Phosphate 5'-phosphosulphate Research Articles

Inflammation-induced transcriptional regulation of Golgi transporters required for the synthesis of sulfo sLex glycan epitopes.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3'-phosphate 5'-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.

Transcriptional regulation of human 3'-phosphoadenosine 5'-phosphosulphate synthase 2.

Sulphonation is a fundamental process that is essential for normal growth and development as well as maintenance of the internal milieu. The universal sulphonate donor molecule essential for all sulphoconjugation reactions is adenosine 3'-phosphate 5'-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the action of bifunctional PAPS synthase. There are two isozymes encoded by genes located on chromosome 4 (PAPS synthase 1) and chromosome 10 (PAPS synthase 2). The promoter for PAPS synthase 2 contains neither a TATAAA nor a CCAAT box, although a consensus initiator motif is present. Three human cell lines were used to examine promoter activity after transfection with various lengths of the 5'-flanking region of the PAPS synthase 2 gene fused to a reporter gene. Proximal promoter activity was located between bp -84 and bp -124 upstream of the purported transcription start site. This region contains two GC/GT boxes that are essential for full promoter activity, as indicated by deletion analysis and supported further by mutagenesis. A nuclear extract of SW13 cells, which highly express PAPS synthase 2, contained proteins that bound to probes possessing promoter-specific GC/GT boxes. Furthermore, the presence of specificity protein (Sp) 1, Sp2 and Sp3 proteins in the nuclear extract was confirmed by supershift analysis. Co-transfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 2 gene; the involvement of Sp2 and/or Sp3 remains to be clarified further.

Transcriptional regulation of human 3′-phosphoadenosine 5′-phosphosulphate synthase 2

Sulphonation is a fundamental process that is essential for normal growth and development as well as maintenance of the internal milieu. The universal sulphonate donor molecule essential for all sulphoconjugation reactions is adenosine 3′-phosphate 5′-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the action of bifunctional PAPS synthase. There are two isozymes encoded by genes located on chromosome 4 (PAPS synthase 1) and chromosome 10 (PAPS synthase 2). The promoter for PAPS synthase 2 contains neither a TATAAA nor a CCAAT box, although a consensus initiator motif is present. Three human cell lines were used to examine promoter activity after transfection with various lengths of the 5′-flanking region of the PAPS synthase 2 gene fused to a reporter gene. Proximal promoter activity was located between bp −84 and bp −124 upstream of the purported transcription start site. This region contains two GC/GT boxes that are essential for full promoter activity, as indicated by deletion analysis and supported further by mutagenesis. A nuclear extract of SW13 cells, which highly express PAPS synthase 2, contained proteins that bound to probes possessing promoter-specific GC/GT boxes. Furthermore, the presence of specificity protein (Sp) 1, Sp2 and Sp3 proteins in the nuclear extract was confirmed by supershift analysis. Co-transfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 2 gene; the involvement of Sp2 and/or Sp3 remains to be clarified further.

The pharmacology of nucleotide receptors on primary rat brain endothelial cells grown on a biological extracellular matrix: effects on intracellular calcium concentration.

1. Brain capillary endothelial cells express a variety of nucleotide receptors, but differences have been reported between culture models. This study reports examination of nucleotide receptors on primary cultured rat brain capillary endothelial cells (RBCEC) grown on a biological extracellular matrix (ECM) to produce a more differentiated phenotype. 2. Fura-2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration [Ca(2+)](i). ATP, UTP, and 2-methylthioATP (2-MeSATP) increased [Ca(2+)](i) to similar levels, while 2-MeSADP, ADP and adenosine gave smaller responses. 3. Removal of extracellular calcium caused no significant change in the [Ca(2+)](i) response to 2-MeSATP, evidence that the response was mediated by a metabotropic (P2Y) receptor. 4. All cells tested responded to ATP, UTP, 2-MeSATP and ADP, while 63% responded to adenosine and 50% to 2-MeSADP. No cells responded to alpha, beta-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca(2+)](i) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The [Ca(2+)](i) response to the P2Y(1)-selective agonist 2-MeSADP was abolished in the presence of the subtype-selective antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS). 6. The P2Y(2) antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%. 7. The A(1) subtype-selective adenosine receptor agonist N(6)-Cyclopentyladenosine (CPA) gave a small but characteristic [Ca(2+)](i) response, while A(2A) and A(2B) subtype-selective agonists failed to generate [Ca(2+)](i) changes. 8. The results are consistent with the presence on RBCEC of a P2Y(2)-like receptor coupled to phospholipase C, and a P2Y(1)-like receptor mobilizing intracellular Ca(2+). The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.

Purification and Characterization of a Lymph Node Sulfotransferase Responsible for 6-O-Sulfation of the Galactose Residues in 2′-Fucosyllactose and Other Sialyl LewisX-Related Sugars

A microsomal galactose-6-O-sulfotransferase (Gal-6-O-Stase) from porcine lymph nodes, able to transfer the sulfate group from adenosine 3′-phosphate 5′-phosphosulphate (PAPS) onto 2′-fucosyllactose (2′-FL) and other sialyl LewisX(sLex)-related sugars, has been purified and characterized. The enzyme was purified to about 35,000-fold by a combination of conventional and affinity chromatographic steps. The purified enzyme preparation exhibited two protein bands at around 80-90 and 170 kDa on 7.5% SDS-PAGE under reducing conditions. Both of these protein bands always comigrated in the gel when peak fractions containing Gal-6-O-Stase activity from the 3′,5′-ADP-agarose column were subjected to 6% SDS-PAGE under reducing conditions. These protein bands also showed similar binding patterns to WGA (wheat germ agglutinin), Con A (concanvalin A), and EBA (elderberry agglutinin). Similarly, when the enzyme preparation after the hydroxylapatite step was photolabeled with 8-azido-[32P]-PAPS, both 80-90 and 170 kDa protein bands were labeled in a specific manner. These results suggest a possible association of these two protein bands with the enzyme activity. The carbohydrate substrate specificity of this enzyme suggests that it is well suited to catalyze the sulphonation at the C-6 position of the galactose residues of oligosaccharides that are structurally similar to sLex. Furthermore, a survey of several porcine organs revealed that this enzyme was selectively expressed in lymphoid tissues such as lymph nodes (peripheral and mesenteric) and spleen. These findings suggest that this enzyme may be involved in the assembly of 3′-sialyl-6′-sulfo Lewisx, the major capping group of HEV-ligands for L-selectin.

Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2'-fucosyllactose: implications in the synthesis of the ligand for L-selectin.

A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.

On the suitability of adenosine 3′-phosphate 5′-phosphosulphate as a selective P2Y receptor antagonist in intact tissues

Agonist and antagonist effects of the putative P2Y 1 receptor antagonist adenosine 3′-phosphate 5′-phosphosulphate (PAPS) were studied in intact tissues. In the carbachol-precontracted guinea-pig taenia coli, PAPS caused prominent relaxation (EC 50 3.3 μM). The response was attenuated by the P2 receptor antagonists 4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS) and reactive red 2 with apparent K d values (0.27 and 0.29 μM) indicating that PAPS acts through the non-P2Y receptor, which is the site of action of α, β-methylene ATP ( α, β-MeATP) in the taenia coli. Incubation with PAPS (10–100 μM) attenuated the P2Y receptor-mediated relaxation caused by 5′- O-(2-thiodiphosphate) (ADP βS); PAPS (100 μM) also attenuated the relaxation caused by α, β-MeATP, as well as the α 1-adrenoceptor-mediated response to noradrenaline. In the noradrenaline-precontracted rat aorta, PAPS caused minor relaxation (EC 50 24.7 μM), which was reduced by the P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonate ( iso-PPADS; 1 μM), indicating that PAPS activates endothelial P2Y receptors. Incubation with PAPS (10 and 100 μM) attenuated the P2Y receptor-mediated relaxation caused by ADP βS; PAPS (100 μM) also attenuated the P2U receptor-mediated relaxation caused by UTP and the muscarine receptor-mediated response to acetylcholine. In rat vas deferens, PAPS (100 μM) attenuated the P2X receptor-mediated contraction elicited by α, β-MeATP but did not alter the α 1-adrenoceptor-mediated response to noradrenaline. The results indicate that PAPS attenuates P2Y receptor-mediated relaxation in intact tissues. However, due to its limited subtype selectivity and non-P2 receptor effects, the nucleotide is not a suitable antagonist for this subtype.

Kinetic mechanism of adenosine 5'-phosphosulphate kinase from rat chondrosarcoma.

Biosynthesis of the activated sulphate donor adenosine 3'-phosphate 5'-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5'-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3'-phosphate 5'-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5'-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released.

Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma

ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction.

Purification and properties of a phenol sulphotransferase from Euglena using l-tyrosine as substrate

A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var. bacillaris. The apparent Km for adenosine 3'-phosphate 5'-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5'-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km values. Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine. D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position. Adenosine 3',5'-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (Ki = 20 microM) and uncompetitive (Ki = 500 microM) inhibition kinetics respectively. This appears to be the first phenol sulphotransferase to accept tyrosine as substrate. This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.

Sulphotransferase and Its Substrate: Adenosine-3'-Phosphate-5'-Phosphosulphate in Human Fetal Liver and Placenta

The activity of sulphotransferase (ST) towards 2-naphthol and the concentration of its endogenous substrate adenosine-3'-phosphate-5'-phosphosulphate (PAPS) were measured in human fetal and adult liver and in the placenta. The activity of ST (mean +/- SD; nmol/min/mg protein) was 0.28 +/- 0.06 (fetal liver); 1.82 +/- 0.44 (adult liver; p less than 0.001) and 0.021 +/- 0.014 (placenta; p less than 0.001). The concentration of PAPS (mean +/- SD; nmol/g wet tissue) was 10.1 +/- 0.9 (fetal liver); 23.4 +/- 2.4 (adult liver; p less than 0.001) and 3.6 +/- 1.1 (placenta; p less than 0.001). Both ST and PAPS were higher in fetal liver than in placenta. The difference between fetal liver and placenta was more marked for ST than for its substrate. Such a consideration was also drawn when fetal and adult liver were compared. Thus, the activity of the ST rather than the concentration of its substrate seems to be the limiting factor in sulphation.

Distribution of 2-naphthol sulphotransferase and its endogenous substrate adenosine 3'-phosphate 5'-phosphosulphate in human tissues.

The activity of sulphotransferase towards 2-naphthol and the concentration of its endogenous substrate, adenosine 3'-phosphate 5'-phosphosulphate (PAPS), have been measured in five specimens of human liver, lung, and kidney, and the mucosa from the ileum and the ascending, descending and sigmoid colon. The activity of 2-naphthol sulphotransferase (mean nmol.min-1.mg-1 protein) was 1.82 (liver); 0.034 (kidney); 0.19 (lung); 0.64 (ileum); 0.47 (ascending colon); 0.50 (descending colon); 0.40 (sigmoid colon). The concentration of PAPS (mean nmol.g-1 wet tissue) was 22.6 (liver); 4.8 (kidney); 4.3 (lung); 12.8 (ileum); 8.1 (ascending colon); 7.5 (descending colon); 6.2 (sigmoid colon). The concentration of PAPS and the activity of 2-naphthol sulphotransferase were higher in the liver than in the extrahepatic tissues. There was significant difference between ileum and ascending colon, both the activity of sulphotransferase and the concentration of PAPS being higher in the former. 2-Naphthol sulphotransferase activity and the concentration of PAPS have consistent distribution patterns. Differences between the tissues studied were more marked for sulphotransferase than for its endogenous substrate.

Characterization of sulphotransferase in human ileum and colon.

The kinetics of sulphotransferase (ST) were studied at varying concentrations of 2-naphthol or adenosine 3'-phosphate-5'-phosphosulphate (PAPS) in specimens of ileum and colon mucosa obtained from 6 subjects. When 2-naphthol was the variable substrate the enzyme obeyed non-Michaelis-Menten kinetics. The enzyme kinetic profile consists of two phases: one at higher and the other at lower activity for 2-naphthol. The maximum velocity of reaction (Vmax, mean +/- SD; pmol/min.mg protein) of the high-affinity phase was 403 +/- 82 (ileum) and 216 +/- 42 (colon) (p less than 0.01). Vmax for the low-affinity phase was 669 +/- 105 (ileum) and 415 +/- 84 (colon) (p less than 0.01). Km (mean +/- SD) of the high affinity phase was 0.034 +/- 0.004 (ileum) and 0.025 +/- 0.001 mmol/l (colon) (NS) and that of the low-affinity phase was 0.101 +/- 0.013 (ileum) and 0.095 +/- 0.014 mmol/l (colon) (NS). At varying concentrations of PAPS the enzyme obeyed Michaelis-Menten kinetics. Vmax (mean +/- SD) was 995 +/- 163 (ileum) and 520 +/- 114 (colon) pmol/min.mg protein (p less than 0.02). Km was 0.096 +/- 0.008 (ileum) and 0.079 +/- 0.012 mmol/l (colon) (NS). The ST of ileum differs from that of colon for Vmax only.

Studies on degradation of adenosine-5′-phosphosulphate (APS) and adenosine-3′-phosphate-5′-phosphosulphate (PAPS) in extracts of Anabaena cylindrica

[ 35S]Adenosine-5′-phosphosulphate ([ 35S]APS) and [ 35S]adenosine-3′-phosphate-5′-phosphasulphate ([ 35S]PAPS) were rapidly degraded by extracts of Anabaena cylindrica. The loss of radioactivity from these sulphur nucleotides resulted in a corresponding increase of free 35SO 4  in the incubation mixture. The soluble fraction of the broken cells (S 75) hydrolysed both PAPS and APS, whereas the pellet fractions (P 20 and P 75) hydrolysed PAPS only. The degradation of [ 35S]PAPS was almost completely suppressed by various 5′-adenine nucleotides, 3′-AMP, nucleotide triphosphates or pyrophosphate, while glucose-6-phosphate, phosphate ions and sodium sulphite were less effective. The hydrolysis of [ 35S]APS was prevented by sodium fluoride and 5′-AMP, but 3′-AMP was ineffective.

Biological sulphate reduction

Sulphate is reduced to thiols by micro-organisms and plants and these are incorporated via amino acids into protein. Higher animals however do not utilize sulphate and get their sulphur thiol groups usually from amino acids. Some bacteria also use sulphate as an alternative to oxygen as a hydrogen acceptor. Biochemical evidence suggests that sulphate is first activated by adenosine triphosphate (ATP) before it is reduced. Two sulphur-containing nucleotides, adenosine-5′-phosphosulphate (APS) and adenosine-3′-phosphate 5′ phosphosulphate (PAPS) have been identified as carriers of sulphur in bacteria and in green plants during sulphate reduction. Enzymes associated with sulphate and sulphite reduction in bacteria and in green plants are described in this paper, and ecological and economic aspects of the dissimilation of sulphate by bacteria are also considered.