Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

Falany, Charles N.; Krasnykh, Victor; Falany, Josie L.

doi:10.1016/0960-0760(95)00015-r

Cited by 196 publications

(205 citation statements)

References 17 publications

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“…In human tissues, several members of the cytosolic sulfotransferase (SULT) family are responsible for the sulfation of estrogens, therapeutic estrogenic compounds and HRT agents (14)(15)(16). As reported previously, SULT1E1 and SULT 2A1 are the major isoforms involved in the sulfation of Tib and its metabolites (10).…”

Section: Introductionmentioning

confidence: 88%

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Falany

2007

The Journal of Steroid Biochemistry and Molecular Biology

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Sulfation is important in the metabolism and inactivation of steroidal compounds and hormone replacement therapeutic (HRT) agents in human tissues. Although generally inactive, many steroid sulfates are hydrolyzed to their active forms by sulfatase activity. Therefore, the specific sulfotransferase (SULT) isoforms and the levels of steroid sulfatase (STS) activity in tissues are important in regulating the activity of steroidal and HRT compounds. Tibolone (Tib) is metabolized to three active metabolites and all four compounds are readily sulfated. Tib and the Δ4-isomer are sulfated at the 17β-OH group by SULT2A1 and the 17-sulfates are resistant to hydrolysis by human placental STS. 3α-OH and 3β-OH Tib can form both 3-and 17-monosulfates as well as disulfates. Only the 3β-sulfates are susceptible to STS hydrolysis. Raloxifene monosulfation was catalyzed by at least seven SULT isoforms and SULT1E1 also synthesizes raloxifene disulfate. SULT1E1 forms both monosulfates in a ratio of approximately 8:1 with the more abundant monosulfate migrating on HPLC identical to the SULT2A1 synthesized monosulfate. The raloxifene monosulfate formed by both SULT isoforms is sensitive to STS hydrolysis whereas the low abundance monosulfate formed by SULT1E1 is resistant. The benzothiophene sulfates of raloxifene and arzoxifene were hydrolyzed by STS whereas the raloxifene 4′-phenolic sulfate was resistant. These results indicate that tissue specific expression of SULT isoforms and STS could be important in the inactivation and regeneration of the active forms of HRT agents.

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Section: Introductionmentioning

confidence: 88%

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Falany

2007

The Journal of Steroid Biochemistry and Molecular Biology

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“…This identification has not been confirmed by the demonstration of sulphation activity with the expressed BR-STL proteins. The BR-STL cDNAs isolated from both rat and human brain cDNA libraries have been expressed in both E. coli and Sf9 insect cells, which in our laboratory have been used previously to generate enzymically active SULTs [11,16,17]. Approximately 20 prototypical SULT substrates have been tested as substrates with the expressed BR-STLs ; however, sulphation activity has not been detected with any of these substrates.…”

Section: Discussionmentioning

confidence: 99%

“…His-tagged hBR-STL protein was purified by Ni# + -affinity chromatography and electroelution for use as an antigen, and as a standard in the immunoblotting procedures. Initial attempts to raise polyclonal antibodies in rabbits using procedures that were effective for other cytosolic SULTs [11,[15][16][17] were unsuccessful. Also attempts to raise rabbit antibodies to hBR-STL by commercial laboratories were unsuccessful.…”

Section: Immunoblot Analysis Of Rbr-stl In Rat Brainmentioning

confidence: 99%

Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain

Falany

Xie

Wang

et al. 2000

Biochemical Journal

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The sulphotransferase (SULT) gene family is involved with the conjugation of many small drugs, xenobiotics and endogenous compounds. In this report, we describe the cloning and expression of novel cDNAs from human and rat brain, which are structurally related to the SULTs. These cDNAs have been termed ‘brain sulphotransferase-like’ (BR-STL), because of their similarity to the SULTs and their selective expression in brain tissue. The proteins encoded by the human and rat BR-STL cDNAs (hBR-STL-1 and rBR-STL cDNA respectively), denoted as hBR-STL and rBR-STL, are 98% identical and 99% similar in sequence. The hBR-STL-1 cDNA contains an 852-nt open reading frame encoding a 284-amino-acid protein with a calculated molecular mass of 33083 Da. Northern-blot analyses of RNA isolated from eight human tissues indicate that the hBR-STL message is selectively expressed in brain. Characterization of different brain regions showed that message levels were highest in cortical brain regions. rBR-STL message levels were relatively low in brains of 1-day-old male and female rats, but increased to adult levels in RNA from 7-day-old rats, and remained at that level in adult animals. The hBR-STL and rBR-STL cDNAs were expressed in both Escherichia coli and Sf9 insect cells in the presence or absence of an N-terminal histidine-affinity tag (His-tag). Polyclonal antibodies were raised in chickens against purified His-tagged hBR-STL, and were used to detect the presence of rBR-STL in adult male and female rat brain cytosol. The high degree of sequence conservation, and the selective localization of the BR-STL message in brain, suggest an important function in the central nervous system.

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“…Photoaffinity Labeling of Human DHEA-ST and EST Expressed in E. coli-The preparations used in these experiments were human DHEA-ST and EST expressed in E. coli and partially purified by DEAE chromatography as described previously (14,21). The results are shown in Fig.…”

Section: Photolabeling Pattern Of Pap-binding Proteins In Cytosolic Amentioning

confidence: 99%

Photoaffinity Labeling of Human Recombinant Sulfotransferases with 2-Azidoadenosine 3′,5′-[5′-32P]Bisphosphate

Radominska

Drake

Zhu

et al. 1996

Journal of Biological Chemistry

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Photoaffinity labeling with 2-azidoadenosine 3 ,5 -[5 -32 P]bisphosphate was used to identify and characterize adenosine 3 ,5 -bisphosphate-binding proteins in human liver cytosol and recombinant sulfotransferase proteins. The sulfotransferases investigated in these studies were the human phenol sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone sulfotransferase, and estrogen sulfotransferase. The cDNAs for these enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was highly dependent on UV irradiation, was protected by co-incubation with unlabeled adenosine 3 ,5 -bisphosphate and phosphoadenosine phosphosulfate, and reached saturation at concentrations above 10 M. To verify that the 31-35-kDa photolabeled proteins were indeed sulfotransferases, specific antibodies known to recognize human sulfotransferases were used for Western blot analyses of photolabeled proteins. It was shown unequivocally that the proteins in the 31-35-kDa region recognized by the antibodies also photoincorporated 2-azidoadenosine 3 ,5 -[5 -32 P]bisphosphate. This is the first application of photoaffinity labeling with 2-azidoadenosine 3 ,5 -[5 -32 P]bisphosphate for the characterization of recombinant human sulfotransferases. Photoaffinity labeling will be also useful in the purification and functional identification of other adenosine 3 ,5 -bisphosphate-binding proteins and to determine amino acid sequences at or near their active sites.Sulfation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, bile acids, and hormones. The sulfate donor for these reactions is 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS). 1 Adenosine 3Ј,5Ј-bisphosphate (PAP) is a product of the reaction catalyzed by all sulfotransferases (STs) and competitively inhibits PAPS binding (1, 2).Direct photoaffinity labeling with radioactively labeled PAPS has been used to identify PAPS-binding proteins. Sylvers et al. (7) and applied to the photoaffinity labeling of Escherichia coli ribosomes.In this paper, we report photoaffinity labeling studies of human and rat liver cytosolic ST and several recombinant human STs with 2-azido-[ 32 P]PAP synthesized and characterized according to Sylvers et al. (7). Studies were performed that demonstrated specific photoinsertion of 2-azido-[ 32 P]PAP into these enzymes and also indicated the potential usefulness of this approach in the purification and molecular characterization of purified native and cloned PAPS-and PAP-binding proteins, including the STs. Moreover, photolabeling with 2-azido-[ 32 P]PAP will be also useful in studies investigating the structure of the ST active sites. MATERIALS AND METHODSPAP, PAPS, 4-nitrophenol, and other reagents were purchased from Sigma. 2,6-Dichloro-4-nitrophenol (DCNP) was obtained from K and K Laboratories (Plainview, NY).Synthesis of 2-Azidoadenosine 3Ј,5Ј-[5Ј-32 P]Bisphosphate-2-Azido-[ 32 P]PAP was synthesized and purified as described previously (7).[␥-32 P]ATP wa...

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Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

Cited by 196 publications

References 17 publications

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Interactions of the human cytosolic sulfotransferases and steroid sulfatase in the metabolism of tibolone and raloxifene

Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain

Photoaffinity Labeling of Human Recombinant Sulfotransferases with 2-Azidoadenosine 3′,5′-[5′-32P]Bisphosphate

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