Bile acid sulfotransferase I from rat liver sulfates bile acids and 3-hydroxy steroids: purification, N-terminal amino acid sequence, and kinetic properties

A bile acid:3'phosphoadenosine-5'phosphosulfate:sulfotransferase (BAST I) from adult female rat liver cytosol has been purified 157-fold by a two-step isolation procedure. The N-terminal amino acid sequence of the 30,000 subunit has been determined for the first 35 residues. The Vmax of purified BAST I is 18.7 nmol/min per mg protein with N-(3-hydroxy-5 beta-cholanoyl)glycine (glycolithocholic acid) as substrate, comparable to that of the corresponding purified human BAST (Chen, L-J., and I. H. Segel, 1985. Arch. Biochem. Biophys. 241: 371-379). BAST I activity has a broad pH optimum from 5.5-7.5. Although maximum activity occurs with 5 mM MgCl2, Mg2+ is not essential for BAST I activity. The greatest sulfotransferase activity and the highest substrate affinity is observed with bile acids or steroids that have a steroid nucleus containing a 3 beta-hydroxy group and a 5-6 double bond or a trans A-B ring junction. These substrates have normal hyperbolic initial velocity curves with substrate inhibition occurring above 5 microM. Of the saturated 5 beta-bile acids, those with a single 3-hydroxy group are the most active. The addition of a second hydroxy group at the 6- or 7-position eliminates more than 99% of the activity. In contrast, 3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) is an excellent substrate. The initial velocity curves for glycolithocholic and deoxycholic acid conjugates are sigmoidal rather than hyperbolic, suggestive of an allosteric effect. Maximum activity is observed at 80 microM for glycolithocholic acid. All substrates, bile acids and steroids, are inhibited by the 5 beta-bile acid, 3-keto-5 beta-cholanoic acid. The data suggest that BAST I is the same protein as hydrosteroid sulfotransferase 2 (Marcus, C. J., et al. 1980. Anal. Biochem. 107: 296-304).     

Partial purification and some properties of rat liver sulfotransferase I,a glucocorticoid sulfotransferase usually restricted to female rats

Three rat liver enzymes, sulfotransferases I, II, and III, sulfate glucocorticoids. This report describes the purification of sulfotransferase I, the glucocorticoid sulfotransferase usually restricted to females, 227 ± 63-fold from cytosol. As shown, this represents a probable 1000- to 1250-fold enrichment compared to liver homogenates. Highly purified sulfotransferase I exhibits a molecular weight of approximately 156,000, determined by sedimentation velocity experiments. Its pH optimum is pH 6.0 ± 0.1. However, its properties are routinely measured at pH 6.8 for reasons enumerated in the text. At this pH it exhibits a sequential mechanism for cortisol sulfation. Its K_m values for cortisol and its coenzyme, 3′-phosphoadenosine-5′-phosphosulfate, are 7.09 ± 0.65 and 10.6 ±1.1 μm, respectively. Sulfotransferase I also appears to contain essential sulfhydryl groups, shown by p-hydroxymercuribenzoate inhibition studies. Furthermore, it is activated by divalent Co, Cr, Mg, or Mn and inhibited by 100 μm Cd²⁺ or 50 μm Zn²⁺. Comparison of the ability of purified sulfotransferase I to sulfate 40 μm cortisol, dehydroepiandrosterone, deoxycorticosterone, estradiol-17β, and testosterone showed that sulfotransferase I is not as specific a glucocorticoid sulfotransferase as is sulfotransferase III (described earlier). Sulfotransferase I was inhibited strongly by a variety of steroids and diethylstilbestrol at 1.0 μm concentrations. Extensive comparisons of the properties of sulfotransferases I and III and their potential significance are made throughout the text.     

4-Hydroxytamoxifen sulfation metabolism

Tamoxifen (TAM) is an important chemotherapeutic agent for the treatment of breast cancer. It has also been shown to decrease breast cancer incidence in healthy women at high risk for the disease. The increased risk of endometrial cancer in women has raised concerns in the use of the drug. Tamoxifen has also been shown to be a potent hepatocarcinogen in rats. The oxidative metabolites of TAM include alpha-hydroxytamoxifen (alpha-OH-TAM) and 4-hydroxytamoxifen (4-OH-TAM). The studies on the sulfation of these metabolites are very limited. It has been reported that alpha-OH-TAM is a substrate for rat hydroxysteroid sulfotransferase a (STa). Our studies on the sulfation of 4-OH-TAM demonstrated that 4-hydroxytamoxifen can be sulfated by human liver and human intestinal cytosols. Human phenol-sulfating sulfotransferase and human estrogen sulfotransferase are the major enzymes for the sulfation of 4-OH-TAM. Human dopamine-sulfating sulfotransferase also has sulfation activity for 4-OH-TAM. In contrast, rat liver and intestine cytosols have no detectable sulfation activity for 4-OH-TAM. The results suggest that the alpha-OH-TAM sulfation pathway leads to bioactivation of TAM, and the 4-OH-TAM sulfation pathway leads to detoxification of TAM. This agrees with the fact that TAM is more toxic for rats than for human beings.     

Hepatic DNA and RNA adduct formation from the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene and its electrophilic sulfuric acid ester metabolite in preweanling rats and mice

DNA and RNA adducts that were chromatographically identical to those formed in vitro on reaction of 7-sulfooxymethyl-12-methyl-benz[a]anthracene with guanine and adenine nucleosides were formed in the livers of rats and mice given i.p. injections of 7-hydroxymethyl- or 7-sulfooxymethyl-12-methyl-benz[a]anthracene. Considerably higher levels of these hepatic adducts were obtained from the latter short-lived electrophilic ester than from the hydroxymethyl compound. These observations are consistent with the finding of rat liver cytosolic sulfotransferase activity for 7-hydroxymethyl-12-methylbenz[a]anthracene (Watabe et al., Science 215, 403, 1982). Formation of these hepatic adducts from 7-hydroxymethyl-12-methylbenz[a]anthracene was inhibited by prior administration to rats of dehydroepiandrosterone, an inhibitor of the sulfotransferase activity for this hydroxymethyl hydrocarbon.     

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.     

Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa

Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.     

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3′-phosphoadenosine-5′-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.     

Enzymatic sulfation of glycochenodeoxycholic acid by tissue fractions from adult hamsters

Using a radiometric assay with glycochenodeoxycholic acid as substrate, bile acid:3'-phosphoadenosine-5'-phosphosulfate sulfotransferase activity was found in 105,000 g supernatant fractions of liver, proximal intestine, and adrenal gland homogenates from adult hamsters. Optimum conditions for measurement of the hepatic enzyme were determined. In both male and female animals sulfation only occurred at the 7 alpha-position. Saturation analysis with glycohenodeoxycholic acid revealed that the higher activity observed in fractions from female compared to male hamsters was due to a 4-fold lower apparent Km (79 muM vs. 317 muM) for this bile acid in the females. The sulfation of glycohenodeoxycholic acid was competitively inhibited by glycolithocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The data are consistent with the concept that sulfation of many, if not all, bile acids can occur in vivo.     

Hepatocyte metabolism of coenzyme Q1 (ubiquinone-5) to its sulfate conjugate decreases its antioxidant activity

Previous studies on the metabolism of coenyzme Q (CoQ) have focused on products found in the urine, bile or feces. However, the metabolites found in these samples were end products from a multitude of catabolic processes which did not necessarily reflect CoQ intracellular metabolism (e.g. in the liver, the major site of CoQ synthesis or metabolism). Using isolated rat hepatocytes, we have found that the sulfation of coenzyme Q1 (CoQ1) was the initial and dominant step following its reduction to the hydroquinone. This metabolic process is important as conjugation may occur on the hydroquinone metabolites of any coenzyme10 scission product retaining the quinone ring. By using rat liver cytosol, we were able to identify the monosulfated metabolite of CoQ1. The CoQ1 sulfate conjugate was identified by mass spectrometry followed by tandem mass spectrometry. The rate of formation of the CoQ1 sulfate conjugate was markedly increased by the addition of NADH and was prevented by dicumarol, a DT-diaphorase (NQO1) inhibitor. CoQ1 sulfate conjugate formation catalysed by cytosol was inhibited by the sulfotransferase 1A (SULT1A) inhibitor, pentachlorophenol (PCP) suggesting that sulfation was carried out by the SULT 1A isoform. CoQ1 sulfation in isolated hepatocytes and inversely CoQ1 hydroquinone formation were dependent on the concentration of inorganic sulfate in the media. Intracellular sulfation also decreased CoQ1 antioxidant and cytoprotective activity towards cumene hydroperoxide (CHP) induced cell death. Sulfotransferases may therefore play a significant role in endogenous CoQ metabolism following its degradation to short chain products.     

Identification and characterization of a novel benzo[a]pyrene-derived DNA adduct

The aim of this study was to generate and identify a novel benzo[a]pyrene (BP)-derived DNA adduct found both in vitro and in vivo. To date, the majority of studies have focused on N(2)-[10 beta(7 beta,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxyguanosine (anti-BPDE-dG), the major adduct generated following bioactivation of BP. However, a second adduct is also formed following bioactivation of BP which has been speculated to result from further metabolism of 9-OH-BP. In order to identify this second reaction pathway, the ultimate DNA binding species, and the DNA base involved, we have synthesized and characterized a dG-derived DNA adduct arising from further bioactivation of 9-OH-BP in the presence of rat liver microsomes. Analysis of the adducted nucleotides was conducted using both the (32)P-postlabeling assay and capillary electrophoresis-mass spectrometry (CE-MS).     

The strong hepatocarcinogenicity of the electrophilic and mutagenic metabolite 6-sulfooxymethylbenzo[a]pyrene and its formation of benzylic DNA adducts in the livers of infant male B6C3F1 mice

6-Hydroxymethylbenzo[a]pyrene was activated to an electrophilic and mutagenic sulfuric acid ester metabolite by rat and mouse liver sulfotransferase activity. The intrinsic mutagenicity of this reactive ester, 6-sulfooxymethylbenzo[a]pyrene, was inhibited by glutathione and glutathione S-transferase. A single i.p. dose of 2.5 nmol/g body wt of 6-sulfooxymethylbenzo[a]pyrene in infant male B6C3F1 mice induced liver tumors in 35 of 36 mice at 10 months with an average multiplicity of 4.4. A comparable dose of the parent hydrocarbon, 6-hydroxymethylbenzo[a]pyrene, was only a tenth as active. The electrophilic sulfuric acid ester produced high levels of benzylic DNA adducts in the livers of these mice that accounted for about 80% of the total DNA adducts. These results strongly suggest that this sulfuric acid ester is an important ultimate electrophilic and carcinogenic metabolite in carcinogenesis by 6-hydroxymethylbenzo[a]pyrene and possibly even by 6-methylbenzo[a]pyrene and benzo[a]pyrene in mouse liver.     

A hydroxysteroid sulfotransferase, st2b2, is a skin cholesterol sulfotransferase in mice

The mRNA of a sulfotransferase (St2b2) mediating cholesterol sulfation was detected in mouse skin. Recombinant St2b2 also mediated the sulfation of pregnenolone, 3beta-hydroxy-5-cholen-24-oic acid, and dehydroepiandrosterone. St2b2 protein was detected in skin cytosols on Western blotting. The addition of 10 nM TPA to skin epidermal cells from newborn mice resulted in a twofold increase in cholesterol sulfation and concomitantly enhanced the St2b2 content after 40 h. Other candidate cholesterol sulfotransferases, St2a4 and St2a9, were not detected in skin by RT-PCR. These results indicate that St2b2 is a cholesterol sulfotransferase in mouse skin.     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.     

Enzymatic sulfation of steroids III. The sulfation of corticosterone by the glucocorticoid sulfotranferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This ‘mosaic’ assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6–9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate.The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20–32% and 25–35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37–55% and 40–60% decreases of sulfation of the twoo steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases.Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferase I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due to mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

Evidence for an ordered reaction mechanism for bile salt: 3'phosphoadenosine-5'-phosphosulfate: sulfotransferase from rhesus monkey liver that catalyzes the sulfation of the hepatotoxin glycolithocholate

The in vivo formation of the sulfate ester of glycolithocholate is a critical step in the elimination of this hepatotoxic bile salt. Rhesus monkeys fed chenodeoxycholate or ursodeoxycholate, the precursors of lithocholate, develop frank cirrhosis in association with accumulation of nonsulfated glycolithocholate in bile. An enzyme catalyzing the formation of glycolithocholate-3-sulfate has been isolated from hepatic cytosol of adult female rhesus monkeys and has been purified 146-fold. When reduced it appears as a 30 kD band on an SDS-polyacrylamide gradient gel. It has a pH optimum of 7.0 and is stimulated by low concentrations of Mg2+ (up to 2 mM), but does not have an absolute requirement for this metal ion. The kinetics of this enzyme have been investigated to ascertain whether its reaction mechanism can account for the poor in vivo rate of glycolithocholate sulfation. Inhibitor studies with an oxidized metabolite of lithocholate, 3-keto-5 beta-cholanoate, showed that the latter is a competitive inhibitor of glycolithocholate and is noncompetitive with the active form of sulfate, 3'phosphoadenosine-5'-phosphosulfate. The monophosphonucleotide 3'-AMP is a competitive inhibitor of 3'phosphoadenosine-5'-phosphosulfate, and is noncompetitive with glycolithocholate. These observations are consistent with a sequentially ordered Bi Bi reaction mechanism in which the bile salt is the first substrate to bind to the enzyme. Such a reaction mechanism for bile salt:3'phosphoadenosine-5'-phosphosulfate:sulfotransferase would be, therefore, the first time in which the sulfate acceptor (the bile salt) is the initial substrate to bind to a sulfotransferase. These studies have shown that although rhesus monkeys have a liver enzyme capable of forming the sulfate ester of glycolithocholate, its reaction mechanism and the potent inhibition caused by simple metabolites, such as 3-keto-5 beta-cholanoate, may serve to under-express the activity of the enzyme in vivo.     

Enzymatic sulfation of steroids. II. The sulfation of corticosterone by the glucocorticoid sulfotransferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This "mosaic" assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6--9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate. The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20--32% and 25--35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37--55% and 40--60% decreases of sulfation of the two steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases. Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferases I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

Enzymatic sulfation of bile salts: III. Enzymatic sulfation of taurolithocholate in human and guinea pig fetuses and adults

The activities of bile salt sulfotransferase, the enzyme responsible for the sulfation of bile salts, were determined in fetal and adult livers of humans and guinea pigs. Fetal enzyme activities in guinea pigs were approximately one-tenth of the adult and increased gradually as the gestation progressed. The bile salt sulfotransferase activities were found in human fetal livers but only 14% of the adult fatty liver activity. The result indicates human or guinea pig fetuses are capable of sulfating lithocholate derived from the mother.     

Purification and characterization of hepatic and intestinal phenol sulfotransferase with high affinity for benzo[a]pyrene phenols from channel catfish, Ictalurus punctatus

Cytosol from channel catfish liver and intestinal mucosa has high sulfotransferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyrene. To further investigate this conjugation pathway, sulfotransferase activity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal and hepatic cytosol by chromatography on anion exchange and PAP-agarose affinity columns. SDS-PAGE of the active fractions showed that one major band with molecular size of about 41,000 Da was isolated from intestine, while two bands of about 41,000 and 31,000 Da were obtained from liver. Antibodies against human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 31,000-Da protein. N-Terminal sequence information could not be obtained from the pure proteins. Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matched a sequence found in several mammalian sulfotransferases. No fish sulfotransferase sequences were available for comparison. The identity of the hepatic 31,000-Da protein was not established. The purified 41,000-Da proteins had very high activities with 3-, 7-, or 9-hydroxybenzo[a]pyrene, with K(m) values in the 40-100 nM range and V(max) 125-300 nmol/min/mg of protein. Substrate inhibition was observed when the concentrations of hydroxylated benzo[a]pyrenes were above 0.5 microM. As well as benzo[a]pyrene phenols, the purified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitrophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracene, dehydroepiandrosterone, estrone, and 17beta-estradiol. Phenolic compounds were the preferred substrates for the purified enzymes.