, which had been
previously characterized as having strong N-OH-2AAF
sulfotransferase activity and reacting positively to an antiserum
raised against ST1C1(23) . Kinetic analysis of the N-OH-2AAF sulfotransferase activities for the three dimers
indicated that the ST1C1 homodimer (Q1) was approximately 40 times more
catalytically efficient than the PST-1 homodimer (Q3) and approximately
1.5-fold more efficient than the heterodimer (Q2). Kinetic analysis of p-nitrophenol sulfotransferase activities among the three
dimers indicated a different pattern of catalytic efficiencies. The
PST-1 homodimer (Q3), was approximately 3-fold more efficient than the
ST1C1 homodimer (Q1) and 6-fold more efficient than the heterodimer
(Q2). These results indicated that subunit-subunit interactions may
have a role in determining dimer catalytic properties. Although these
studies indicated that sulfoconjugation of N-OH-2AAF to its
highly reactive sulfuric acid ester form was most efficiently catalyzed
by a ST1C1-containing sulfotransferase dimer, the actual in vivo contribution of the various dimer forms was less predictable since
other factors, including relative dimer abundance and tissue
localization, must be taken into account. Studies reported here
involving analysis of cytosol suggested that the heterodimer (Q2) was a
principal source of N-OH-2AAF sulfotransferase activity in
vivo. Identification of Q2 as an ST1C1 The existence of heterodimeric sulfotransferases also
introduces a new level of structural complexity as an enzyme family.
Heterodimer formation among glutathione S-transferases is
typically restricted to subunits with homologies of
70-90%(33, 34) . In contrast, the amino acid
sequence homology between the subunits for the PST-1 and ST1C1 is
reported to be only 51%(24) . To date, there have been no
investigations of the requirements for sulfotransferase dimer
association and dissociation, and little is known about the
sulfotransferase domains responsible for dimerization. Questions such
as whether sulfotransferase heterodimer formation is restricted to
specific subclasses of sulfotransferases or is a common property of all
sulfotransferases are subjects for future investigations.
Volume 270,
Number 32,
Issue of August 11, pp. 18941-18947, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)-dependent sulfonation of a wide variety of
hydroxylated endobiotic and xenobiotic compounds (1, 2, 3, 4) . Whereas the primary
function of xenobiotic sulfoconjugation is to permit detoxication of
the compound, it occasionally results in the production of highly
reactive intermediates capable of causing genotoxic and cytotoxic
damage to cells. One sulfotransferase activity that has been studied
with great interest is that responsible for the sulfoconjugation of N-hydroxyarylamic acids and N-hydroxyarylamines(5, 6, 7) . The
sulfuric acid esterification of these compounds results in bioactivated
forms reported to cause liver cancer in
rodents(6, 7, 8, 9, 10) . A
model compound for this class of carcinogens that has been extensively
investigated is N-OH-2AAF. Sulfuric acid esters of N-OH-2AAF have been implicated in the in vivo production of the N-(deoxyguanosin-8-yl)- and
3-(deoxyguanosin-N
-yl)-2AAF DNA adducts in rat
liver (11, 12) and the N-(deoxyguanosin-8-yl)-2-aminofluorene DNA adduct in mouse
liver(10) . In addition, carcinogen-mediated loss of this
activity among rat liver cells, putatively initiated for
carcinogenesis, has been suggested to contribute to the development of
a resistance phenotype to carcinogen toxicity(9, 13) .
This phenotype contributes to the selective clonal proliferation of
initiated cells into preneoplastic nodules during the promotion stage
of carcinogenesis (14) .
Chemicals and Materials
DEAE-Sepharose Fast
Flow, MonoQ HR 5/5 anion exchange column, PhastGel 10-15%
gradient polyacrylamide gels, and nitrocellulose membranes were
purchased from Pharmacia Biotech Inc. Ceramic hydroxylapatite,
Tris-HCl, and all Western blot immunochemical reagents were purchased
from Bio-Rad. A Reliasil C18 300-Å, 5 µM (2 mm
15 cm) reversed phase HPLC column was purchased from Michrom
Bioresources (Pleasanton, CA). A BioSep SEC-S2000 HPLC column (7.8
300 mm) was obtained from Phenomenex (Torrance, CA). Waters
Protein PAK DEAE-5PW (2.15
15 cm) HPLC column was purchased
from Millipore (Bedford, MA). N-OH-2AAF was purchased from
Chemsyn Science Laboratories (Lenexa, KS). PAPS was obtained from Dr.
Sanford Singer (University of Dayton, Dayton, OH). Dithiothreitol,
sucrose, 2-mercaptoethanol, glycerol, trifluoroacetic acid, methanol,
and acetonitrile were purchased from Fisher. Tris-hydrochloride was
purchased from Amresco (Solon, OH). Monobasic potassium phosphate was
purchased from J.T. Baker. 2-Naphthol was purchased from Aldrich.
ATP-agarose and all other chemicals were obtained from Sigma.
Sulfotransferase Purification
Aryl
sulfotransferases were purified from livers of male Sprague-Dawley rats
(Sasco Inc., Omaha, NE) by a modified procedure of Ringer et
al.(17) . In brief, chromatography was performed using
previously reported gradient conditions but substituting DEAE-Sepharose
Fast Flow and ceramic hydroxylapatite for DE52 and Bio-Gel HT,
respectively. Following hydroxylapatite chromatography, fractions
enriched in N-OH-2AAF sulfonation activity were pooled (HA1),
and fractions enriched in 2-naphthol sulfonation activity were pooled
(HA2). HA1 and HA2 fractions were dialyzed and applied individually to
ATP-agarose, as described previously(17) . Following
ATP-agarose chromatography, HA1 and HA2 fractions were dialyzed against
1 liter of buffer A (25 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose, 5% glycerol, and 1 mM dithiothreitol) and
individually applied to a MonoQ HR 5/5 anion exchange column.
Chromatography was done using FPLC (Pharmacia) at room temperature,
with a gradient elution of buffer A and buffer A containing 1.0 M NaCl (buffer B). The gradient elution was performed as follows;
sample injection was followed by an 8-min wash with buffer A, 0% B to
20% B in 55 min, 20% B to 100% B in 5 min, 3 min wash with 100% B,
followed by reequilibration in buffer A. Flow rate for analysis was 1
ml/min, and absorbance was monitored at 280 nm. Fractions of 1 ml were
collected, and peaks were pooled, concentrated, and final protein
concentration determined by absorbance at 215 nm. An extinction
coefficient of 15 at 215 nm was used for a 1 mg/ml protein
solution(25) .Sulfotransferase Assays
N-OH-2AAF
sulfonation activity was determined by HPLC using the procedure of
Yamazoe et al.(26) with minor modifications. In
brief, a typical reaction mixture contained 50 µM Tris-HCl, pH 7.8, 250 µM PAPS, 1 mM
dithiothreitol, 20 µMN-OH-2AAF, and 0.5 µg
of purified sulfotransferase or 30 µl of a chromatography fraction
in a final volume of 100 µl. Reactions were incubated for 10 min at
37 °C and terminated by the addition of 200 µl of acetonitrile. N-OH-2AAF sulfoconjugation was quantitated by HPLC. When
indicated, N-OH-2AAF sulfotransferase activity was determined
using a trapping assay in which the sulfuric acid ester of
[
C]N-OH-2AAF reacted with
methionine-agarose beads(17) . The colorimetric determination
of 2-naphthol sulfotransferase activity at pH 5.5 described by Jacoby (27) was routinely used to monitor elution of sulfotransferases
during column chromatography. p-Nitrophenol sulfotransferase
activity was determined at pH 5.5 essentially as described by Gong et al.(23) . Reactions were initiated by the addition
of enzyme (0.5-1.0 µg) and incubated for 10 min at 37 °C,
and the product was subsequently quantitated by HPLC. The apparent
kinetic values for sulfotransferases were determined from a single time
point under initial velocity conditions, compensating for substrate
inhibition kinetics using the formula v = VA/(K + A + (A/K))(28) .
The Q1 and Q3 samples used for kinetic analysis contained less than
0.05% 33.9-kDa and 35.7-kDa subunit contamination, respectively.
Reversed Phase HPLC Characterization of
Sulfotransferases
Purified fractions of ASTs were analyzed by
C18 reversed phase HPLC using a Waters 625 LC system equipped with a
column heater, 486 variable wavelength detector, and the Millennium
2010 chromatography manager. Chromatography was performed at a flow
rate of 0.2 ml/min and a column temperature of 30 °C. Absorbance
was monitored at 215 nm. The column was equilibrated before analysis
with an aqueous solution of 2% acetonitrile and 0.1% trifluoroacetic
acid (C). A gradient elution was done using 90% acetonitrile with 0.09%
trifluoroacetic acid in water (D) as follows. Sample was injected
followed by a 3 min wash with 100% C, gradient was started from 0% D to
50% D in 7 min, 50% D to 75% D in 25 min, 75% D for 3 min, followed by
reequilibration with C.Determination of Molecular Mass
Purified
sulfotransferases were analyzed using an electrospray tandem quadropole
mass spectrometer (Perkin Elmer Sciex model API III, Toronto, Canada).
Some samples were analyzed by first isolating purified isoenzyme peaks
from C18 reversed phase HPLC analysis, followed by direct injection
into the mass spectrometer. Alternatively, samples were analyzed in the
LC/mass spectrometry mode by injection of samples onto an Ultrafast
Microprotein Analyzer microbore HPLC system (Michrom Bioresources Inc.,
Pleasanton, CA) with a 1 mm 15-cm 300-Å Reliasil C18
reversed phase column. The column was equilibrated with 0.1%
trifluoroacetic acid and 2% acetonitrile in water, and the sample was
eluted directly into the mass spectrometer nebulizer with a 20-min
45-75% gradient of 0.09% trifluoroacetic acid and 90%
acetonitrile in water.
Size Exclusion HPLC Chromatography
Size exclusion
HPLC analysis was done using a Waters 510 pump, 486 variable wavelength
detector, and the Millennium Chromatography Manager. Proteins were
eluted isocratically with a buffer containing 25 mM Tris-HCl,
pH 8.0, 0.25 M sucrose, 5% glycerol, and 1 mM dithiothreitol. The flow rate was 0.5 ml/min, and absorbance was
monitored at 215 nm. Fractions of 0.5 ml were collected for activity
and A analysis.
DEAE-HPLC of Male Rat Liver Cytosol
Cytosol (5 ml)
prepared as described above was fractionated by preparative DEAE-HPLC
using the procedure of Gong et al.(23) . Two Waters
510 pumps controlled by the Millennium Chromatography Manager were used
for gradient delivery and absorbance at 280 nm was monitored using a
Waters 486 detector. Fractions of 3 ml were collected and analyzed for
2-naphthol and N-OH-2AAF sulfoconjugation activity as
described above.Western Blot Immunochemical Analysis
Single
dimension SDS-PAGE of proteins was performed using a Pharmacia
PhastSystem following the procedure of Ringer et
al.(17) . Samples of 1-4-µl aliquots containing
0.1-2.0 µg of protein were applied to the gel. Proteins were
transferred to a nitrocellulose membrane using the Pharmacia
PhastTransfer electrophoretic transfer system. Before transfer, the
nitrocellulose membrane was prewetted with 25 mM Tris, pH 8.3,
containing 192 mM glycine, and 20% (v/v) methanol. Transfer
conditions were 12.5 mA/gel for 5 V-h at 15 °C. Following transfer,
immunochemical detection of sulfotransferases was performed using the
Bio-Rad Immun-Blot assay kit and gold enhancement kit as described
previously(17) . Immunochemically stained bands were
quantitated using a digital image analysis system (Fotodyne
Foto/Eclipse, Hartland, WI). In some experiments the sulfotransferase
polyclonal primary antibody was pretreated with purified 33.9- or
35.7-kDa protein to confer immunostaining specificity. Typically,
0.1-2 µg of the 33.9- or 35.7-kDa subunit/ml of antibody
buffer was used to pretreat the polyclonal antibody at room temperature
for 1 h before incubating the membrane with the antiserum.Tryptic Digestion and Amino Acid
Sequencing
Aliquots containing 50 µg of C18 reversed phase
HPLC-purified protein were digested with trypsin (30:1, w/w) for 17 h
at 37 °C in the presence of 200 mM ammonium bicarbonate,
pH 8.5. Amino acid sequence analyses were performed using a model 470A
gas-phase protein sequencer equipped with a model 120A on-line
phenylthiohydantoin amino acid analyzer (Applied Biosystems, Inc.)
according to standard procedures(29) . Sulfotransferase
identification was based on alignment of tryptic peptide amino acid
sequences with the previously published deduced amino acid sequences
for PST-1 (21) and ST1C1(24) .
Characteristics of AST IV Purified by Conventional
Chromatography
The principal features for the conventional
purification of AST IV (16) involved fractionation of AST IV
from the AST I and II isoforms by DEAE-cellulose chromatography,
followed by separation of AST IV from the AST III form by
hydroxylapatite chromatography. Final separation of AST IV from the
remaining protein contaminants was accomplished by PAPS elution from an
ATP-agarose affinity column. Shown in Fig.1a is a
typical protein elution profile from the hydroxylapatite column
(Bio-Rad Bio-Gel HT) in which two peaks of sulfotransferase activity
are observed, HA1 and HA2. As defined previously(16) , the
first eluting peak (HA1) corresponds to AST IV, whereas the second peak
(HA2) may correspond to the previously identified but uncharacterized
AST III. The HA1 and HA2 peak regions were further purified by
ATP-agarose affinity chromatography, and their characteristics are
shown in Fig.1b. Both fractions showed
sulfotransferase activity for 2-naphthol and N-OH-2AAF, with
AST IV (HA1) having relatively greater activity for N-OH-2AAF
and HA2 having relatively greater activity for 2-naphthol. Furthermore,
upon SDS-PAGE, each peak produced a single band of approximately 34 kDa
when either silver-stained for protein or immunochemically stained with
polyclonal antiserum raised against AST IV in agreement with previous
reports (17, 30) (Fig.1b).
). Pooled fractions
representing HA1 and HA2 are shown with longdashedverticallines. Panel b,
characterization of HA1 and HA2 pools following ATP-agarose
purification. 2-Naphthol activity at pH 5.5 (hatchedbox) and methionine-agarose bead assay of N-OH-2AAF sulfoconjugation activity (stripedbox) were determined as described under
``Experimental Procedures.'' The relative intensity following
image analysis of Western blots of HA1- and HA2-purified pools stained
with a polyclonal antiserum to AST IV is also shown (filledbox).
Identification of AST IV (HA1) and HA2 Heterogeneity
following High Performance Chromatography
To assess the possible
existence of multiple forms of AST IV, ATP-agarose affinity-purified
HA1 and HA2 fractions were subjected to MonoQ anion exchange
chromatography. As shown in Fig.2, HA1 (AST IV) and HA2 were
subsequently resolved into three peaks of sulfotransferase activity,
named Q1, Q2, and Q3 on the basis of elution order. Evaluation of peaks
for N-OH-2AAF sulfotransferase activity showed strong activity
for Q1 and Q2, whereas Q3 had low but detectable levels. In contrast,
2-naphthol sulfotransferase activity was found to be lower in Q1 than
in Q2 and Q3. Despite the distinct chromatographic and enzymatic
characteristics, SDS-PAGE analysis of Q1, Q2, and Q3 showed each was
comprised of a single 34-kDa band and were thus indistinguishable from
AST IV.
)
and 2-naphthol () sulfoconjugation was measured following MonoQ
FPLC fractionation of HA1 and HA2 fractions as described under
``Experimental Procedures.'' Labels indicating the elution
positions of Q1, Q2, and Q3 are shown above for
reference.
Structural Basis for AST IV Heterogeneity
To
determine whether the basis for the heterogeneity in AST IV and HA2
represented different aggregation states of the sulfotransferase dimer,
the protein elution profiles for Q1, Q2, and Q3 were determined by high
performance size exclusion chromatography. As shown in Fig.3,
the results of this analysis indicated that the activity profiles of
all three forms, although showing slightly different retention times,
co-migrated with the dimer position.
) and carbonic anhydrase (29 kDa) (
). The enzyme
activity profiles for Q1-Q3 are shown for the sulfoconjugations
of 2-naphthol () and N-OH-2AAF
(
).
ST1C1 homodimer, Q2 is an ST1C1PST-1 heterodimer,
and Q3 is a PST-1PST-1 homodimer.
Kinetic Analysis of Q1, Q2, and Q3
The relative
sulfotransferase kinetic capacities of the three dimers comprising AST
IV were determined using N-OH-2AAF and p-nitrophenol
as sulfonyl acceptors (Table2). Samples of Q1 and Q3 used for
kinetic analysis contained less than 0.05% 33.9-kDa and 35.7-kDa
contaminating subunit, respectively. For N-OH-2AAF, Q1-Q3 Kvalues were similar, whereas V
values ranged over a 30-fold span. Q1 had the
highest V value, whereas Q2, the heterodimer,
had values approximately 50% those of Q1, and Q3 had a V value that was about 10-fold lower than that
of Q2. The kinetic values using p-nitrophenol as substrate
show a slightly different pattern. The K value for Q3 was about 10-fold lower than those for Q1 and
Q2. The p-nitrophenol V
values were
grouped over 4-fold range with Q1>Q2>Q3. The high level of N-OH-2AAF sulfotransferase activity observed for Q1, the ST1C1
homodimer, and the relatively low activity found in Q3, the PST-1
homodimer, indicated that the ST1C1 subunit was the primary source for
this activity in AST IV.
Analysis of the Distribution of Q1, Q2, and Q3 in Male
Rat Liver Cytosol Using DEAE-HPLC
Experiments were performed to
assess the general contribution of Q1, Q2, and Q3 to cytosolic N-OH-2AAF activity and confirm the presence of heterodimer
(Q2) in cytosol prior to purification procedures. Fresh liver cytosol
was fractionated on a DEAE-HPLC column and evaluated for the presence
of peaks corresponding to Q1, Q2, and Q3. As shown in Fig.6,
analysis of chromatographic fractions for N-OH-2AAF and
2-naphthol sulfotransferase activity showed three well resolved peaks.
These peaks eluted in regions corresponding to the previously observed
positions for Q1, Q2, and Q3. The relative contributions to total
cytosolic N-OH-2AAF sulfotransferase activity were 44%, 46%,
and 10% for the Q1, Q2, and Q3 regions, respectively. The identities of
the Q1-Q3 peaks were confirmed by immunochemical analysis of
fractions corresponding to the apex of each of the three DEAE-HPLC
peaks (Fig.7). These fractions were subjected to SDS-PAGE and
Western blot immunochemical analysis using a polyclonal antibody to AST
IV that had been preabsorbed with either the ST1C1 (35.7 kDa) or PST-1
(33.9 kDa) subunit before use. As shown in Fig.7a,
preabsorption of the polyclonal antiserum to AST IV with purified ST1C1
or PST-1 subunit was able to confer immunostaining specificity to the
antiserum for the detection of each subunit. Furthermore, these
conditions were shown to be applicable for use in liver cytosols (Fig.7a, lanes6 and 7),
where preabsorption of the antiserum with both subunits lowered
immunostaining of sulfotransferase in cytosol to background levels. As
shown in Fig.7(b and c), the first DEAE
activity peak (labeled Q1), which showed high N-OH-2AAF sulfonation activity and lower 2-naphthol
sulfonation, stained only for the presence of the ST1C1 subunit. The
second peak (labeled Q2), which showed intermediate levels of
both sulfotransferase activities, stained for the presence of both the
ST1C1 and PST-1 subunits. The last peak (labeled Q3), which
showed high 2-naphthol sulfonation activity and low levels of N-OH-2AAF sulfonation activity, stained only for the presence
of the PST-1 subunit. These results established the existence of Q2 in
the freshly prepared cytosol and supported the position that Q2 is an
important source of N-OH-2AAF sulfotransferase activity in
vivo.
) and 2-naphthol () sulfonation
activities. Fractions were also analyzed for absorbance at 280 nm(- -
-) and are shown for reference. The elution positions for Q1, Q2, and
Q3 are indicated by labels.
PST-1 heterodimer
constitutes the first report of a functional heterodimeric
sulfotransferase. Heterodimeric proteins have been shown to be
important in cellular functions ranging from the regulation of gene
expression by heterodimeric nuclear transcription factors (31, 32) to the metabolism of xenobiotics by
heterodimeric detoxication enzymes(33) . For nuclear
transcription factors, heterodimer interactions change the availability
or specific activity of factors for modulating gene expression. In the
case of xenobiotic metabolizing enzymes such as glutathione S-transferases, heterodimeric interactions produce new dimers
that increase the detoxifying functions of cells(34) . The
xenobiotic detoxifying or bioactivating properties of glutathione S-transferases in rats have been shown to be a result of
tissue-specific (35) and/or sex-specific (36) patterns
of subunit expression, resulting in formation of catalytic homodimers
and permitted heterodimers. Similarly, the existence of heterodimeric
sulfotransferases introduces a new level of functional diversity for
this family of detoxication enzymes. Furthermore, there is considerable
evidence that expression of sulfotransferases in rat liver are also
regulated in a sex-specific
manner(8, 17, 23, 24, 37) .
Interestingly, the rat liver N-hydroxyarylamine
sulfotransferases HAST I and HAST II have been reported to be
male-dominant and male-specific, respectively(23) . If these
sulfotransferases, as suggested above, correspond to Q1 (ST1C1
homodimer) and Q2 (PST-1ST1C1 heterodimer), respectively, then it
may be concluded that the higher N-OH-2AAF sulfotransferase
activity observed in male liver cytosols (8, 17, 23) arises from the presence of the
male-dominant (homodimer) and male-specific (heterodimer) forms. Future
studies to assess the regulatory basis for sex-specific
sulfotransferase subunit expression and the extent to which
homodimer/heterodimer formation occurs are needed to provide further
insight into the role(s) that sulfotransferases have in xenobiotic
metabolism.
Mass spectrometry analysis and protein sequence
determinations were performed by The Molecular Biology Resource
Facility of the William K. Warren Medical Research Institute, Oklahoma
City, OK. We gratefully acknowledge the help of Dr. Ken Jackson with
the mass spectrometry and peptide sequencing studies. We thank Dr. Paul
Cook for helpful discussions concerning the kinetic analysis. We also
thank Dr. Akbar S. Khan for computer manipulation of sequences. We
gratefully acknowledge the assistance of Laura Smith in the preparation
of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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