Abstract: Alpha-lipoic acid has been shown to possess cancer-cell-killing activity via activation of the apoptosis pathway. In this study, the cytotoxic activities of alpha-lipoic and dihydro-alpha-lipoic acid were compared in HL-60 cells. The cell-killing activity of dihydro-alpha-lipoic acid was higher than that of alpha-lipoic acid. Both alpha-lipoic and dihydro-alpha-lipoic acid induced caspase-3 cleavage and internucleosomal DNA fragmentation in treated cells. On the other hand, apparent necrotic or late-stage apoptotic cell populations could be detected in dihydro-alpha-lipoic acid cells but not in those treated with alpha-lipoic acid. Moreover, dihydro-alpha-lipoic acid, but not alpha-lipoic acid, induced marked mitochondrial permeability transition. Antioxidants could not prevent dihydro-alpha-lipoic- or alpha-lipoic-acid-induced cell death. In addition, dihydro-alpha-lipoic and alpha-lipoic acid did not up-regulate cellular reactive oxygen level. These results indicated that dihydro-alpha-lipoic acid exerts more potent cytotoxicity than alpha-lipoic acid through different cytotoxic actions.
Abstract: The mouse cytosolic sulfotransferase, mSULT1D1, catalyzes the sulfonation of a wide range of phenolic molecules including p-nitrophenol (pNP), alpha-naphthol (alphaNT), serotonin, as well as dopamine and its metabolites. To gain insight into the structural basis for its broad range substrate specificity, we solved two distinct ternary crystal structures of mSULT1D1, complexed with 3'-phosphoadenosine-5'-phosphate (PAP) plus pNP or PAP plus alphaNT. The structures revealed that the mSULT1D1 contains an L-shaped accepter-binding site which comprises 20 amino acid residues and four conserved water molecules. The shape of the accepter-binding site can be adjusted by conformational changes of two residues, Ile148 and Glu247, upon binding with respective substrates.
Abstract: Mammalian sulfotransferases (STs) utilize exclusively the sulfuryl group donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to catalyze the sulfurylation reactions based on a sequential transfer mechanism. In contrast, the commensal intestinal bacterial arylsulfate sulfotransferases (ASSTs) do not use PAPS as the sulfuryl group donor, but instead catalyze sulfuryl transfer from phenolic sulfate to a phenol via a Ping-Pong mechanism. Interestingly, structural comparison revealed a similar spatial arrangement of the active site residues as well as the cognate substrates in mouse ST (mSULT1D1) and Escherichia coli CFT073 ASST, despite that their overall structures bear no discernible relationship. These observations suggest that the active sites of PAPS-dependent SULT1D1 and phenolic sulfate-utilizing ASST represent an example of convergent evolution.
Abstract: We report the crystal structure of mouse sulfotransferase, mSULT1D1, complexed with donor substrate 3'-phosphoadenosine 5'-phosphosulfate and accepter substrate p-nitrophenol. The structure is the first report of the native Michaelis complex of sulfotransferase. In the structure, three proposed catalytic residues (Lys48, Lys106, and His108) were in proper positions for engaging in the sulfuryl transfer reaction. The data strongly support that the sulfuryl transfer reaction proceeds through an S(N)2-like in-line displacement mechanism.
Abstract: A simple methodology for the identification of hemostatic proteins that are subjected to posttranslational tyrosine sulfation was developed. The procedure involves sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm, followed by [(35)S]sulfate labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [(35)S]sulfate-labeled hemostatic proteins, and tyrosine O-[(35)S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins-protein S, prekallikrein, and plasminogen-were identified. Such a target-specific approach will allow investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understanding of the functional role of posttranslational tyrosine sulfation.
Abstract: By searching the mouse EST database, we identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (RIKEN cDNA 2410078J06). Sequence analysis revealed that this new SULT belongs to the cytosolic SULT6 gene family. The recombinant form of this newly identified SULT, designated SULT6B1, was expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Purified mouse SULT6B1 exhibited sulfonating activity toward thyroxine and bithionol among a variety of endogenous and xenobiotic compounds tested as substrates. pH optimum of purified mouse SULT6B1 was determined to be 8.0. Tissue-specific expression of mouse and human SULT6B1 was examined by RT-PCR. While human SULT6B1 was specifically expressed in kidney and testis, mouse SULT6B1 was detected in brain, heart, kidney, thymus, lung, liver and testis. Further studies are needed in order to clarify the role of SULT6B1 in the metabolism of thyroxine and possibly some xenobiotics in mouse.
Abstract: Conjugation reactions catalyzed by the cytosolic sulfotransferase, SULT1A3, or catechol-O-methyltransferase (COMT) are known to be involved in the regulation and homeostasis of dopamine and other monoamine neurotransmitters. Whether different conjugation reactions may act in a concerted manner, however, remains unclear. The current study aimed to investigate the concerted action of SULT1A3 and COMT in dopamine metabolism. Analysis of the medium of SK-N-MC cells, metabolically labeled with [(35)S]sulfate in the presence of dopamine, revealed the generation and release of predominantly [(35)S]sulfated 3-methyldopamine and, to a lesser extent [(35)S]sulfated dopamine. Addition to the labeling medium of tropolone, a COMT inhibitor, enhanced the production of [(35)S]sulfated dopamine, with a concomitant decrease of [(35)S]sulfated 3-methyldopamine. Enzymatic assays using the eleven known human cytosolic SULTs revealed SULT1A3 as the major enzyme responsible for the sulfation of both dopamine and 3-methyldopamine. Kinetic analysis showed that the catalytic efficiency of SULT1A3 with 3-methyldopamine was 1.6 times than that with dopamine. Using subcellular fractions prepared from SK-N-MC cells, the majority of COMT dopamine-methylating activity was found to be present in the cytosol. Collectively, these results imply a concerted action of sulfation and methylation in the irreversible inactivation and disposal of excess dopamine in SK-N-MC cells.
Abstract: Cruciferous vegetables and their isothiocyanates are promising foods and agents for cancer prevention. We focus here on the effects of mustard oil (SMO) in a variety of the Japanese radish, Shibori Daikon (Raphanus sativus), on the proliferation of 3Y1 rat fibroblasts and the H-ras-transformed derivative, HR-3Y1-2. SMO (1.6 microg/ml) inhibited the proliferation of HR-3Y1-2, but not 3Y1 after 24 h after treatment. A cell cycle analysis showed that SMO induced G2/M arrest after 6 h, although this effect was not observed 24 h after the treatment. SMO transiently decreased the cellular reduced glutathione level accompanied with up-regulation of the intracellular reactive oxygen species 2-3 h post-treatment. Glutathione ethyl ester and N-acetyl-L-cysteine prevented the growth inhibitory effect of SMO. This mustard oil extract consisted of 95.6% 4-methylthio-3-butenyl isothiocyanate and 4.4% 4-methylthiobutyl isothiocyanate. SMO selectively inhibited H-ras-transformed 3Y1 cells associated with transient oxidative stress via reduced glutathione (GSH) depletion.
Abstract: In vertebrates, sulfation as catalyzed by members of the cytosolic sulfotransferase (SULT) family has been suggested to be involved in the homeostasis of steroids. To establish the zebrafish as a model for investigating how sulfation functions to regulate steroid metabolism during the developmental process, we have embarked on the identification of steroid-sulfating SULTs in zebrafish. By searching the GenBank database, we identified two putative cytosolic SULT sequences from zebrafish, designated SULT3 ST1 and ST2. The recombinant proteins of these two zebrafish SULT3 STs were expressed in and purified from BL21 (DE3) cells transformed with the pGEX-2TK expression vector harboring SULT3 ST1 or ST2 cDNA. Upon enzymatic characterization, purified SULT3 ST1 displayed the strongest sulfating activity toward 17beta-estradiol among the endogenous substrates tested, while SULT3 ST2 exhibited substrate specificity toward hydroxysteroids, particularly dehydroepiandrosterone (DHEA). The pH-dependence and kinetic constants of these two enzymes with 17beta-estradiol and DHEA were determined. A developmental expression study revealed distinct patterns of the expression of SULT3 ST1 and ST2 during embryonic development and throughout the larval stage onto maturity. Collectively, these results imply that these two steroid-sulfating SULT3 STs may play differential roles in the metabolism and regulation of steroids during zebrafish development and in adulthood.
Abstract: By searching the GenBank database, we recently identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (IMAGE Clone ID 679629) and a novel mouse SULT gene (LOC 215895). Sequence analysis revealed that both mouse SULTs belong to the cytosolic SULT3 gene family. The recombinant form of these two newly identified SULTs, designated SULT3A1 and SULT3A2, were expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Both purified SULT3A1 and SULT3A2 exhibited strong amine N-sulfonating activities toward 1-naphthylamine among a variety of endogenous and xenobiotic compounds tested as substrates. Kinetic constants of the sulfation of 1-naphthylamine and 1-naphthol by these two enzymes were determined. Collectively, these results imply that these two amine-sulfonating SULT3s may play essential roles in the metabolism and detoxification of aromatic amine compounds in the body.
Abstract: In mammals, sulfonation as mediated by specific cytosolic sulfotransferases (SULTs) plays an important role in the homeostasis of dopamine and other catecholamines. To gain insight into the structural basis for dopamine recognition/binding, we determined the crystal structure of a mouse dopamine-sulfating SULT, mouse SULT1D1 (mSULT1D1). Data obtained indicated that mSULT1D1 comprises of a single alpha/beta domain with a five-stranded parallel beta-sheet. In contrast to the structure of the human SULT1A3 (hSULT1A3)-dopamine complex previously reported, molecular modeling and mutational analysis revealed that a water molecule plays a critical role in the recognition of the amine group of dopamine by mSULT1D1. These results imply differences in substrate binding between dopamine-sulfating SULTs from different species.
Abstract: Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is known to play an important role in the regulation and homeostasis of monoamine neurotransmitters. The current study was designed to examine the occurrence of the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine by human cytosolic SULTs and to investigate the inhibitory effects of these hydroxylated derivatives on the sulfation of their unhydroxylated counterparts, serotonin and dopamine. A systematic study using 11 known human cytosolic SULTs revealed SULT1A3 as the responsible enzyme for the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine. The pH-dependence and kinetic constants of SULT1A3 with 7-hydroxyserotonin or 6-hydroxydopamine as substrate were determined. The inhibitory effects of 7-hydroxyserotonin and 6-hydroxydopamine on the sulfation of serotonin and dopamine were evaluated. Kinetic analyses indicated that the mechanism underlying the inhibition by these hydroxylated monoamine derivatives is of a competitive-type. Metabolic labeling experiments showed the generation and release of [(35)S]sulfated 7-hydroxyserotonin and [(35)S]sulfated 6-hydroxydopamine when SK-N-MC human neuroblastoma cells were labeled with [(35)S]sulfate in the presence of 7-hydroxyserotonin or 6-hydroxydopamine. Upon transfection of the cells with siRNAs targeted at SULT1A3, diminishment of the SULT1A3 protein and concomitantly the sulfating activity toward these hydroxylated monoamines was observed. Taken together, these results indicated clearly the involvement of sulfation in the metabolism of 7-hydroxyserotonin and 6-hydroxydopamine. By serving as substrates for SULT1A3, these hydroxylated monoamines may interfere with the homeostasis of endogenous serotonin and dopamine.
Abstract: Adult T-cell leukemia occurs in human T-lymphotropic virus type I-infected individuals and is endemic to the south-western area of Kyushu in Japan. In this communication, we examined the effect of soy isoflavones on the growth of adult T-cell leukemia cells in vitro and in vivo. In the in vitro study, daidzein and genistein but not glycitein significantly inhibited the proliferation of ED-40515 and Hut102 cells in a dose-dependent manner. Among the isoflavones studied, genistein had the highest growth-inhibitory effect; however, genistein did not exert an apparent growth-inhibitory effect on Jurkat and Molt-4 cells, which were non-adult T-cell leukemia cells. Genistein prevented the G(1)/S or G(2)/M transition at 3 and 10 or 30 microM, respectively. Genistein upregulated p21 protein expression together with p53 accumulation. In addition, treatment with 30 microM genistein strongly induced phosphorylation of checkpoint kinase (CHK) 2 and p53 at serines 15, 20 and 37. Caffeine, an inhibitor of ataxia-telangiectasia mutated protein kinase, alleviated the genistein-induced p53 and CHK2 phosphorylation, suggesting the involvement of DNA damage at 30 microM. However, marked phosphorylation of CHK2 and p53 could not be detected at 3 and 10 microM genistein. These data indicate that genistein has biphasic growth-inhibitory properties. The in vivo studies demonstrated that soy-derived isoflavones significantly inhibit ED-40515 cell growth and infiltration into various organs in non-obese diabetic severe combined-immunodeficiency common gamma-chain knockout mice. Taken together, it is evident that soy isoflavones might serve as a promising compound for the treatment of adult T-cell leukemia.
Abstract: Protein tyrosine sulfation is emerging as a widespread post-translational modification in multicellular eukaryotes. The responsible enzyme, named tyrosylprotein sulfotransferase (TPST), catalyzes the sulfate transfer from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues of proteins. Two distinct TPSTs, designated TPST-1 and TPST-2, had previously been identified. In the present study, we cloned human TPST-1 and TPST-2 expressed and characterized the recombinant enzymes using peptide substrates. These enzymes displayed distinct acidic pH optima and stimulatory effects of Mn(2+). Additionally, the activity of TPST-2, but not TPST-1, was stimulated in the presence of Mg(2+). Compared with TPST-2, TPST-1 displayed considerably lower K(m) and V(max) for the majority of the tested peptide substrates, implying their differential substrate specificity. Quantitative real-time PCR analysis showed that although the two TPSTs were co-expressed in all 20 human tissues examined, the levels of expression of TPST-1 and TPST-2 varied significantly among different tissues. These latter findings may imply distinct physiological functions of TPST-1 and TPST-2.
Abstract: By employing RT-PCR in conjunction with 3'-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (designated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. Its pH optima were 6.0 and 9.0 with 3,3',5-triiodo-l-thyronine (l-T3) as substrate and 6.0 with beta-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of divalent metal cations on the l-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ exhibited dramatic inhibitory effects, whereas Mn2+ showed a significant stimulation. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity.
Abstract: By employing the reverse transcriptase-polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.
Abstract: Cytosolic sulfotransferases (STs) are generally thought to be involved in detoxification of xenobiotics, as well as homeostasis of endogenous compounds such as thyroid/steroid hormones and catecholamine hormones/neurotransmitters. We report here the identification and characterization of a zebrafish estrogen-sulfating cytosolic ST. The zebrafish ST was bacterially expressed, purified, and examined for enzymatic activities using a variety of endogenous compounds as substrates. Results showed that the enzyme displayed much higher activities toward two endogenous estrogens, estrone (E(1)) and 17beta-estradiol (E(2)), in comparison with thyroid hormones, 3,3',5-triiodothyronine (T(3)) and thyroxine (T(4)), dopamine, dihydroxyphenylalanine (Dopa), and dehydroepiandrosterone (DHEA). The kinetic parameters, K(m), and V(max), with estrogens and thyroid hormones as substrates were determined. The calculated V(max)/K(m) for E(1), E(2), T(3), and T(4) were, respectively, 31.6, 16.7, 1.5, and 0.8 nmol min(-1) mg(-1) microM(-1), indicating clearly the estrogens being preferred physiological substrates for the enzyme. The inhibitory effects of isoflavone phytoestrogens on the sulfation of E(2) by this zebrafish ST were examined. The IC(50) determined for quercetin, genistein, and daidzein were 0.7, 2.5, and 8 microM, respectively. Kinetic analyses revealed that the mechanism underlying the inhibition by these isoflavones to be of the competitive type.
Abstract: The gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens beta-1,3-glucanase BGN1, but no significant similarity to fungal beta-1,6-glucanases or beta-1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo-beta-1,3:1,6-glucan hydrolases as a subclass of endo-beta-1,3-glucanases in some mycoparasitic fungi is suggested.
Abstract: The human monoamine-form phenol sulfotransferase (PST), SULT1A3, has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is stereospecific for their d-form enantiomers and can be stimulated dramatically by Mn(2+). This activity is not present in the simple phenol-form PST, SULT1A1, which is otherwise >93% identical to SULT1A3 in amino acid sequence. The majority of the differences between these two proteins reside in two variable regions of their sequences. Through the characterization of chimeric PSTs where these two regions were exchanged between them, it was demonstrated that variable Region II of SULT1A3 is required for the stereospecificity of its Dopa/tyrosine-sulfating activity, whereas variable Region I of SULT1A3 is required for the stimulation by Mn(2+) of this activity. Further studies using point-mutated SULT1A3s mutated at amino acid residues in these two regions and deletional mutants missing residues 84-86 and 84-90 implicate residue Glu-146 (in variable Region II of SULT1A3), as well as the presence of residues 84-90 of variable Region I, in the stereospecificity in the absence of Mn(2+). Residue Asp-86 (in variable Region I of SULT1A3), on the other hand, is critical in the Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3. A model is proposed, with reference to the reported x-ray crystal structure of SULT1A3, to explain how the normal role of SULT1A3 in dopamine regulation may be subverted in the presence of Mn(2+). These studies could be relevant in understanding the stereoselective action of SULT1A3 on chiral drugs.
Abstract: Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.
Abstract: As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.
Abstract: At least three extracellular laminaran hydrolases which hydrolyzed laminaran (beta-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a beta-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for beta-1,3-linkages, but lam B was specific for beta-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.
Abstract: Environmental estrogen-like chemicals are increasingly recognized as a potential hazardous factor for wildlife as well as humans. We have recently embarked on developing a zebrafish model for investigating the role of sulfonation in the metabolism and adverse functioning of environmental estrogens. Here, we report on a systematic investigation of the sulfonation of representative environmental estrogens (bisphenol A, 4-n-octylphenol, 4-n-nolylphenol, diethylstilbestrol, and 17 alpha-ethynylestradiol) by zebrafish cytosolic sulfotransferases (STs). Of the seven enzymes tested, four zebrafish STs (designated ZF ST #2, ZF ST #3, ZF ST #4, and ZF DHEA ST) exhibited differential sulfonating activities toward the five environmental estrogens tested, with ZF ST #3 being more highly active than the other three. It was further demonstrated that bisphenol A, 4-n-octylphenol, and 4-n-nonylphenol exerted concentration-dependent inhibition of the sulfonation of 17 beta-estradiol, implying a potential role of these environmental estrogens in interfering with the sulfonation, and possibly homeostasis, of endogenous estrogens. Kinetic studies revealed that the mechanism underlying the inhibition by bisphenol A or 4-n-nonylphenol to be of the competitive type.
Abstract: By employing reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'-rapid amplification of cDNA ends technique, we have cloned a novel mouse sulfotransferase cDNA. Database search led to the identification of a human gene encoding the homologue of this newly discovered mouse sulfotransferase. RT-PCR technique was employed to clone the cDNA encoding the human enzyme. Sequence analysis revealed that the novel mouse and human sulfotransferases display nearly 98% identity in their amino acid sequences. Their amino acid sequence identity to other known cytosolic sulfotransferases, however, was found to be below 36%. These two highly conserved sulfotransferases therefore appear to belong to a family different from the two major mammalian cytosolic sulfotransferase gene families. Northern blot analysis revealed the neuronal tissue-specific expression of these two novel sulfotransferases. Recombinant mouse and human brain sulfotransferases, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as 33 kD proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified mouse and human brain sulfotransferases displayed enzymatic activities toward endogenous and xenobiotic compounds, including L-triiodothyronine, thyroxine, estrone, p-nitrophenol, 2-naphthylamine, and 2-naphthol. Using mouse brain filtrate as substrate, both brain sulfotransferases were shown to catalyze specifically the sulfation of only a few compounds.
Abstract: Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.
Abstract: Human monoamine (M) form and simple phenol (P) form phenol sulfotransferases (PSTs) are greater than 93% identical in their primary sequences and yet display distinct substrate specificities and other enzymatic properties. Through the generation and characterization of a series of chimeric PSTs, we have previously demonstrated two highly variable regions within their sequences to be responsible for determining their substrate phenotypes (Sakakibara, Y., Takami, Y., Nakayama, T., Suiko, M., and Liu, M.-C. (1998) J. Biol. Chem. 273, 6242-6247). By employing the site-directed mutagenesis technique, the present study aims to identify and quantitatively evaluate the specific amino acid residues critical to the substrate binding and catalysis in these two enzymes. Twelve mutated M-PSTs and seven mutated P-PSTs were generated, expressed, and purified. Enzymatic characterization showed that, of the twelve mutated M-PSTs, mutations at residues Asp-86, Glu-89, and Glu-146 resulted in a dramatic decrease in V(max)/K(m) with dopamine as substrate, being greater than 450 times for the D86A/E89I/E146A mutated M-PST. With p-nitrophenol as substrate, the V(max)/K(m) determined for the D86A/E89I/E146A-mutated M-PST increased more than 25 times and approached that determined for the wild-type P-PST. These results indicated that the concerted action of the three mutated residues (D86A, E89I, and E146A) is sufficient for the conversion of the substrate phenotype of M-PST to that of P-PST. Among the mutated P-PSTs, the I89E- and A146E-mutated P-PSTs displayed considerable deviations in V(max)/K(m) with dopamine or p-nitrophenol as substrate. No corresponding changes, however, were detected with the opposite compound as substrate. These results indicated that, in contrast to M-PST, mutations at Ala-86, Ile-89, and Ala-146 to the corresponding residues in M-PST are not sufficient for rendering the change of P-PST substrate phenotype to that of M-PST. For both M-PSTs and P-PSTs, mutations at Lys-48 or His-108 led to the loss of sulfotransferase activities, indicating their importance in the catalytic mechanism.
Abstract: 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.
Abstract: A reliable HPLC method was used for the identification of positional isomerism and stereoisomerism of sulfated tyrosine residues in human urine. Upon separation of human urine by ion-pair HPLC on a reverse-phase column, p-tyrosine-O-sulfate (p-TyrS) was identified. Differentiation of the L and D forms was done by using a column with a chiral stationary phase. It was concluded that L-p-tyrosine (L-p-Tyr) which is the predominant tyrosine isomer in the human body, was sulfated and excreted in human urine as a normal constituent. The sulfated forms of D-p-Tyr and m-Tyr could not be detected under these analytical conditions.
Abstract: The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.
Abstract: Although dispensable, costimulation through CD28 facilitates activation of naïve T lymphocytes. CD28 engagement led to the redistribution and clustering of membrane and intracellular kinase-rich raft microdomains at the site of T cell receptor (TCR) engagements. Although not affecting TCR down-regulation, this process led to higher and more stable tyrosine phosphorylation of several substrates and higher consumption of Lck. These results may provide a general mechanism for amplifying receptor signaling by reorganization of membrane microdomains.
Abstract: Analysis of the nucleotide sequence of a recently cloned mouse sulfotransferase cDNA (clone 679153) revealed the presence in its 3'-untranslated sequence of an AT-rich region which contains four ATTTA motifs and an TTATTTAT-like sequence, commonly found among those encoding inflammation-related proteins. The recombinant enzyme expressed in Escherichia coli and purified to near electrophoretic homogeneity displayed strong sulfotransferase activities toward various prostaglandins, thromboxane B2, and leukotriene E4. These results mark the first discovery of the sulfation of eicosanoids catalyzed by a distinct sulfotransferase.
Abstract: Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes. An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA. To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells. Recombinant SRP54 was purified from both sources. The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin. Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction. A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54. Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.
Abstract: Human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases (PSTs), which are greater than 93% identical in their primary sequences, were used as models for investigating the structural determinants responsible for their distinct substrate specificity and other enzymatic properties. A series of chimeric PSTs were constructed by reciprocal exchanges of DNA segments between cDNAs encoding M-form and P-form PSTs. Functional characterization of the recombinant wild-type M-form, P-form, and chimeric PSTs expressed in Escherichia coli and purified to homogeneity revealed that internal domain-spanning amino acid residues 84-148 contain the structural determinants for the substrate specificity of either M-form or P-form PST. Data on the kinetic constants (Km, Vmax, and Vmax/Km) further showed the differential roles of the two highly variable regions (Region I spanning amino acid residues 84-89 and Region II spanning amino acid residues 143-148) in substrate binding, catalysis, and sensitivity to the inhibition by 2,6-dichloro-4-nitrophenol. In contrast to the differential sulfotransferase activities of M-form and P-form PSTs toward dopamine and p-nitrophenol, the Dopa/tyrosine sulfotransferase activities were found to be restricted to M-form, but not P-form, PST. Furthermore, the variable Region II of M-form PST appeared to play a predominant role in determining the Dopa/tyrosine sulfotransferase activities of chimeric PSTs. Kinetic studies indicated the role of manganese ions in dramatically enhancing the binding of D-p-tyrosine to wild-type M-form PST. Taken together, these results pinpoint unequivocally the sequence encompassing amino acid residues 84-148 to be the substrate specificity/catalytic domain of both M-form and P-form PSTs and indicate the importance of the variable Regions I and II in determining their distinct enzymatic properties.
Abstract: A cDNA encoding the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase was cloned and sequenced. The enzyme contains an APS kinase domain in its N-terminal portion and an ATP sulfurylase domain in its C-terminal portion. Recombinant full-length enzyme and its constituent APS kinase and ATP sulfurylase domains were individually expressed, purified, and shown to have their respective enzymatic activities.
Abstract: Upon sulfonation, carcinogenic hydroxyarylamines such as N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) can be further activated to form ultimate carcinogens in vivo. Previous studies have shown that a SULT1C1 sulfotransferase is primarily responsible for the sulfonation of N-OH-2AAF in rat liver. In the present study, two novel human sulfotransferases shown to be members of the SULT1C sulfotransferase subfamily based on sequence analysis have been cloned, expressed, and characterized. Comparisons of the deduced amino acid sequence encoded by the human SULT1C sulfotransferase cDNA 1 reveal 63.7, 61.6, and 85.1% identity to the amino acid sequences of rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. In contrast, the deduced amino acid sequence of the human SULT1C sulfotransferase 2 cDNA displays 62.9, 63.1, 63.1, and 62.5% identity to the amino acid sequences of the human SULT1C sulfotransferase 1, rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. Recombinant human SULT1C sulfotransferases 1 and 2, expressed in Escherichia coli and purified to near electrophoretic homogeneity, were shown to cross-react with the antiserum against the rat liver SULT1C1 sulfotransferase and exhibited sulfonating activities with N-OH-2AAF as substrate. Tissue-specific expression of these novel human SULT1C sulfotransferases were examined by employing the Northern blotting technique. The results provide a foundation for the investigation into the functional relevance of these new SULT1C sulfotransferases in different human tissues/organs.
Abstract: Upon two-dimensional thin-layer separation, the sulfated L-3, 4-dihydroxyphenylalanine (L-DopaS) generated enzymatically was found to co-migrate with only one of the two ninhydrin-stained spots corresponding to the two sulfated forms (3-O-sulfate and 4-O-sulfate) of synthetic L-DopaS. To clarify precisely the identity of the enzymatically generated L-DopaS, the two sulfated forms of synthetic L-DopaS were separated and purified using high performance liquid chromatography. Purified L-Dopa 3-O-sulfate and L-Dopa 4-O-sulfate were identified by 1H-nuclear magnetic resonance (NMR) spectrometry and used as standards in the analysis of the L-DopaS generated during metabolic labeling of HepG2 human hepatoma cells or enzymatic assay using recombinant human monoamine (M)-form phenol sulfotransferase. The results obtained demonstrated unequivocally the generation of L-Dopa 3-O-sulfate, indicating the specificity of the M-form phenol sulfotransferase being for the meta-hydroxyl group of L-Dopa.
Abstract: Nucleotide sequences of two mouse cDNAs encoding new sulfotransferase enzymes were determined. Analysis of the deduced amino acid sequences revealed that one represents a novel member of the phenol sulfotransferase family and the other is highly homologous to human SULT2B1 hydroxysteroid sulfotransferases. The recombinant enzymes, transiently expressed in COS-7 cells, were characterized with respect to their substrate specificity using a variety of substrates for different types of sulfotransferases. The tissue-specific expression of these two new mouse sulfotransferases was examined by Northern blot analysis.
Abstract: A mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3,4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3,3',5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.
Abstract: Tissue-specific and developmental stage-dependent expression of a novel Dopa/tyrosine sulfotransferase in Sprague-Dawley rats was examined. Both immunoblot and Northern blot analyses showed that the enzyme was expressed predominantly in liver and to a lesser extent in kidney. Its expression could not be detected in nine other organs tested. Livers from different age groups of male or female rats were examined for the developmental regulation of the expression of the Dopa/tyrosine sulfotransferase. Results from immunoblot and Northern blot analyses revealed that the enzyme was present at a very low level in livers of 1-day-old to 2-week-old rats, and gradually increased to a maximum level in rats older than 2 months. Data from the enzymatic assays also showed a similar trend of expression in both male and female rats. The Dopa/tyrosine sulfotransferase activities detected in liver samples of the 8-week-old male and female rats were, respectively, 8.6 and 6.6 times that of the activities detected in liver samples of the 1-day-old male and female rats. These data provide a foundation for the future investigation of the cis- and trans-acting factors involved in the regulation of the tissue-specific and developmental stage-dependent expression of this enzyme.
Abstract: Using [35S]PAPS as the sulfate donor, we have detected a sulfotransferase from bovine heart which catalyzes the sulfation of tyrosine-containing peptides. The enzyme displayed optimal activity at pH 5.75 and 35 degrees C in a one-hour reaction. The addition of 10 mM Mn2+ or Co2+ to the reaction mixture increased the sulfotransferase activity by 3.4- and 3.5-fold, respectively. In contrast, the maximum increment stimulated by Mg2+ was only 1.75-fold at 15 mM concentration, and instead of exerting an enhancement effect, Ca2+ was found to be a potent inhibitor. The addition of 50 mM NaF to the reaction mixture resulted in an increase in sulfotransferase activity of 3.3-fold. The K(m) for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) was determined to be 2 microM at a constant 0.5 mM Boc-Glu-Asp-Tyr-Val. Among the 10 peptides tested as substrates, Boc-Glu-Asp-Tyr-Val and Boc-Asp-Asp-Tyr-Val provided the highest activities.
Abstract: Human monoamine (M)-form phenol sulfotransferase (PST) was PCR-cloned and transiently expressed in COS-7 cells. The recombinant enzyme was demonstrated to display not only the previously reported sulfotransferase activity toward dopamine, but also novel manganese-dependent Dopa/tyrosine sulfotransferase activities. These results imply a new functional role of the human M-form PST in the homeostatic regulation of Dopa and tyrosine.
Abstract: HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified.
Abstract: Three mammalian and eight non-mammalian arylsulfatases were investigated for their activities toward tyrosine-O-sulfate (TyrS) in peptides. None of the mammalian arylsulfatases exhibited detectable activities toward TyrS-containing peptides. Of the non-mammalian arylsulfatases tested, Types VII, VIII, and H-1, 2, and 5 displayed strong activity on endo-TyrS residues. The prokaryotic sulfatase, Type VI, was active only on free TyrS and N-terminal TyrS of Leu-enkephalin. All the sulfatases were active on p-nitrophenyl sulfate and p-nitrocatechol sulfate.
Abstract: Chang liver cells, Caco-2 human intestinal epithelial cells and Madin-Darby canine kidney (MDCK) cells, labelled with [35S]sulphate in the presence of different concentrations of cycloheximide, produced 87.7-95.3%, 35.8-41.1% and 23.2-25.9%, respectively, of the amounts of free tyrosine O-[35S]-sulphate (Tyr[35S]) formed by corresponding cells labelled in the absence of cycloheximide. Homogenates prepared from the three kinds of cells showed the presence of enzymic activities catalysing the sulphation of L-tyrosine, with specific activities in the order: Caco-2 cells > MDCK cells > Chang liver cells. In all three cases, most of the tyrosine sulphotransferase' activity was found in the cytosolic fraction, indicating the enzyme to be a cysolic protein. A tyrosine-dependence experiment revealed that, for all three kinds of cells labelled with [35S]sulphate, the production of free Tyr[35S] was proportional to the concentration of L-tyrosine present in the culture medium. These results imply an involvement of sulphation in removing excess intracellular L-tyrosine.
Abstract: A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000. Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.
Abstract: HepG2 human hepatoma cells, labeled with [35S]sulfate in the presence of 10-30 micrograms/ml of cycloheximide, released up to 64% of the amount of free tyrosine-O-[35S]sulfate produced and released by cells labeled in the absence of cycloheximide. A time-course study revealed that, in cells incubated in medium containing [3H]tyrosine, free [3H]tyrosine-O-sulfate was produced within 5 min of incubation, whereas no [3H]tyrosine-sulfated proteins were detected until 20 min after the incubation had begun. Using 3'-phosphoadenosine, 5'-phospho[35S]sulfate as the sulfate donor, HepG2 cell homogenate was shown to contain enzymic activity catalyzing the sulfation of L-tyrosine with the formation of tyrosine-O-[35S]sulfate. Upon subcellular fractionation, the majority of the enzyme activity was found in the cytosolic fraction. The enzyme, designated tyrosine sulfotransferase, displayed the optimum activity at pH 8.0 in the presence of 10 mM Mn2+. Under optimum conditions, the apparent Km of the enzyme for L-tyrosine, at 4.5-microM concentration of 3'-phosphoadenosine, 5'-phosphosulfate, was determined to be 1.95 mM, while that for 3'-phosphoadenosine, 5'-phosphosulfate, at 1 mM L-tyrosine concentration, was 8.3 microM. The Vmax determined under these conditions was 1.05 pmol.min-1.mg protein-1. A tyrosine-dependence study showed that, for cells labeled with [35S]sulfate, the production and release of free tyrosine-O-[35S]sulfate appeared to proceed actively and increase proportionally to the L-tyrosine concentration when it was raised above a threshold level in the culture medium. These results may imply a possible involvement of sulfation in removing excess intracellular L-tyrosine.
Abstract: A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatography, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.
Abstract: A novel phenol sulfotransferase (PST) was detected in bovine liver microsomal membrane fraction. The enzyme was found to be capable of catalyzing the sulfation of simple phenolic compounds, with 3'-phosphoadenosine-5'-phosphosulfate as the sulfate donor. Detergent extracted PST showed a pH optimum of 5.7 and, among the simple phenols tested, the PST exhibited highest activity toward alpha-naphthol. No activities were detected when tyrosine and its derivatives were used as substrates. Both 2,6-dichloro-4-nitrophenol and chlorpromazine were capable of inhibiting the activity of the PST toward p-nitrophenol with inhibition Coefficient50 values of 100 nM and 4 mM, respectively.
Abstract: In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.
