Adenosine-5'-phosphosulfate--a multifaceted modulator of bifunctional 3'-phospho-adenosine-5'-phosphosulfate synthases and related enzymes.

All sulfation reactions rely on active sulfate in the form of 3'-phospho-adenosine-5'-phosphosulfate (PAPS). In fungi, bacteria, and plants, the enzymes responsible for PAPS synthesis, ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase, reside on separate polypeptide chains. In metazoans, however, bifunctional PAPS synthases catalyze the consecutive steps of sulfate activation by converting sulfate to PAPS via the intermediate APS. This intricate molecule and the related nucleotides PAPS and 3'-phospho-adenosine-5'-phosphate modulate the function of various enzymes from sulfation pathways, and these effects are summarized in this review. On the ATP sulfurylase domain that initially produces APS from sulfate and ATP, APS acts as a potent product inhibitor, being competitive with both ATP and sulfate. For the APS kinase domain that phosphorylates APS to PAPS, APS is an uncompetitive substrate inhibitor that can bind both at the ATP/ADP-binding site and the PAPS/APS-binding site. For human PAPS synthase 1, the steady-state concentration of APS has been modelled to be 1.6 µM, but this may increase up to 60 µM under conditions of sulfate excess. It is noteworthy that the APS concentration for maximal APS kinase activity is 15 µM. Finally, we recognized APS as a highly specific stabilizer of bifunctional PAPS synthases. APS most likely stabilizes the APS kinase part of these proteins by forming a dead-end enzyme-ADP-APS complex at APS concentrations between 0.5 and 5 µM; at higher concentrations, APS may bind to the catalytic centers of ATP sulfurylase. Based on the assumption that cellular concentrations of APS fluctuate within this range, APS can therefore be regarded as a key modulator of PAPS synthase functions.

Participation of ATP in nonadrenergic, noncholinergic relaxation of longitudinal muscle of wistar rat jejunum.

A role of ATP in nonadrenergic, noncholinergic (NANC) relaxations was examined in the Wistar rat jejunum. Electrical field stimulation (EFS) induced NANC relaxation of longitudinal muscle of the jejunal segments in a frequency-dependent manner. A purinoceptor antagonist, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, 100 muM) inhibited the relaxation: relaxations induced by EFS at lower or higher frequencies were either completely or partially inhibited, respectively. After the jejunal segments had been desensitized to ATP, the relaxations were decreased to the same extent as those inhibited by A3P5PS. An inhibitor of small conductance Ca(2+)-activated K(+) channels (SK channels), apamin (100 nM), completely inhibited EFS-induced relaxations. Treatment of the segments with an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, thapsigargin (1 muM), significantly inhibited the relaxations. The exogenous ATP-induced relaxation of longitudinal muscle occurred with a concomitant decrease in intracellular Ca(2+) levels. Apamin and thapsigargin abolished these ATP-induced responses. A3P5PS significantly inhibited the inhibitory junction potentials which were induced in the longitudinal muscle cells. In addition, apamin significantly inhibited the hyperpolarization that was induced by exogenous ATP in the cells. These findings in the Wistar rat jejunum suggest that ATP participates in the NANC relaxation via activation of SK channels induced by Ca(2+) ions that are released from the thapsigargin-sensitive store site.

Crystallization and preliminary crystallographic studies of the cofactor-binding domain of the LysR-type transcriptional regulator Cbl from Escherichia coli.

Cbl (CysB-like protein) is a member of the family of LysR-type transcriptional regulators (LTTRs) and controls genes engaged in sulfur assimilation in Escherichia coli. It has been postulated that adenosine 5-phosphosulfate (APS) is responsible for abolishing Cbl-activated transcription from the ssu promoter (Bykowski et al., 2002). To elucidate the structural basis of Cbl function and to confirm the role of APS as an anti-inducer, the cofactor-binding domain of Cbl (c-Cbl, MW = 26 kDa) was cloned, purified and crystallized in the presence of APS. The crystals belong to space group C222(1), but show substantial variation of the unit-cell parameters and diffraction anisotropy. Despite this, X-ray data extending to 3.0 A resolution have been collected and solution of the structure by molecular replacement is in progress.

Effects of AMP derivatives on cyclic AMP levels in NG108-15 cells.

1. In NG108-15 neuroblastomaxglioma hybrid cells, ATP stimulates intracellular cyclic AMP formation, which is inhibited by both adenosine (P(1)) and P2 receptor antagonists. In the present study, we examined the effects of several AMP derivatives in NG108-15 cells and mouse neuroblastoma N18TG-2 cells. 2. Adenosine 2'-monophosphate (A2P), adenosine 3'-monophosphate (A3P) and adenosine 5'-phosphosulphate (A5PS) increased cyclic AMP levels with similar concentration-dependencies in NG108-15 cells. 3. Increases in cyclic AMP by AMP derivatives were inhibited by the P2 receptor antagonist PPADS, but not by suramin. Effects of AMP derivatives were also inhibited by P(1) receptor antagonists ZM241385, XAC, DPCPX and partially by alloxazine. The ecto-nucleotidase inhibitor alpha, beta-methyleneADP was without effect. 4. In contrast, AMP derivatives did not change cyclic AMP levels in N18TG-2 cells. Accumulation of cyclic AMP in N18TG-2 cells was stimulated by adenosine A(2) receptor agonists CGS21680 and NECA, but not by ATP or beta, gamma-methyleneATP, agonists for cyclic AMP production in NG108-15 cells. 5. Reverse transcription-coupled polymerase chain reaction (RT - PCR) analyses revealed that N18TG-2 cells express both A(2A) and A(2B) receptors, while NG108-15 cells express mainly A(2A) receptors. 6. AMP derivatives did not affect the P2X and P2Y receptors expressed in NG108-15 cells. 7. These results suggest that A2P, A3P and A5PS act as agonists for cyclic AMP production and that these compounds are valuable tools for determinating the mechanism of ATP-stimulated cyclic AMP response in NG108-15 cells.

Effect of rat liver cytosolic enzymes and cofactors on mutagenicity of 1-amino-8-nitropyrene.

1-Amino-8-nitropyrene (1,8-ANP), a product of 1,8-dinitropyrene metabolism by either bacterial or mammalian enzymes, is weakly mutagenic to the 'classical nitroreductase'-deficient Salmonella tester strain TA98NR. The addition to the test system of rat liver cytosol without cofactors did not produce any effect on the 1,8-ANP mutagenic response toward TA98NR strain. Conversely, when both rat hepatic cytosol and NADPH (1 mM) were added to the mutagenicity assay, a 10-fold increase in 1,8-ANP mutagenic activity was observed. This suggests the involvement of rat hepatic cytosolic NADPH-dependent nitroreductase(s) in 1,8-ANP mutagenic activation. The addition to the mutagenesis assay of pentachlorophenol, an inhibitor of O-acetyltransferase and sulfotransferase, produced a dose-dependent decrease of 1,8-ANP mutagenic activation, whereas 2,6-dichloro-4-nitrophenol, a more specific inhibitor of sulfotransferase than O-acetyltransferase, did not affect the activation of 1,8-ANP to a mutagen at concentrations that selectively inhibit only bacterial sulfotransferase. This indicates that bacterial O-acetyltransferase but not sulfotransferase plays a role in the mutagenic activation of 1,8-ANP. Addition of acetyl co-enzyme A (AcCoA) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), cofactors for O-acetyl-transferase and sulfotransferase respectively, to the test system caused a dose-dependent inhibition of 1,8-ANP mutagenic activation by rat liver cytosol and NADPH, probably due to the formation of highly reactive O-acetoxy and N-sulfate ester derivatives of 1,8-ANP, which react with nucleophilic sites before reaching bacterial DNA. This hypothesis was confirmed by DNA covalent binding in in vitro experiments showing that both the cofactors AcCoA and PAPS enhanced the NADPH/rat liver cytosol-mediated covalent binding of 1,8-ANP to DNA from calf thymus 10- and 3-fold respectively. It seems likely that rat hepatic cytosolic nitroreductases activate 1,8-ANP to an N-hydroxyarylamine derivative which can be further metabolized to mutagenic species by either bacterial or mammalian O-acetyltransferase.

Synthesis and properties of a nonhydrolyzable adenosine phosphosulfate analog.

Initial activation of inorganic sulfate for subsequent synthesis of sulfated biomolecules requires the action of ATP-sulfurylase to generate adenosine 5'-phosphosulfate (APS). This activated sulfate intermediate is both chemically labile and susceptible to enzymatic degradation. Consequently, it has not proven useful as a ligand for either purification or characterization of the various APS-utilizing enzymes. For these purposes, a stable analog of APS was required. This paper describes the simple and efficient synthesis and structural confirmation of a nonhydrolyzable APS analog, beta-methylene APS, with an overall molar yield of 40-50%. The method involves nucleophilic substitution of the chlorine moiety of a 5'-chloromethylphosphonate ester of 2',3'-O-isopropylidene adenosine by a sulfite ion. We also report the initial utilization of this compound as an inhibitor in kinetic trials of both ATP-sulfurylase and APS kinase and as an affinity ligand for the purification of these two APS-utilizing enzymes from cartilaginous tissue.