Sulfotransferase Reaction Research Articles

Interacting polymer-modification enzymes in heparan sulfate biosynthesis

Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcAβ1–4GlcNSO3α1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluorescence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a functional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O-sulfated glucosamine residues. The inability to achieve simultaneous 2-O- and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme interactions in heparan sulfate biosynthesis.

Skeletal Dysplasias Caused by Sulfation Defects.

Proteoglycans (PGs) are macromolecules present on the cell surface and in the extracellular matrix that confer specific mechanical, biochemical, and physical properties to tissues. Sulfate groups present on glycosaminoglycans, linear polysaccharide chains attached to PG core proteins, are fundamental for correct PG functions. Indeed, through the negative charge of sulfate groups, PGs interact with extracellular matrix molecules and bind growth factors regulating tissue structure and cell behavior. The maintenance of correct sulfate metabolism is important in tissue development and function, particularly in cartilage where PGs are fundamental and abundant components of the extracellular matrix. In chondrocytes, the main sulfate source is the extracellular space, then sulfate is taken up and activated in the cytosol to the universal sulfate donor to be used in sulfotransferase reactions. Alteration in each step of sulfate metabolism can affect macromolecular sulfation, leading to the onset of diseases that affect mainly cartilage and bone. This review presents a panoramic view of skeletal dysplasias caused by mutations in genes encoding for transporters or enzymes involved in macromolecular sulfation. Future research in this field will contribute to the understanding of the disease pathogenesis, allowing the development of targeted therapies aimed at alleviating, preventing, or modifying the disease progression.

Enzymatic Conversion of Cypridina Luciferyl Sulfate to Cypridina Luciferin with Coenzyme A as a Sulfate Acceptor in Cypridina (Vargula) hilgendorfii.

In the luminous ostracod Cypridina (presently Vargula) hilgendorfii, Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) is converted to Cypridina luciferin by a sulfotransferase with 3'-phosphoadenosine-5'-phosphate (PAP) as a sulfate acceptor. The resultant Cypridina luciferin is used for the luciferase-luciferin reaction of Cypridina to emit blue light. The luminescence stimulation with major organic cofactors was examined using the crude extracts of Cypridina specimens, and we found that the addition of coenzyme A (CoA) to the crude extracts significantly stimulated luminescence intensity. Further, the light-emitting source in the crude extracts stimulated with CoA was identified as Cypridina luciferyl sulfate, and we demonstrated that CoA could act as a sulfate acceptor from Cypridina luciferyl sulfate. In addition, the sulfate group of Cypridina luciferyl sulfate was also transferred to adenosine 5'-monophosphate (5'-AMP) and adenosine 3'-monophosphate (3'-AMP) by a sulfotransferase. The sulfated products corresponding to CoA, 5'-AMP and 3'-AMP were identified using mass spectrometry. This is the first report that CoA can act as a sulfate acceptor in a sulfotransferase reaction.

The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5'-phosphosulfate to the cytosol.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.

Golgi-resident PAP-specific 3′-phosphatase-coupled sulfotransferase assays

Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3′-phosphoadenosine-5′-phosphosulfate (PAPS)1Abbreviations used: 5′-AMP, 5′-adenosine monophosphate; 3′-AMP, 3′-adenosine monophosphate; APS, adenosine 5′-phosphosulfate; CHST, carbohydrate sulfotransferase; PAP, 3′-phosphoadenosine-5′-phosphate; gPAPP, Golgi-resident PAP 3′-phosphatase; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; SULT, cytosolic sulfotransferase; ALPL, tissue non-specific alkaline phosphatase.1, to various acceptor substrates, generating 3′-phosphoadenosine-5′-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3′-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3′-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis–Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.

Chondrodysplasia and Abnormal Joint Development Associated with Mutations in IMPAD1, Encoding the Golgi-Resident Nucleotide Phosphatase, gPAPP

We used whole-exome sequencing to study three individuals with a distinct condition characterized by short stature, chondrodysplasia with brachydactyly, congenital joint dislocations, cleft palate, and facial dysmorphism. Affected individuals carried homozygous missense mutations in IMPAD1, the gene coding for gPAPP, a Golgi-resident nucleotide phosphatase that hydrolyzes phosphoadenosine phosphate (PAP), the byproduct of sulfotransferase reactions, to AMP. The mutations affected residues in or adjacent to the phosphatase active site and are predicted to impair enzyme activity. A fourth unrelated patient was subsequently found to be homozygous for a premature termination codon in IMPAD1. Impad1 inactivation in mice has previously been shown to produce chondrodysplasia with abnormal joint formation and impaired proteoglycan sulfation. The human chondrodysplasia associated with gPAPP deficiency joins a growing number of skeletoarticular conditions associated with defective synthesis of sulfated proteoglycans, highlighting the importance of proteoglycans in the development of skeletal elements and joints.

Expression profile of Papss2 (3′‐phosphoadenosine 5′‐phosphosulfate synthase 2) during cartilage formation and skeletal development in the mouse embryo

Sulfation of proteoglycans is a very important posttranslational modification in chondrocyte growth and development. The enzyme 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS) catalyzes the biosynthesis of PAPS (3'-phosphoadenosine 5'-phosphosulfate), which serves as the universal sulfate donor compound for all sulfotransferase reactions (Schwartz and Domowicz [2002] Glycobiology 109:143-151). Two major isoenzymes, PAPS synthase 1 (PAPSS1) and PAPS synthase 2 (PAPSS2) were identified in higher organisms for the synthesis of PAPS. PAPSS1 is the more prominent isoform and is ubiquitously expressed in human adult tissues, including cartilage, while PAPSS2 shows a more restricted expression pattern and appears to be the major variant in growth plate cartilage (Fuda et al. [2002] Biochem J 365(Pt 2):497-504). Mutations within the murine and the human PAPSS2 genes are responsible for diseases affecting the skeletal system (Kurima et al. [1998] Proc Natl Acad Sci USA 95:8681-8685; ul Haque et al. [1998] Nat Genet 20:157-162), like the spondyloepimetaphyseal dysplasia (SEMD) Pakistani type. To further elucidate the function of Papss2 within the developing skeleton, we investigated the expression pattern of the murine gene at different developmental stages. We detected Papss2 mRNA starting from 11.5 days post coitum (dpc) at the sites of first chondrogenic condensations and the expression continued in all cartilaginous elements tested of 12.5 dpc, 13.5 dpc, 16.5 dpc embryos, and newborn mice. Papss2 transcripts were also observed in other tissues such as heart, tongue, kidney, and neuronal tissues. However, the most significant levels of Papss2 mRNA were found in condensing and proliferating chondrocytes, whereas hypertrophic chondrocytes show a dramatic down-regulation of Papss2 mRNA expression, indicating an important role of the gene product for cartilage growth and development in mouse embryo.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

34S isotope effect on sulfate ester hydrolysis: mechanistic implications.

In this communication, we report the first determination of 34S kinetic isotope effects (KIEs) for the hydrolysis of sulfate monoesters. The method involves the conversion of the inorganic sulfate, acquired at partial extent of reaction, to SO2, followed by isotope ratio determination by mass spectrometry. The KIEs determined for p-nitrophenyl sulfate and p-acetylphenyl sulfate are 1.0154 (+/-0.0002) and 1.0172 (+/-0.0003), respectively. These results, together with previous peripheral 18O KIE values, are inconsistent with an associative mechanism. The isotope effect method we report should also prove useful for studying the mechanism of other sulfuryl group transfers, including sulfatase and sulfotransferase reactions, as well as sulfate hydrolyses under other conditions.

Development of a simple homogeneous assay to screen for inhibitors of N-acetylglucosamine-6-sulfotransferases

L-selectin, a leukocyte adhesion molecule, plays a central role in lymphocyte homing to secondary lymphoid tissue and to certain sites of inflammation. Carbohydrate sulfation was implicated in this process, when it was demonstrated that carbohydrate sulfotransferase-mediated sulfation of N-acetylglucosamine (GlcNAc) within sialyl Lewis X of cognate endothelial ligands for L-selectin was an essential modification for L-selectin binding. The recently identified GlcNAc-6-sulfotransferases GlcNAc6ST-1 and -2, which facilitate GlcNAc sulfation by catalyzing the transfer of a sulfonyl group from 3 ′-phosphoadenosine 5 ′-phosphosulfate (PAPS) to the 6-hydroxy group of the acceptor GlcNAc moiety, contribute to the biosynthesis of the 6-sulfosialyl Lewis X motif. Due to their pivotal role in L-selectin ligand biosynthesis, this enzyme class has recently emerged as an important and relatively unexplored class of potential targets for anti-inflammatory therapy. However, no inhibitors have been reported to date and screening for lead inhibitors has been hampered by the lack of simple assay formats suitable for high-throughput screening. Here, we report the development of a simple homogeneous in vitro sulfotransferase assay using a newly synthesized biotinylated glycoside as a substrate. The assay is based on GlcNAc6ST-2-mediated [ 35S]sulfate transfer from [ 35S]PAPS to the biotinylated glycoside and subsequent detection using streptavidin-coated SPA beads. K m values with partially purified GlcNAc6ST-2 for PAPS and the biotinylated glycoside were estimated to be 8.4 and 34.5 μM, respectively. The sulfotransferase reaction could be inhibited by 3 ′,5 ′-ADP with an IC 50 of 2.1 μM. The assay can be operated in 384-well format; is characterized by a high signal-to-noise ratio, low variation, and excellent Z factors; and is highly suitable for high-throughput screening.

Human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase: biochemistry, molecular biology and genetic deficiency.

3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase (PAPSS) catalyzes the biosynthesis of PAPS which serves as the universal sulfonate donor compound for all sulfotransferase reactions. PAPSS forms PAPS in two sequential steps. First inorganic sulfate combines with ATP to form adenosine 5'-phosphosulfate (APS) and pyrophosphate catalyzed by ATP sulfurylase domain and in the second step, APS combines with another molecule of ATP to form PAPS and ADP catalyzed by APS kinase domain. The bifunctional PAPSS1 is comprised of NH2-terminal APS kinase domain (approximately 1-260 aa), and a COOH-terminal ATP sulfurylase domain (approximately 220-623 aa). In humans there are two major isoforms PAPSS1 and PAPSS2. In brain and skin PAPSS1 is the major expressed isoform, whereas in liver, cartilage and adrenal glands PAPSS2 isoform expression predominates and in various other tissues the proportions of the isoform expressions is purported to vary. The deduced amino acid sequences of the two isoforms reveal 77% identity between PAPSS1 and PAPSS2. In addition there is a splice variant PAPSS2b which contains notably an extra five amino acid sequence GMALP. From human tissues PAPSS1 and a splice variant PAPSS2b has been molecularly cloned, overexpressed, purified and have been biochemically characterized partially. PAPSS2b exhibited an apparent difference towards varying ATP concentration showing a sigmoidal response, with a 0.5 [v/Vmax] at 1.4 mM ATP whereas PAPSS1 exhibited a hyperbolic response with a 0.5 [v/Vmax] at 0.25 mM ATP. Although this being the case, comparison of PAPSS1 and PAPSS2 crude extracts, did not show marked difference in the kinetic properties with either substrates ATP or sulfate leading to speculate that the extra GMALP pentapeptide present in PAPSS2b could be altering the kinetic behavior. The ATP binding sites of the alpha-beta-ATP hydrolysis, active site motif HxxH (425-428 aa) is present in the ATP sulfurylase domain and the beta-gamma-hydrolase motif GxxGxxK (59-65 aa) is present in the APS kinase domain. The motifs are highly conserved between both isoforms. Gene sequence analysis of PAPSS1 (approximately 106 kB) and PAPSS2 (approximately 86.5 kB), revealed a total of 12 exons. Among exons 2-11 the sizes are highly conserved, although intron sizes varied remarkably. Exons 1 and 12 varied in sizes, contained 5'-UTR and 3'-UTR respectively. PAPSS1 and PAPSS2 contained no putative TATA box and CCAAT box. However both PAPSS1 and PAPSS2 possessed many GC boxes. From promoter analysis, it is apparent that both PAPSS1 and PAPSS2 are inducible, perhaps at various time periods, regulated by specific transcription factors. The deficiency of PAPSS2 results in osteochondrodysplasias. Osteochondrodysplasias are genetically heterogeneous group of disorders that affects skeletal development, linear growth, and the maintenance of cartilage and bone. A large inbred family with a distinct form of recessively inherited, spondyloepimetaphyseal dysplasia (SEMD) was mapped to PAPSS2 isoform located in the chromosome region of 10q23-24. PAPSS1 located in the chromosome 4q23 deficiency and consequent effect in lymphocyte recruitment in High Endothelial Venules has been reported. Several single nucleotide polymorphism (SNP) of PAPSS has been identified, some of which are in the coding region (cSNPs), has been shown to have altered enzyme activity.

Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily?

The reaction steps leading from the intermediate adenosine 5′-phosphosulfate (APS) to sulfide within the higher plant reductive sulfate assimilation pathway are the subject of controversy. Two pathways have been proposed: a ‘bound intermediate’ pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a ‘free intermediate’ pathway in which APS is further activated to 3′-phosphoadenosine 5′-phosphosulfate (PAPS) by APS kinase followed by reduction of the sulfo group to free sulfite by PAPS reductase. Sulfite is then reduced to free sulfide by sulfite reductase. Sulfide, either free or bound, is then incorporated into organic form (as cysteine) by the enzyme O-acetylserine (thiol) lyase. In order to better characterize the pathway we attempted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy. We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily. We show here that the thioredoxin domain of these proteins is functional. We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to sulfite by APS reductase and the subsequent reduction of sulfite to sulfide by sulfite reductase. In this scheme the product of the APS kinase reaction, PAPS, is not a direct intermediate in the pathway but rather acts as a substrate for sulfotransferase action and perhaps as a store of activated sulfate that can be returned to the pathway as APS via phosphohydrolase action on PAPS. Interactions between enzyme isoforms within the chloroplast stroma may bring about substrate channeling of APS and contribute to the partitioning of APS between sulfotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the other.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Non-radioactive adenosine 5'-phosphosulfate sulfotransferase assay by coupling with sulfite reductase and O-acetylserine(thiol)lyase.

Adenosine 5'-phosphosulfate (APS) sulfotransferase is thought to be an enzyme that transfers the sulfo-group of APS to a carrier compound with a thiol group in the assimilatory sulfate reduction pathway of higher plants. We developed a rapid, non-radioactive assay for APS sulfotransferase. Sulfite released by APS sulfotransferase reaction in the presence of excess dithiothreitol was further converted to cysteine by coupling with yeast sulfite reductase and cabbage O-acetylserine(thiol)lyase. The cysteine thus formed was measured colorimetrically. By this method, 5 to 300 nmol of sulfite could be assessed. When the method was applied to APS sulfotransferase, the enzyme activity was APS-dependent with the partially purified enzyme. We could also detect APS sulfotransferase activity in some higher plants by this method.

An Enzymatic Procedure for the Preparation and Purification of 3′-Phosphoadenosine 5′-Phospho- [35S]Sulfate ([35S]PAPS): Applications in Syntheses of 8-Azido and 8-Bromo Derivatives of [35S]PAPS

This paper describes a rapid and an efficient procedure for the enzymatic synthesis of 3′-phosphoadenosine 5′-phospho[35S]sulfate ([35S]PAPS). [35S]PAPS was synthesized by incubating ATP and a carrier-free [35S]Na235SO4with ATP sulfurylase, a recombinant APS kinase and inorganic pyrophosphatase. The transfer of35SO4group from [35S]Na2SO4to [35S]PAPS proceeded more efficiently in the presence of an ATP-regenerating system composed of pyruvate kinase and phosphoenol pyruvate. About 90% of the radioactivity present in the starting material [35S]Na2SO4was trans- ferred to [35S]PAPS within a 2-h reaction incubation. The reaction products were applied to a Mono Q column, and [35S]PAPS was eluted by a step-wise gradient of triethylamine bicarbonate buffer (pH 7.5). Under these conditions, [35S]PAPS eluted as a sharp peak at 0.7Mtriethylammonium bicarbonate and it was very well separated from other contaminants. The purified [35S]PAPS (yield 85%, purity >95%) was functional in donating sulfate to an oligosaccharide acceptor in a standard sulfotransferase reaction. The enzymatic procedure described above was particularly useful for the synthesis of [35S]PAPS at a wide range of concentrations and specific activities (up to 1500 Ci/mmol). This generally useful approach was also found to be successful in the syntheses of 8-azido and 8-bromo derivatives of [35S]PAPS. Applications of these two derivatives of PAPS, for purification and identification of sulfotransferases, have also been discussed.

Characterization of guinea pig estrogen sulfotransferase expressed by Chinese hamster ovary cell-K1 stable transfectants.

Estrogen sulfotransferase (EST) activity expressed by Chinese hamster ovary (CHO)-K1 cells stably transfected with a plasmid containing a guinea pig EST complementary DNA insert was subjected to biochemical characterization, and the EST protein was further examined by nondenaturing isoelectric focusing and immunoblot analysis. CHO-K1 cells transfected with the same plasmid without the EST complementary DNA insert as well as untransfected CHO-K1 cells did not demonstrate either EST activity or the presence of an immunologically related protein. The EST expressed by the stably transfected CHO-K1 cells was found to manifest Michaelis-Menten kinetics and would use only estrogenic steroids as substrates, whereas other forms of steroids, such as pregnenolone, dehydroepiandrosterone, cortisol, and testosterone, were not acted on. When 17 beta-estradiol was used as a substrate, sulfonation occurred exclusively at the 3 position; 17-sulfonate was not formed. Thus, the expressed EST acted selectively on the 3-hydroxyl group of phenolic steroids. The apparent Km values for estrone, 17 beta-estradiol, and estriol were 60, 70, and 40 nM, respectively. The maximum velocity (Vmax) determinations for estrone and 17 beta-estradiol were equivalent, whereas the Vmax for estriol was reduced by 33%. Of the three estrogens, only 17 beta-estradiol caused substrate inhibition at a high concentration. Steroid sulfonation requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the active sulfonate donor, and the Km value for PAPS was 1.2 microM. In steroid sulfotransferase reactions, two products are formed: the sulfonated steroid product and the desulfonated cofactor, 3'-phosphoadenosine-5'-phosphate (PAP). The sulfonation of 17 beta-estradiol was inhibited by PAP in a dose-dependent manner. In addition, the Km for PAPS was increased by PAP, whereas the Vmax was unaffected, indicating competitive inhibition (Ki, approximately 0.52 microM). The EST protein expressed by the CHO-K1 cell stable transfectants demonstrated a mol wt of 34 kilodaltons, as determined by sodium dodecyl sulfate-gel electrophoresis. Additionally, when the expressed EST protein was subjected to isoelectric focusing, it was found to consist of multiple charge isoforms. These findings are comparable to what has been previously reported for native guinea pig adrenocortical EST. Furthermore, the charge isoform pattern that was demonstrated for the expressed EST was similar to the pattern observed for the native protein.

Kinetic studies on a sulfotransferase from Klebsiella K-36, a rat intestinal bacterium.

Sulfotransferase purified from Klebsiella K-36, a rat intestinal bacterium, stoichiometrically catalyzed the transfer of a sulfate group of phenylsulfate esters to phenolic compounds. One of the reaction products, p-nitrophenol (PNP), non-competitively inhibited the enzyme as to p-nitrophenylsulfate (PNS), a donor substrate, but competitively inhibited the enzyme as to an acceptor substrate, alpha-naphthol. The other reaction product, alpha-naphthol-O-sulfate, non-competitively inhibited the enzyme with regard to both these substrates. These kinetic data suggest that the sulfotransferase reaction proceeds according to an ordered bi bi reaction mechanism. The natural phenolic substances, gallic acid, quercetin, tannic acid, and serotonin were good substrates of K-36 sulfotransferase.

Cholecystokinin activation: Evidence for an ordered reaction mechanism for the tyrosyl protein sulfotransferase responsible for the peptide sulfation

The kinetics of the forward tyrosyl protein sulfotransferase (TPS) reaction were examined using an assay based on the 35SO 4 transfer from 3′-phosphoadenosine 5′-phospho( 35S sulfate ([ 35S]PAPS) to tyrosyl residues of the non-sulfated cholecystokinin derivative, BocCCK-8(ns). TPS present in the microsomal membranes from rat cerebral cortex was used for these studies. Initial velocity measurements performed over a wide range of PAPS, BocCCK-8(ns), 3′-PAP and BocCCK-8(s) concentrations, indicated that the reaction follows an ordered mechanistic pathway. The K M value determined for BocCCK-8(ns) was 160 ± 18 μM, and that for [ 35S]PAPS was 0.15 ± 0.03 μM. 3′-Phosphoadenosine 5′-phosphate (3′-PAP) was found to be a product inhibitor with a Ki = 0.30 ± 0.02 μM. BocCCK-8(s) produced an uncompetitive inhibition pattern on the TPS reaction. Adenosine 5′-phosphosulfate (APS) behaved as a competitive inhibitor versus PAPS with a Ki = 3.0 ± 0.3 μM. ATP inhibited competitively the reaction when PAPS was the varied substrate with a Ki = 3.6 ± 0.5 μM. The results of product and substrate inhibition studies and the patterns of dead end inhibition obtained with APS are best fit by an ordered Bi-Bi reaction mechanism where PAPS is the first substrate to bind and 3′-PAP is the last product to be released.

Biosynthesis of neutral glucocerebroside homologues in the absence of myelin assembly after nerve transection.

The biosynthesis of myelin-associated glycolipids during various stages of myelination was studied by in vitro incorporation of [3H]Gal, [3H]Glc, or [35S]sulfate into the endoneurium of rat sciatic nerve. In the normal adult nerve, where the level of myelin assembly is substantially reduced and Schwann cells are principally involved in maintaining the existing myelin membrane, [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG) and the galactocerebrosides (GalCe) with lower levels of incorporation into the sulfatides. Such incorporation was enhanced 35 days after crush injury of the adult rat sciatic nerve, which is characterized by active myelin assembly. In contrast, at 35 days after permanent nerve transection where there is no axonal regeneration or myelin assembly, the incorporation of [3H]Gal or [3H]Glc into GalCe was nearly undetected whereas the incorporation of [3H]Gal into MGDG was completely inhibited. Instead, the 3H-labeled glycolipids in transected nerve were identified as the glucocerebrosides (GlcCe) and oligohexosylceramide derivatives with tetrahexosylceramide being a major product. In contrast, [35S]sulfate was incorporated into endoneurial sulfatides in the transected nerve, which suggests that endogenous GalCe rather than newly synthesized GalCe served as the substrate for the sulfotransferase reaction. The GlcCe homologues are not considered as constituents of the myelin membrane but are likely plasma membrane components synthesized in the absence of myelin assembly. It is likely that the cells responsible for GlcCe biosynthesis are Schwann cells, since they comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and high-performance liquid chromatography of 3′-phosphoadenosine-5′-phospho[ 35S]sulfate with a predetermined specific activity

3′-Phosphoadenosine-5′-phospho[ 35S]sulfate (PAP 35S) was prepared by incubating ATP and carrier-free H 2 35SO 4 with a 100,000 g supernatant fraction prepared from chick embryo chondrocytes. The product was partially purified by paper by paper electrophoresis and mixed with unlabeled PAPS to give a solution of PAP 35S with a specific activity and a concentration approximating those required for the desired metabolic studies. The product was analyzed by high-performance liquid chromatography on an anion-exchange column to determine the proportion of the 35SO 4 cpm and A 260 material found in the PAPS and other contaminating nucleotides. The PAP 35S was purified further by preparative high-performance liquid chromatography. The exact specific activity of the PAP 35S was then determined by using this PAP 35S preparation as the SO 4 donor in a sulfotransferase reaction using a microsomal preparation from the chick embryo chondrocytes as the enzyme and an 3H-labeled oligosaccharide as the SO 4 acceptor. The sulfated oligosaccharide was then isolated and the number of 3H and 35SO 4 counts per minute in this product were used to calculate the specific activity of the donor. The features of this generally useful approach for preparing PAP 35S of any desired specific activity and concentration are discussed.