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.     

Hepatic triiodothyronine sulfation and its regulation by growth hormone and triiodothyronine in rats.

The regulatory mechanism of cytosolic sulfation of T3 has been studied in rat liver. Sulfation of T3 is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 times higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T3 sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T3 was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T3 (50 micrograms/kg daily for 7 days) to thyroidectomized rats. Administration of T3 in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T3 sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2,6-dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T3 was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat liver aryl sulfotransferase-catalyzed sulfation and rearrangement of 9-fluorenone oxime

The role of hepatic cytosolic aryl sulfotransferase (3'-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1) in the enzymic rearrangement of 9-fluorenone oxime to phenanthridone was investigated. 9-Fluorenone oxime was found to be an excellent substrate for a partially purified rat liver aryl sulfotransferase preparation. This compound was in fact superior to 2-naphthol, the standard assay substrate. This is the first reported observation of aryl oxime sulfation by the aryl sulfotransferases. 9-Fluorenone oxime sulfation exhibited pronounced substrate inhibition at high substrate concentrations. However, despite virtually complete conversion of 9-fluorenone oxime to the corresponding N-O-sulfate conjugate in enzyme incubation mixtures, only small amounts of rearrangement product were detected after long-term incubations. In addition, 9-fluorenone oxime-O-sulfonic acid was chemically synthesized and tested for stability. The results showed that rearrangement was pH-dependent and occurred slowly over several hours. It is therefore concluded that aryl sulfotransferase-catalyzed sulfation likely plays an important role in the in vitro and in vivo disposition of 9-fluorenone oxime. Moreover, sulfation facilitates the Beckmann-like conversion of 9-fluorenone oxime to phenanthridone. Sulfation alone, however, appears insufficient to account for all of the previously reported in vitro and in vivo rearrangement.     

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.     

Partial purification and characterization of 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases

Two 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases were purified 600-fold and 340-fold, respectively, from isolated bovine cornea cells. Sulfotransferase I exhibited an apparent Mr = 220,000, whereas an Mr = 140,000 was calculated for sulfotransferase II. The final preparations were both devoid of chondroitin sulfate sulfotransferase activity. The position of sulfation was determined by proton nuclear magnetic resonance spectroscopy. Sixty per cent of the sulfate ester groups formed by sulfotransferase I were linked to the C-6 atom of galactosyl residues, the other ones to the C-6 atom of N-acetylglucosamine. Sulfotransferase II showed a different specificity: 23% of the newly formed sulfate ester groups were on galactosyl and 77% on N-acetylglucosaminyl residues. Both sulfotransferase preparations acted in a cooperative manner. In the presence of both sulfotransferases, the incorporation of [35S]sulfate into keratan sulfate was up to 75% higher than could be expected from the sum of individual activities. From the specific radioactivities of the oligosaccharides produced by digestion with endo-beta-galactosidase, it was also concluded that both enzyme species reacted best with keratan sulfate segments exhibiting a relatively high degree of sulfation.     

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.     

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.     

Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol

A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that in the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent Km value of the enzyme for mucus glycoprotein was 10.5 X 10(-6) M (21 mg/ml), and the KI in the presence of ethanol was 4.7 x 10(-1) M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligo-saccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetyl-glucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H----6GlcNAc beta 1----3Gal beta 1----3GalNAc-ol.     

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.     

肝素硫酸转移酶优化表达及其在动物源肝素硫酸化中的应用

肝素是一种重要的凝血药物,目前主要依赖于动物小肠粘膜的提取。动物源肝素含有的抗凝血活性五糖单位GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S少,抗凝血活性低下。文中提出并验证了一种基于酶法催化动物源肝素,提高其硫酸化程度和抗凝血活性的方法。通过比较3种硫酸转移酶肝素-2-硫酸转移酶(Heparan sulfate-2-O-sulfotransferase,HS2ST)、肝素-6-硫酸转移酶(Heparan sulfate-6-O-sulfotransferase,HS6ST)、肝素-3-硫酸转移酶(Heparan sulfate-3-O-sulfotransferase,HS3ST)在重组大肠杆菌及重组毕赤酵母中表达,确定了毕赤酵母作为3种硫酸转移酶的表达宿主;进一步通过N端融合麦芽糖融合蛋白MBP和硫氧还蛋白Trx A,HS2ST和HS6ST的酶表达水平分别提高至(839?14) U/L和(792?23) U/L。通过3种硫酸转移酶HS2ST、HS6ST和HS3ST共同催化动物源肝素,其抗凝血活性由(76?2) IU/mg提高至(189?17) IU/mg。     

Sulfation of environmental estrogen-like chemicals by human cytosolic sulfotransferases

To investigate whether sulfation, a major Phase II detoxification pathway in vivo, can be employed as a means for the inactivation/disposal of environmental estrogens, recombinant human cytosolic sulfotransferases were prepared and tested for enzymatic activities with bisphenol A, diethylstilbestrol, 4-octylphenol, p-nonylphenol, and 17alpha-ethynylestradiol as substrates. Of the seven recombinant enzymes examined, only SULT1C sulfotransferase #1 showed no activities toward the environmental estrogens tested. Among the other six sulfotransferases, the simple phenol (P)-form phenol sulfotransferase and estrogen sulfotransferase appeared to be considerably more active toward environmental estrogens than the other four sulfotransferases. Metabolic labeling experiments revealed the sulfation of environmental estrogens and the release of their sulfated derivatives by HepG2 human hepatoma cells. Moreover, sulfated environmental estrogens appeared to be incapable of penetrating through the HepG2 cell membrane.     

Purification and characterization of galactocerebroside sulfotransferase from rat kidney

Galactocerebroside sulfotransferase (EC 2.8.2.11) was purified to apparent homogeneity from rat kidneys. The purified protein is stable at -20 degrees C, and has an estimated molecular weight of 64,000 and a pI of 5.1. In contrast to other known sulfotransferases, the enzyme appears not to require divalent metal ions for activity. The Km for the donor, 3'-phosphoadenosine 5'-phosphosulfate, is 5.2 microM. Structural studies on this "active" sulfate donor show the requirement of a phosphate group at the 3' position of the ribose moiety. Modification of the amino group at either the 6 or 8 position on the purine ring renders the corresponding compounds poor substrates. Both galactosylceramide and lactosylceramide are effective acceptors for this enzyme, while galactosylsphingosine and galactosylglycerolipids are sulfated only poorly, suggesting that the in vivo sulfation of these glycolipids is carried out by different sulfotransferases. The active site of the enzyme contains arginine residues which appear to be important in binding the sulfate donor. The enzyme protein is hydrophobic and binds 0.17 mg [3H]Triton X-100/mg protein. The purified enzyme contains bound lipids, consisting primarily of cholesterol and phosphatidylcholine. The lipid environment affects the activity of the enzyme which, in turn, regulates the sulfation of glycolipids.     

Continuous fluorometric assay of phenol sulfotransferase.

Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.     

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.     

Effects of detergents on the sulfation of chondroitin sulfate by sulfotransferase from chicken embryo epiphyseal cartilage

Homogenates of chicken embryo epiphyseal cartilage were prepared in buffered saline. The bulk of the sulfotransferase was found in the supernatant. However, small amounts of sulfotransferase were consistently found in the particulate fraction. Detergents (Triton X-100 and C12E8) added to the incubation mixture activated the sulfation of exogenous sulfate acceptor by the particulate fraction, whereas detergent treatment during homogenization increased sulfotransferase activity in the supernatant at the expense of that in the particulate fraction. Since sulfotransferase activities of the supernatant and particulate fractions had similar properties concerning specificity, affinity for chondroitin with different degrees of sulfation, thermal stability and activation by protamine, we conclude that the same enzyme is present in both fractions and that detergent activates indirectly, by releasing it to the medium.     

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.     

Tyrosine sulfation of statherin

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.     

Enzymatic sulfation of galactosyl- and lactosylceramides in cell lines derived from renal tubules.

1. The renal cell lines, JTC-12 and MDCK, not only synthesize galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate in vivo, but also contain enzymes that catalyze the transfer of sulfate to galactosylceramide and lactosylceramide in vitro. 2. Concentration of cations necessary for maximum sulfotransferase activity occurred at 40 mM Ca2+ with galactosylceramide and 15 mM Ca2+ with lactosylceramide as the substrate. Na+ was also found to stimulate the sulfation of galactosylceramide, but was slightly inhibitory for the sulfation of lactosylceramide. 3. The products of the in vitro assay mixture were characterized as galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate by a variety of TLC separations. 4. The apparent Km of JTC-12 cells for galactosylceramide was 17 microM, while that for lactosylceramide was 82 microM. The Km values of MDCK cells were comparable to those of JTC-12 cells. Competition studies suggested that galactosylceramide and lactosylceramide were sulfated by a single enzyme in both cell lines.     

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.     

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.