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PubMed1995

Hydrolysis of estrone sulfate in uterine minces of the 6-days pregnant rat.

In view of the possible participation of estrogen 3-sulfoconjugates in reproductive events, such as reproductive cycles, gestation and parturition, an experiment was carried out where the conversion of labeled estrone sulfate ([3H]E1S) to estrone ([3H]E1) was measured during in vitro incubation with minced uterine tissues representing implantation sites (IS) and non-implanted areas (NIS), from pregnant rats at the time of blastocyst implantation. Significant hydrolysis of the 3-sulfate, by the action of the estrogen sulfatase, was found in both tissues being less in IS than in NIS, when expressed either as pmol of E1 formed/mg wet tissue/h (238 +/- 37 vs 337 +/- 15, respectively) or as pmol of E1 formed/mg protein/h (1278 +/- 198 vs 1773 +/- 81). Both differences are statistically significant at the 0.001 level. The results obtained here suggest that E1S present in uterine fluids may be taken up and hydrolyzed by the sulfatase present in both intrauterine tissues of the 6-days pregnant rat. However, the decreased formation of E1 found in IS suggests that rat blastocyst is able to regulate the local concentration of unconjugated estrogens required at IS by modulating the activity of the estrogen sulfatase.

Animals↗
PubMed2006

Estradiol as an anti-aromatase agent in human breast cancer cells.

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.

Aromatase↗
PubMedDate not supplied

The effect of 17beta estradiol withdrawal on the level of brain and peripheral neurosteroids in ovarectomized rats.

Dehydroepiandrosterone (DHEA), pregnenolone (P) and their sulfate derivatives are neuroactive neurosteroids synthesized endogenously in the brain and in steroidogenic organs and influence or are influenced by a variety of physiological processes. Since parturition is followed by a rapid drop in estrogen levels in serum and brain it may be hypothesized that the drastic drop in the brain exposure to estrogens may cause a disturbance in the neurosteroid-to-neurosteroid-sulfate equilibrium with clinical relevance. In order to develop a rat animal model for human postpartum rapid estrogen decline conditions, the present study investigated effects of sudden withdrawal of hyperphysiological estrogens levels on levels of DHEA, DHEAS, P and PS in peripheral blood and brain tissue as well as cortical sulfatase activity. Twenty-four 3-month-old female rats were ovarectomized followed by either no estrogen, high levels of estrogen alone, or followed by sudden withdrawal after high-administered estrogen levels. Results indicated elevated brain cortical DHEA-S and reduced cortical sulfatase in ovarectomized rats following sudden estrogen withdrawal. No significant alterations in DHEA, P or PS were noted. Study observations suggest the marked influence estrogen withdrawal states may have on cortical DHEA-S levels in particular, the precise mechanism of which remains unknown but which may be related to the paralleled decrease in sulfatase activity. This DHEA-S increase may lead to attenuated GABAergic tone and may be relevant to post-natal behavioral disturbances (e.g. depression, anxiety).

Animals↗
PubMed2002

Synthesis and utility of sulfated chromogenic carbohydrate model substrates for measuring activities of mucin-desulfating enzymes.

A chromogenic substrate, 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate was synthesized and used in combination with beta-N-acetylhexosaminidase for detection of the sulfatase, MdsA, by release of 4-nitrophenol. MdsA was originally isolated from the bacterium Prevotella strain RS2 and is believed to be involved in desulfation of sulfomucins, major components of the mucus barrier protecting the human colon surface. The exo nature of the MdsA sulfatase was indicated by its inability to de-esterify the disaccharide 4-nitrophenyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. This latter compound was prepared from monosaccharide precursors by two different methods, the shorter requiring just six steps from 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and giving an overall yield of 26.4%. The syntheses of 4-nitrophenyl beta-D-galactopyranoside 3-triethylammonium sulfate and 6-triethylammonium sulfate and their use in combination with beta-galactosidase as chromogenic substrates for detecting Bacteroides fragilis sulfatases with different specificities was also demonstrated.

Aspergillus oryzae↗
PubMedDate not supplied

The selective estrogen enzyme modulator (SEEM) in breast cancer.

Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol biosynthesis. This tissue also contains sulfotransferase for the formation of the biologically inactive estrogen sulfates. In the last years, it was demonstrated that various progestins (promegestone, nomegestrol acetate, medrogestone), as well as tibolone and its metabolites are potent inhibitors of sulfatase and 17beta-hydroxysteroid dehydrogenase activities. It was also shown that medrogestone, nomegestrol acetate, promegestone or tibolone can stimulate the sulfotransferase activity for the local production of estrogen sulfates. All these data, in addition to numerous agents, which can block the aromatase action, lead to the new concept of selective estrogen enzyme modulators (SEEM), which can largely apply to breast cancer tissue. The exploration of various progestins and other active agents in trials with breast cancer patients, showing an inhibitory effect on sulfatase and 17beta-hydroxysteroid dehydrogenase, or a stimulatory effect on sulfotransferase, will provide a new possibility in the treatment of this disease.

17-Hydroxysteroid Dehydrogenases↗
PubMed1998

Crystal structure of human arylsulfatase A: the aldehyde function and the metal ion at the active site suggest a novel mechanism for sulfate ester hydrolysis.

Human lysosomal arylsulfatase A (ASA) is a prototype member of the sulfatase family. These enzymes require the posttranslational oxidation of the -CH2SH group of a conserved cysteine to an aldehyde, yielding a formylglycine. Without this modification sulfatases are catalytically inactive, as revealed by a lysosomal storage disorder known as multiple sulfatase deficiency. The 2.1 A resolution X-ray crystal structure shows an ASA homooctamer composed of a tetramer of dimers, (alpha 2)4. The alpha/beta fold of the monomer has significant structural analogy to another hydrolytic enzyme, the alkaline phosphatase, and superposition of these two structures shows that the active centers are located in largely identical positions. The functionally essential formylglycine is located in a positively charged pocket and acts as ligand to an octahedrally coordinated metal ion interpreted as Mg2+. The electron density at the formylglycine suggests the presence of a 2-fold disordered aldehyde group with the possible contribution of an aldehyde hydrate, -CH(OH)2, with gem-hydroxyl groups. In the proposed catalytic mechanism, the aldehyde accepts a water molecule to form a hydrate. One of the two hydroxyl groups hydrolyzes the substrate sulfate ester via a transesterification step, resulting in a covalent intermediate. The second hydroxyl serves to eliminate sulfate under inversion of configuration through C-O cleavage and reformation of the aldehyde. This study provides the structural basis for understanding a novel mechanism of ester hydrolysis and explains the functional importance of the unusually modified amino acid.

Aldehydes↗
PubMed1998

Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A.

Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue. The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative. In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis. The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties. Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50%. Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis. A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.

Alanine↗
PubMedDate not supplied

Solid-phase parallel synthesis of 17alpha-substituted estradiol sulfamate and phenol libraries using the multidetachable sulfamate linker.

We report an application of the multidetachable sulfamate linker in the synthesis of two model libraries of N-derivatized 17alpha-piperazinomethyl estradiols (phenols and sulfamates) by solid-phase parallel chemistry. The solid-phase precursor, a 3-sulfamoyl-17alpha-(N-trifluoroacetyl-piperazinomethyl) estradiol, was synthesized in solution from estrone and loaded efficiently onto trityl chloride resin as polymeric support. After cleavage of the trifluoroacetyl protecting group, sequential acylation reactions with five Fmoc-protected amino acids and five carboxylic acids were performed to introduce two levels of molecular diversity. Finally, the resins were split into two parts, and acidic (5% trifluoroacetic acid in dichloromethane) and nucleophilic (piperazine in tetrahydrofuran) cleavages were used to generate libraries A (5 x 5 sulfamates) and B (5 x 5 phenols) members in overall yields of 18-66% and high HPLC purities (87-96%) without purification steps. A preliminary screening test for inhibition of steroid sulfatase showed that the phenols were clearly weaker inhibitors, as compared to their sulfamate analogues. The most potent inhibitors were those with suitable hydrophobic amino acid and carboxylic acid substituents. Thus, compounds with a phenylalanine residue as the first element of diversity inhibited over 90% of steroid sulfatase activity at a concentration of 1 nM in homogenates of HEK-293 transfected cells, being as potent as the leading inhibitor 17alpha-tert-butylbenzyl estradiol 3-O-sulfamate previously reported. These results suggest that the steroid sulfatase inhibitory potency of estradiol derivatives, sulfamoylated or not, can be increased by the hydrophobic effect of a suitable substituent introduced in the proximity of the D ring of the steroid. The present work also demonstrated the efficiency and the cleavage versatility of the sulfamate linker to generate libraries of compounds with relevant biological importance, phenols and sulfamates.

Amino Acids↗
PubMed1989

Demonstration of acid hydrolase activity in primary cultures of bovine brain microvessel endothelium.

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood-brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and beta-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69-77%). The majority of beta-galactosidase (approximately 48%) and total sulfatase (approximately 58%) activity was associated with the lysosome fraction of the BMECs. In contrast, approximately 52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

Acid Phosphatase↗
PubMed1999

Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding.

Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.

Amino Acid Sequence↗
PubMed2005

Molecular characterization of the human Calpha-formylglycine-generating enzyme.

Calpha-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calcium-binding protein containing an N-terminal (residues 86-168) and a C-terminal (residues 178-374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the N-terminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocross-linking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.

Amino Acid Sequence↗
PubMed2005

Crystal structure of human pFGE, the paralog of the Calpha-formylglycine-generating enzyme.

In eukaryotes, sulfate esters are degraded by sulfatases, which possess a unique Calpha-formylglycine residue in their active site. The defect in post-translational formation of the Calpha-formylglycine residue causes a severe lysosomal storage disorder in humans. Recently, FGE (formylglycine-generating enzyme) has been identified as the protein required for this specific modification. Using sequence comparisons, a protein homologous to FGE was found and denoted pFGE (paralog of FGE). pFGE binds a sulfatase-derived peptide bearing the FGE recognition motif, but it lacks formylglycine-generating activity. Both proteins belong to a large family of pro- and eukaryotic proteins containing the DUF323 domain, a formylglycine-generating enzyme domain of unknown three-dimensional structure. We have crystallized the glycosylated human pFGE and determined its crystal structure at a resolution of 1.86 A. The structure reveals a novel fold, which we denote the FGE fold and which therefore serves as a paradigm for the DUF323 domain. It is characterized by an asymmetric partitioning of secondary structure elements and is stabilized by two calcium cations. A deep cleft on the surface of pFGE most likely represents the sulfatase polypeptide binding site. The asymmetric unit of the pFGE crystal contains a homodimer. The putative peptide binding site is buried between the monomers, indicating a biological significance of the dimer. The structure suggests the capability of pFGE to form a heterodimer with FGE.

Amino Acid Motifs↗
PubMed1999

The SEEM: selective estrogen enzyme modulators in breast cancer.

Human breast cancer tissue contains all the enzymes (estrone sulfatase, 17 beta-hydroxysteroid dehydrogenase, aromatase) involved in the last steps of estradiol biosynthesis. This tissue also contains sulfotransferase for the formation of the biologically inactive estrogen sulfates. In the past years, it has been demonstrated that various progestins (promegestone, nomegestrol acetate, medrogestone) as well as tibolone and its metabolites are potent inhibitors of sulfatase and 17 beta-hydroxysteroid dehydrogenase activities. It was also shown that medrogestone, nomegestrol acetate, promegestone or tibolone can stimulate the sulfotransferase activity for the local production of estrogen sulfates. All these data, in addition to numerous agents which can block the aromatase action, lead to the new concept of Selective Estrogen Enzyme Modulators (SEEM) which can largely apply to breast cancer tissue. The exploration of various progestins and other active agents in trials with breast cancer patients, showing an inhibitory effect on sulfatase and 17 beta-hydroxysteroid dehydrogenase, or a stimulatory effect on sulfotransferase, will provide a new option in the treatment of this disease.

Antineoplastic Agents, Hormonal↗
PubMed2005

De novo calcium/sulfur SAD phasing of the human formylglycine-generating enzyme using in-house data.

Sulfatases are a family of enzymes essential for the degradation of sulfate esters. Formylglycine is the key catalytic residue in the active site of sulfatases and is generated from a cysteine residue by FGE, the formylglycine-generating enzyme. Inactivity of FGE owing to inherited mutations in the FGE gene results in multiple sulfatase deficiency (MSD), which leads to early death in infants. Human FGE was crystallized in the presence of traces of the protease elastase, which was absolutely essential for crystal growth, and the structure of FGE was determined by molecular replacement. Before this model was completed, the FGE structure was re-determined by SAD phasing using in-house data based on the anomalous signal of two calcium ions bound to the native enzyme and intrinsic S atoms. A 14-atom substructure was determined at 1.8 A resolution by SHELXD; SHELXE was used for density modification and phase extension to 1.54 A resolution. Automated model building with ARP/wARP and refinement with REFMAC5 yielded a virtually complete model without manual intervention. The minimal data requirements for successful phasing and the relative contributions of the Ca and S atoms are discussed and compared with the related FGE paralogue, pFGE. This work emphasizes the usefulness of de novo phasing using weak anomalous scatterers and in-house data.

Calcium↗
PubMed1988

Steroid metabolism by epidermal keratinocytes.

The metabolism of various radiolabeled steroids by cultured human epidermal keratinocytes was studied in an attempt to identify the steroid-metabolizing enzymes present in these cells. Sulfatase activity was demonstrated in keratinocytes with either E1S or DS as substrates. The products of sulfatase action were E1 and DHEA, respectively. The specific activity of the enzyme was approximately 5- to 14-fold greater with E1S as the substrate compared with DS, and the rates of hydrolysis were linear with incubation time up to 3 h. The metabolism of DHEA by the keratinocyte 17 beta-HSOR-catalyzed reaction resulted in the predominant formation of 5-androstene-3 beta,17 beta-diol. The rate of formation of 5-androstene-3 beta,17 beta-diol was linear with time of incubation up to 18 h, and the specific activity of 17 beta-HSOR, with DHEA as the substrate, was greater in keratinocytes maintained in culture for 4 weeks compared with keratinocytes kept in culture for 1 week. Androstenedione was a minor product of DHEA metabolism. The metabolism of DHT by epidermal keratinocytes resulted in the formation of 5 alpha-androstanedione, 5 alpha-androstane-3 alpha,17 beta-diol, 5 alpha-androstane-3 beta,17 beta-diol, androsterone, and isoandrosterone: the rates of formation of 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol were linear with incubation time up to 24 h, and the specific activities of 3 alpha-HSOR and 3 beta-HSOR did not appear to change with keratinocyte time in culture up to 3 weeks. The metabolism of DOC by epidermal keratinocytes resulted in 5 alpha-dihydrodeoxycorticosterone production: the rate of formation of this metabolite was linear with incubation time up to 4 h. The metabolism of E1 by epidermal keratinocytes yielded E2, and that of E2 resulted in the formation of E1. The rate of E1 formation from E2, was approximately 10-fold greater than the rate of formation of E2 from E1; these rates were linear with incubation time up to 4 h. Epidermal keratinocytes maintained in culture did not metabolize androstenedione to either E1 or E2, and pregnenolone was not metabolized by these cells. This study serves to ascertain that epidermal keratinocytes express steroid 5 alpha-reductase, 17 beta-HSOR, 3 beta-HSOR, 3 alpha-HSOR, 3 beta-hydroxysteroid oxidoreductase-delta 5----4-isomerase, and sulfatase activities.

Arylsulfatases↗
PubMed1979

Non-inactivation of an x-chromosome locus in man.

Cloned fibroblasts from women heterozygous for X-linked ichthyosis (steroid sulfatase deficiency) were examined to see whether or not this locus is subject to X-inactivation. Of 103 clones examined, all had normal levels of steroid sulfatase activity. Two of the women studied were also heterozygous for glucose-6-phosphate dehydrogenase deficiency. This allowed the demonstration that both X chromosomes were represented as the active X in various clones and that selection did not account for these findings. Thus, the steroid sulfatase locus, like the Xga locus to which it is linked, appears to escape X-inactivation in man.

Dehydroepiandrosterone↗
PubMed1985

The in vitro formation of sulfates and glucuronides of estrogens by adult and fetal ovine tissues.

Incubation of nanomolar concentrations of [3H]estrone with ovine liver slices from adult and fetal animals demonstrated, in particular, the production of estrogen sulfates together with smaller amounts of glucuronides, even although microsomal estrogen glucuronyltransferase (GT) and sulfatase activities were high, especially in adult tissue. [3H]Estriol was conjugated almost exclusively as sulfate under the same experimental conditions. Slices of maternal and fetal kidney medulla were also strikingly active in promoting estrogen sulfate production as were slices of fetal kidney cortex. Adult kidney cortex conjugated estrogen only in the glucuronide form. These data indicate the possibility that maternal and fetal liver and kidney might contribute to the high circulating level of estrone sulfate in the pregnant sheep. Through the use of [3H]estrone and [3H]estrone sulfate as substrates, it was possible to demonstrate that adult slices of kidney medulla possessed relatively low sulfatase, considerable sulfotransferase (ST), and virtually no GT activity, whereas cortex had high sulfatase, little or no ST, and low, though demonstrable, GT activity. The ST activity of kidney high-speed supernatants was stimulated by the presence of sulfhydryl groups, whereas that in liver was not. Enzymic reduction of estrone and (or) estrone sulfate by liver and kidney slices indicated that, in the former, 17 alpha-reduction prevailed and, in the latter with the exception of the maternal medulla, 17 beta-reduction was the main pathway, particularly in the fetus.

Animals↗
PubMed1986

Steroid metabolism by cells from human chorion laeve isolated on Percoll gradients.

Collagenase-dispersed cells from human chorion laeve were examined on Percoll gradients. The 3 beta-hydroxysteroid dehydrogenase (a trophoblast marker) and steroid sulfatase activities of the cells were measured and a system was developed to isolate enriched preparations of the trophoblast cells. No cells were found to sediment at Percoll concentrations greater than 50%, and using continuous gradients of Percoll there appeared to be cells with different 3 beta-hydroxysteroid dehydrogenase (3 beta HSD): steroid sulfatase ratios sedimenting in different regions of the gradient. Cells with a high ratio were found in the denser region of the gradient. Continuous gradients provided inadequate separations of distinct populations of cells, thus to obtain a more reproducible system to isolate cells, discontinuous gradients of Percoll were studied. A discontinuous gradient composed of 5, 20, 40, and 60% Percoll was developed and three bands of cells were found sedimenting at the 20, 40 and 60% interfaces, respectively. The number and appearance of cells at the 20 and 60% interfaces varied from tissue to tissue. In contrast, the cells sedimenting at the 40% interface were less variable, a substantial number was found to be present in every tissue studied, they were similar in appearance to the trophoblast cells and had high 3 beta HSD:sulfatase ratios.

3-Hydroxysteroid Dehydrogenases↗