Purification and characterization of bile salt sulfotransferase from human liver

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Alkaline butanol extraction of bile salt and steroid sulfate esters: application to the assay of sulfotransferases

A butan-1-ol solvent-extraction procedure has been evaluated for the assay of 3'-phosphoadenosine-5'-phosphosulfate:sulfotransferase activity with various bile salt and steroid substrates. Although butanol extracted the sulfate esters of steroids and bile salts from aqueous solution at neutral pH, extraction at basic pH gave optimum recovery which was independent of protein in the sample. Greater than 99.9% of unreacted 3'-phosphoadenosine-5'-phospho[35S]sulfate remained in the aqueous phase. The data for sulfotransferase activities obtained with this solvent-extraction assay were not significantly different from those obtained with a standard thin-layer chromatography method. Solvent extraction has enabled multiple, rapid assays of several steroid and bile salt sulfotransferases during chromatographic purification of these enzymes from tissue fractions.     

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.     

A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV.

We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from the desulfated 3'-phosphoadenosine-5'-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure beta-form during purification, and utilized for the regeneration. The activity of betaAST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. betaAST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotransferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and other tissues for specific sulfotransferase enzyme activity and substrate concentration.     

Effect of high and low sulfate concentrations on adenosine 5'-phosphosulfate sulfotransferase activity from Lemna minor

The effect of Na₂SO₄ concentrations from 0 to 17.6 m M in the nutrient solution of Lemna minor L. strain 6580 on adenosine 5'-phosphosulfate sulfotransferase activity was examined. Routinely, the plants were cultivated on 0.88 mA SO₄²⁻. The enzyme activity was increased by 50 to 100% after transfer to 0 or 0.0088 m M SO₄²⁻. Transfer back to 0.88 m M rapidly decreased the enzyme activity to the initial level. Cultivation on 17.6 mM Na₂SO₄ redueed extractable adenosine 5'-phosphosulfate sulfotransferase by 50%. The original level was rapidly re-established on 0,88 m M . In control experiments, a decrease in adenosine 5'-phosphosulfate sulfotransferase activity was also induced by K₂ SO₄, whereas NaCl caused a small increase. This indicates that the observed effects are dependent on the sulfate ion. ATP-sulfurylase activity measured for comparison was only significantly affected by the omission of sulfate, which induced a 20% increase, indicating that this enzyme activity from Lemna minor is less suseeptible to changes in medium sulfate than adenosine 5'-phosphosulfate sulfotransferase. A close relationship between adenosine 5'-phosphosulfate sulfotransferase activity and the content of asparagine, glutamine, non-protein thiols and sulfate in the tissue was detected, indicating a positive control mechanism induced by amides and a negative mechanism induced by thiols and sulfate.     

Novel flavonol 3-sulfotransferase. Purification, kinetic properties, and partial amino acid sequence.

A flavonol sulfotransferase (EC 2.8.2.-), which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the 3-hydroxyl group of flavonol aglycones, has been purified to apparent homogeneity from Flaveria chloraefolia. The specific activity of flavonol 3-sulfotransferase was enriched 2000-fold, as compared with the homogenate, with a recovery of 9%. The molecular mass of the native and denatured enzyme was found to be 34.5 kDa, suggesting that the active from of the enzyme is a monomer. The enzyme exhibited expressed specificity for position 3 of flavonol aglycones, showed two activity optima at pH 6.0 and 8.5, did not require divalent cations, and was not inhibited by either EDTA or sulfhydryl group reagents. The results of substrate interaction kinetics and product inhibition are consistent with an Ordered Bi Bi mechanism where 3'-phosphoadenosine 5'-phosphosulfate is the first substrate to bind to the enzyme and 3'-phosphoadenosine 5'-phosphate is the final product to be released. The amino acid sequence of two peptides representing 17 and 33 amino acids showed no significant sequence similarity with the amino acid sequences reported for animal sulfotransferases. Antibodies raised against F. chloraefolia 3-sulfotransferase were found to cross-react with the 3'- and 4'-sulfotransferase activities of the same plant, suggesting that the three enzymes are structurally related.     

Structure-activity relationship of the cholestatic activity of dihydrotestosterone glucuronide, allo bile acids and lithocholate

Dihydrotestosterone glucuronide (DHTG), a series of 5 alpha-bile acids, or allo-bile acids (3 alpha-hydroxy-5 alpha-cholanic acid, 3-keto-5 alpha-cholanic acid and 3 beta-hydroxy-5 alpha-cholanic acid) and their normal bile acid analogues (3 alpha-hydroxy-5 beta-cholanic acid or lithocholate, 3-keto-5 beta-cholanic acid and 3 beta-hydroxy-5 beta-cholanic acid) were administered intravenously to female rats in order to determine their effects on bile flow. All agents caused a rapid and profound inhibition of bile flow which was dose-dependent. The logarithm of the dose vs the cholestatic response curve for DHTG, the allo-bile acids and lithocholate were all parallel. DHTG was the most potent congener and was two times more potent than 3-keto-5 alpha-cholanic acid and 5 times more potent than lithocholate. These data indicate that the glucuronic acid moiety and the trans configuration of the A and B rings of the steroid nucleus confer the greatest cholestatic potency.     

Enzymatic assay for flavonoid sulfotransferase

A novel enzyme assay for flavonoid sulfotransferase is described. It makes use of tetrabutylammonium dihydrogen phosphate which forms a pair of ions with the flavonoid sulfate esters formed. This renders the sulfate ester soluble in organic solvents such as ethyl acetate, whereas the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, remains in the aqueous reaction mixture. The procedure is simple, rapid, and reproducible. It eliminates the need for chromatographic separation of the reaction products, except when their identification is required, and is suitable for use in the purification and kinetic studies of sulfotransferases.     

Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat

Sulfate esterification has been shown previously to be a prominent feature of lithocholate metabolism in man. These studies were undertaken to ascertain whether this metabolic pathway is also present in rats, and to investigate the physiological significance of bile acid sulfate formation. Lithocholic acid-24-(14)C was administered to bile fistula rats, and sulfated metabolites were identified in bile by chromatographic and appropriate degradative procedures. They constituted only a small fraction (2-9%) of the total metabolites but a more significant fraction (about 20%) of the secreted monohydroxy bile acids, most of the lithocholate having been hydroxylated by the rat liver. When sulfated glycolithocholate was administered orally, it was absorbed from the intestine without loss of the sulfate, presumably by active transport, and secreted intact into the bile. In comparison with non-sulfated lithocholate, an unusually large fraction (24%) of the sulfated bile acid was excreted in the urine, and fecal excretion took place more rapidly. Both the amino acid and sulfate moieties were extensively removed prior to excretion in the feces. Hydroxylation of bile acid sulfates or sulfation of polyhydroxylated bile acids did not occur to any great extent, if at all.     

Demonstration of a novel sulfotransferase in fetal bovine serum, which transfers sulfate to the C6 position of the GalNAc residue in the sequence iduronic acid alpha1-3GalNAc beta1-4iduronic acid in dermatan sulfate.

A novel sulfotransferase activity was discovered in fetal bovine serum using pig skin dermatan sulfate as an acceptor and [35S]3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor. The enzyme was separated from chondroitin:GalNAc 6-O-sulfotransferase by chromatographic techniques. Enzymatic analysis of the reaction products demonstrated that the enzyme transferred sulfate to the C6 position of the GalNAc residue in the sequence -iduronic acid alpha1-3GalNAc beta1-4iduronic acid-. Thus, the enzyme has been identified as a hitherto unreported dermatan sulfate:GalNAc 6-O-sulfotransferase. The finding is in sharp contrast to the current concept that in dermatan sulfate biosynthesis GalNAc 4-O-sulfation is a prerequisite for iduronic acid formation by C5 epimerase.     

A novel type of aryl sulfotransferase obtained from an anaerobic bacterium of human intestine

A novel type of aryl sulfotransferase is produced by an anaerobic bacterium of human intestine, Eubacterium A-44. Aryl sulfotransferase separated from this bacterium differs from the sulfotransferase which uses 3'-phosphoadenosine 5'-phosphosulfate as a donor. The enzyme catalyzes stoichiometric transfer of a sulfate group from a phenol sulfate ester to other phenolic compounds, with strict specificity. The optimal pH and molecular weight were 8-9 and 315,000, respectively.     

An Ecteola-cellulose chromatography assay for 3′-phosphoadenosine 5′-phosphosulfate: Phenol sulfotransferase

A new assay procedure for phenol sulfotransferase which employs [35S]-3'-phosphoadenosine-5'-phosphosulfate as a sulfate donor and a variety of phenols as sulfate acceptors was developed. The appearance of the 35S-sulfated products or the disappearance of the [35S]-3'-phosphoadenosine-5'-phosphosulfate are determined simultaneously by chromatography of the assay incubation mixtures on Ecteola-cellulose columns, eluting with an NH4HCO3 step gradient. Various acidic, neutral, and basic phenols can be employed as substrates for phenol sulfotransferase using this procedure.     

Adenosine 5'-phosphosulfate sulfotransferase and adenosine 5'-phosphosulfate reductase are identical enzymes

Adenosine 5'-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M(r) of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K(m) = 6.5 microM) and not adenosine 3'-phosphate 5'-phosphosulfate as sulfonyl donor. The V(max) of recombinant Lemna APS sulfotransferase (40 micromol min(-1) mg protein(-1)) was about 10 times higher than the previously published V(max) of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO(3)(2-). Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO(3)(2-) was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.     

Identifying androsterone (ADT) as a cognate substrate for human dehydroepiandrosterone sulfotransferase (DHEA-ST) important for steroid homeostasis: structure of the enzyme-ADT complex

In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 A resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.     

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.     

Endocrine steroid sulfotransferases: porcine endometrial estrogen sulfotransferase

Porcine endometrial estrogen sulfotransferase has been isolated and its properties examined. This enzyme only appeared in uteri from ovariectomized gilts which had been primed with estrogen and treated with progesterone. The most stable form of the enzyme was obtained via chromatofocusing of the 100,000 g supernatant from secretory endometrium. A molecular weight of 31 KDa was determined for this sulfotransferase by molecular sieve (Sephadex G-200 Superfine) and disk-gel electrophoresis. The active protein displayed a pI of 6.1, pH optimum of 7.6-7.8 and a requirement of 10 mM Mg2+ for maximum transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to estrone (E1). Km of the reaction was 24 +/- 4.7 microM for PAPS and 24 +/- 9.8 nM for E1 as substrate. Porcine endometrial sulfotransferase thus displayed a much greater affinity for E1 than a similar enzyme previously isolated from bovine adrenals. As has been observed of sulfotransferases from other tissues, an endogenous substrate (presumed to be E1) accompanies the enzyme throughout its purification.     

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).     

Comparison of estrogen sulfotransferase and pregnenolone sulfotransferase of guinea pig.

Guinea pig adrenal estrogen sulfotransferase from either sex was eluted as a single peak, irrespective of buffer salt concentration, when subjected to fast protein liquid chromatography on gel filtration columns. The same enzyme was consistently eluted in two distinct peaks during chromatofocusing. Adrenal pregnenolone sulfotransferase was eluted during gel filtration in a heterogeneous pattern, dependent on salt concentration. These properties have made possible almost complete separation of the two sulfotransferases in one step, although adrenal estrogen sulfotransferase may possess a minute intrinsic ability to catalyze sulfation of pregnenolone. Pregnenolone sulfotransferase had no measurable activity toward estrone. Pregnenolone sulfotransferase from both sexes yielded variable elution patterns during chromatofocusing. Estrogen sulfotransferase from the adrenal, as well as that of guinea pig chorion, was strongly inhibited by N-ethylmaleimide and to a lesser degree by iodoacetamide and iodoacetate. Adrenal and chorion estrogen sulfotransferases were thermolabile and were activated, although not protected from the effect of heat, by binding to 3'-phosphoadenosine 5'-phosphosulfate. Adrenal pregnenolone sulfotransferase was inhibited only by high concentrations of N-ethylmaleimide and not at all by iodoacetamide or iodoacetate. It was more thermostable than the estrogen sulfotransferase and was not activated by binding to 3'-phosphoadenosine 5'-phosphosulfate.     

Slalom encodes an adenosine 3'-phosphate 5'-phosphosulfate transporter essential for development in Drosophila

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate. Here we report the first molecular identification of a gene that encodes a transmembrane protein required to transport adenosine 3'-phosphate 5'-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which we call slalom, display defects in Wg and Hh signaling, which are likely due to the lack of sulfation of glycosaminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal-ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal-ventral determinant pipe.     

Effect of SO2, on the activity of adenosine 5'-phosphosulfate sulfotransferase from spruce trees (Picea abies) in fumigation chambers and under field conditions

The effect of SO₂ on adenosine 5'-phosphosulfate sulfotransferase activity and various other parameters of needles from spruce ( Picea abies L.) was studied using potted grafts in outdoor fumigation chambers and trees growing near a factory. In summer and autumn fumigation of grafted spruce, SO₂, decreased the extractable activity of adenosine 5'-phosphosulfate sulfotransferase to 12–50% of the controls, and reduced the amount of ³⁵S from sulphate incorporated into protein by excised branches to a comparable degree. SO₂ treatment in January and February inhibited the increase in adenosine 5'phosphosulfate sulfotransferase activity measured in the controls during this time. ATP-sulfurylase activity was less affected by SO₂. fumigation. In trees growing near a factory with high SO₂. emission, the activity of adenosine 5'-phosphosulfate sulfotransferase was about 35% of that of trees from a control area. The low enzyme activity was correlated with a high content of sulfate and compounds containing thiol groups.