©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Homodimeric and Heterodimeric Aryl Sulfotransferases Catalyze the Sulfuric Acid Esterification of N-Hydroxy-2-acetylaminofluorene (*)

(Received for publication, January 24, 1995; and in revised form, April 13, 1995)

Charles C. Kiehlbauch Yim F. Lam David P. Ringer (§)

From theOklahoma Medical Research Foundation, Noble Center for Biomedical Research, Oklahoma City, Oklahoma 73104-5046

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Cytosolic sulfotransferases have been shown to catalyze the PAPS(^1)-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^2-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) .

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.


EXPERIMENTAL PROCEDURES

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 [^14C]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^2/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) .


RESULTS

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).


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 (). 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.


Figure 2: MonoQ FPLC resolution of three sulfotransferase activity peaks in the HA1 and HA2 sulfotransferase fractions. Sulfotransferase activity for N-OH-2AAF (bullet) 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.


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) () and carbonic anhydrase (29 kDa) (black square). The enzyme activity profiles for Q1-Q3 are shown for the sulfoconjugations of 2-naphthol () and N-OH-2AAF (bullet).



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 ST1C1bulletST1C1 homodimer, Q2 is an ST1C1bulletPST-1 heterodimer, and Q3 is a PST-1bulletPST-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(max) values ranged over a 30-fold span. Q1 had the highest V(max) value, whereas Q2, the heterodimer, had values approximately 50% those of Q1, and Q3 had a V(max) 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(max) 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.


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 (bullet) 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.




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.




DISCUSSION

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 ST1C1bulletPST-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-1bulletST1C1 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.

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.


FOOTNOTES

*
This work was supported by funds from the Samuel Roberts Noble Foundation, Inc., Ardmore, OK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Oklahoma Medical Research Foundation, Noble Center for Biomedical Research, 825 N.E. 13th St./Mailstop 38, Oklahoma City, OK 73104-5046. Tel.: 405-271-1615; Fax: 405-271-1554.

^1
The abbreviations used are: PAPS, 3`-phosphoadenosine-5`-phosphosulfate; AST, aryl sulfotransferase; HAST, hydroxylamine sulfotransferase; HPLC, high performance liquid chromatography; N-OH-2AAF, N-hydroxy-2-acetylaminofluorene; LC, liquid chromatography; PST, phenol sulfotransferase; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


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