Human Platelet Phenol Sulfotransferase: Partial Purification and Detection of Two Forms of the Enzyme

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.     

Purification and kinetic characterization of a phenol-sulfating form of phenol sulfotransferase from human brain

The identification of three forms of phenol sulfotransferase (PST) in human brain and the subsequent purification and kinetic characterization of a phenol-sulfating form of the enzyme are described. Two forms of PST which were capable of conjugating phenol and a third form which sulfated dopamine were resolved from one another using DEAE-cellulose chromatography. One of the phenol-sulfating forms (P1-PST) was subsequently purified on Affi-Gel blue and Sephacryl S-200, giving a final purification of almost 390-fold, with an overall yield of approximately 5%. The purified enzyme was sensitive to NaCl and showed an optimum for phenol conjugation at pH 8.5. Kinetic analysis demonstrated that sulfation by P1-PST proceeds via a sequential ordered, bi-substrate reaction mechanism, where 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is the leading substrate. The true Km and Kia values for PAPS were both 0.35 microM, while the true Km value for phenol was 2.8 microM.     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.     

Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.     

Sulfation of Dopamine and Other Biogenic Amines by Human Brain Phenol Sulfotransferase

Abstract: Phenol sulfotransferase was isolated in 100,000g supernatant fractions prepared from postmortem samples of human brain. Since phenol sulfotransferase (PST) has been shown to conjugate the amine neurotransmit-ters in vivo , the abilities of eight different biogenic amines and structurally related compounds to act as substrates for PST were studied. These experiments demonstrate that at a concentration of 20 μM, dopamine (DA) was the best substrate examined and was followed in decreasing order of activity by 3-methoxytyramine (3-MT), tyramine, norepinephrine, 3-methoxy-4-hydroxyphenylethyleneglycol, octopamine, 5-hydroxytryptamine and dihydroxyphenylethyleneglycol. At a substrate concentration of 100 /UM the relative order of activity was altered, so that tyramine became the most rapidly conjugated substrate while the activity of DA and 3-MT relative to the other substrates tested was diminished. This change in substrate affinity with differing substrate concentrations can be explained, at least for DA, by the occurrence of apparent substrate inhibition at concentrations above 25 to 30 μM. Using PST isolated in 100,000g supernatant fractions from human brain, the K_m value for DA was found to be 5.0 μM, while the K_m value for the sulfate-donor 3'-phosphoadenosine-5'-phosphosulfate was 0.25 μM. The ratio of 3- O - to 4- O -DA-sulfate formed in vitro by human brain PST was found to be about 4: 1. In addition, both the 3- O - and 4- O -esters were found not to be deaminated by human brain mitochondrial MAO. The relative role of PST with respect to MAO and catechol- O -methyltransferase in the degradation of the biogenic amine neurotransmitters in human brain is discussed.     

Ribose modified nucleosides and nucleotides as ligands for purine receptors

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Synthesis, SAR, and preliminary mechanistic evaluation of novel antiproliferative N⁶,5'-bis-ureido- and 5'-carbamoyl-N⁶-ureidoadenosine derivatives

We have developed efficient methods for the preparation of N(6),5'-bis-ureidoadenosine derivatives and their 5'-carbamoyl-N(6)-ureido congeners. Treatment of 5'-azido-5'-deoxy-N(6)-(N-alkyl or -arylurea)adenosine derivatives (6a-d) with H(2)/Pd-C or Ph(3)P/H(2)O, followed by N-methyl-p-nitrophenylcarbamate gave N(6),5'-bis-ureido products 7a-d in 49-78% yield. Analogous derivatives in the 5'-carbamoyl-N(6)-ureido series were prepared by treatment of 2',3'-bis-O-TBS-adenosine (11) with N-methyl-p-nitrophenylcarbamate followed by acylation with appropriate isocyanates which gave 13a-d in 45-69% yield. A more versatile route for obtaining potentially vast libraries of compounds from both series was achieved by treatment of 5'-N-methylureido- or 5'-N-methylcarbamoyladenosine derivatives with ethylchlorformate to give N(6)-ethoxycarbonyl derivatives (9 and 14) in 55-63% yields, respectively. Simple heating of 9 or 14 in the presence of primary alkyl- or arylamines gave the corresponding N(6),5'-bis-ureido- or 5'-carbamoyl-N(6)-ureidoadenosine derivatives in good yields (33-72% and 39-83%; 10a-e and 15a-e, respectively). Significant antiproliferative activities (IC(50)≈4-10 μg/mL) were observed for a majority of the N(6),5'-bis-ureido derivatives, whereas the 5'-carbamoyl-N(6)-ureido derivatives were generally less active (IC(50) >100 μg/mL). A 2',3'-O-desilylated derivative (5'-amino-5'-deoxy-5'-N-methylureido-N(6)-(N-phenylcarbamoyl)adenosine, 16) was shown to inhibit binding of 16 of 441 protein kinases to immobilized ATP-binding site ligands by 30-40% in a competitive binding assay at 10 μM. Compound 16 was also shown to bind to bone morphogenetic protein receptor 1b (BMPR1b) with a Kd=11.5 ± 0.7 μM.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3′-phosphate 5′-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

4-Coumarate:coenzyme A ligase has the catalytic capacity to synthesize and reuse various (di)adenosine polyphosphates

4-Coumarate:coenzyme A ligase (4CL) is known to activate cinnamic acid derivatives to their corresponding coenzyme A esters. As a new type of 4CL-catalyzed reaction, we observed the synthesis of various mono- and diadenosine polyphosphates. Both the native 4CL2 isoform from Arabidopsis (At4CL2 wild type) and the At4CL2 gain of function mutant M293P/K320L, which exhibits the capacity to use a broader range of phenolic substrates, catalyzed the synthesis of adenosine 5'-tetraphosphate (p(4)A) and adenosine 5'-pentaphosphate when incubated with MgATP(-2) and tripolyphosphate or tetrapolyphosphate (P(4)), respectively. Diadenosine 5',5',-P(1),P(4)-tetraphosphate represented the main product when the enzymes were supplied with only MgATP(2-). The At4CL2 mutant M293P/K320L was studied in more detail and was also found to catalyze the synthesis of additional dinucleoside polyphosphates such as diadenosine 5',5'-P(1),P(5)-pentaphosphate and dAp(4)dA from the appropriate substrates, p(4)A and dATP, respectively. Formation of Ap(3)A from ATP and ADP was not observed with either At4CL2 variant. In all cases analyzed, (di)adenosine polyphosphate synthesis was either strictly dependent on or strongly stimulated by the presence of a cognate cinnamic acid derivative. The At4CL2 mutant enzyme K540L carrying a point mutation in the catalytic center that is critical for adenylate intermediate formation was inactive in both p(4)A and diadenosine 5',5',-P(1),P(4)-tetraphosphate synthesis. These results indicate that the cinnamoyl-adenylate intermediate synthesized by At4CL2 not only functions as an intermediate in coenzyme A ester formation but can also act as a cocatalytic AMP-donor in (di)adenosine polyphosphate synthesis.     

Interaction of myosin subfragment 1 with fluorescent ribose-modified nucleotides. A comparison of vanadate trapping and SH1-SH2 cross-linking

The environment near the ribose binding site of skeletal myosin subfragment 1 (S1) was investigated by use of two adenosine 5'-diphosphate analogues with fluorescent groups attached at the 2'- and 3'-hydroxyls of the ribose ring. We have compared steady-state and time-resolved fluorescent properties of the reversibly bound S1-nucleotide complexes and the complexes generated by N,N'-p-phenylenedimaleimide (pPDM) thiol cross-linking or vanadate (Vi) trapping. A new fluorescent probe, 2'(3')-O-[N-[2-[[[5-(dimethylamino)naphthyl]sulfonyl] amino]ethyl]carbamoyl]adenosine 5'-diphosphate (DEDA-ADP), which contains a base-stable carbamoyl linkage between the ribose ring and the fluorescent dansyl group, was synthesized and characterized. For comparison, we performed parallel experiments with 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (MANT-ADP) [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. Solute quenching studies indicated that both analogues bound reversibly to a single cleft or pocket near the ribose binding site. However, steady-state polarization measurements indicated that the probes were not rigidly bound to the protein. The quantum yields of both fluorophores were higher for the complexes formed after trapping with pPDM or Vi than for the reversibly bound complexes. Both DEDA-ADP and MANT-ADP, respectively, had nearly homogeneous lifetimes free in solution (3.65 and 4.65 ns), reversibly bound to S1 (12.8 and 8.6 ns), and trapped on S1 by pPDM (12.7 and 8.7 ns) or Vi (12.8 and 8.6 ns). In contrast to the quantum yields, the lifetimes were not increased upon trapping, compared to those of the reversibly bound states. These results suggested that static quenching in the reversibly bound complex was relieved upon trapping. Taken together, the results suggest that there was a conformational change near the ribose binding site upon trapping by either pPDM or Vi. On the basis of the quantum yield, lifetime, polarization, and solute accessibility studies, we could not detect differences between the S1-pPDM-nucleotide analog complex and the S1-Vi-nucleotide analogue complex for either analogue. Thus, previously observed differences with the adenine modified nucleotide analogue 1,N6-ethenoadenosine diphosphate (epsilon ADP) could not be detected with these ribose-modified probes, indicating that structural differences may be localized to the adenine binding site and not transmitted to the region near the ribose ring.     

Mapping adenosine cyclic 3',5'-phosphate binding sites on type I and type II adenosine cyclic 3',5'-phosphate dependent protein kinases using ribose ring and cyclic phosphate ring analogues of adenosine cyclic 3',5'-phosphate

A series of adenosine cyclic 3',5'-phosphate (cAMP) derivatives containing modifications or substitutions in either the 2',3',4', or 5' position or the phosphate were examined for their abilities to activate type I isozymes of cAMP-dependent protein kinase (PK I) from rabbit or porcine skeletal muscle and type II isozymes of cAMP-dependent protein kinase (PK II) from bovine brain and heart. The studies revealed that the activation of both PK I and PK II isozymes requires a 2'-hydroxyl group in the ribo configuration, a 3' oxygen in the ribo configuration, and a charged cyclic phosphate. The two isozymes appeared to differ in those portions of their respective cAMP-binding sites that are adjacent to the 4' position of the ribose ring and the 3' position, 5' position, and phosphate portion of the cyclic phosphate ring.     

Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS and the Rp and Sp isomers adenosine 3', 5'-monophosphodimethylamidate. cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)-cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.     

Introduction of alkynyl chains on C-8 of adenosine led to very selective antagonists of the A(3) adenosine receptor.

Some 8-alkynyladenosines were synthesized and evaluated for their adenosine receptor activity, utilizing radioligand binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity assays (A(2B)). Furthermore, the maximal induction of guanosine 5'-(gamma-thio)triphosphate ([35S]GTPgammaS) binding to G proteins and the inhibition of NECA-stimulated binding, in membranes of CHO cells which express the human A(3) receptor, were used to determine the intrinsic activity of these nucleosides at the A(3) adenosine receptor. The results showed that these new adenosine derivatives are very selective ligands for the A(3) receptor subtype and behave as adenosine antagonists, since they do not stimulate basal [35S]GTPgammaS binding, but inhibit NECA-stimulated binding. This is the first report that adenosine derivatives, with unmodified ribose moiety, are adenosine receptor antagonists.     

Metabolite gene regulation: imidazole and imidazole derivatives which circumvent cyclic adenosine 3'',5''-monophosphate in induction of the Escherichia coli L-arabinose operon

Imidazole, histidine, histamine, histidinol phosphate, urocanic acid, or imidazolepropionic acid were shown to induce the L-arabinose operon in the absence of cyclic adenosine 3',5'-monophosphate. Induction was quantitated by measuring the increased differential rate of synthesis of L-arabinose isomerase in Escherichia coli strains which carried a deletion of the adenyl cyclase gene. The crp gene product (cyclic adenosine 3',5'-monophosphate receptor protein) and the araC gene product (P2) were essential for induction of the L-arabinose operon by imidazole and its derivatives. These compounds were unable to circumvent the cyclic adenosine 3',5'-monophosphate in the induction of the lactose or the maltose operons. The L-arabinose regulon was catabolite repressed upon the addition of glucose to a strain carrying an adenyl cyclase deletion growing in the presence of L-arabinose with imidazole. These results demonstrated that several imidazole derivatives may be involved in metabolite gene regulation (23).     

A slowly dissociating form of the cell surface cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum.

Experiments using a phosphodiesterase-minus mutant of Dictyostelium discoideum indicate that ligand-induced loss of cell surface cyclic adenosine 3':5'-monophosphate binding sites (down regulation) can be evoked with concentrations of cyclic adenosine 3':5'-monophosphate as low as 10(-8) M. The loss of receptor sites is observed after 5 min of cell preincubation with cyclic adenosine 3':5'-monophosphate and can be as extensive as 75 to 80%. This decrease in binding sites is correlated with the appearance of a slowly dissociating cyclic adenosine 3':5'-monophosphate binding component. Radioactive cyclic adenosine 3':5'-monophosphate bound to this form of receptor cannot be competed for by nonradioactive cyclic adenosine 3':5'-monophosphate or adenosine 5'-monophosphate and is not accessible to hydrolysis by cyclic adenosine 3':5'-monophosphate phosphodiesterase. The extent of appearance of this binding component is dependent upon the concentration of cyclic adenosine 3':5'-monophosphate used to elicit the down regulation response and the temperature of the incubation medium.     

High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3',5'-ADP, 2',5'-ADP, 5'-ADP, U-2'-p and U-3'-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5'-ADP (Ki = 1.2 microM), adopts a syn conformation (in contrast to 3',5'-ADP and 2',5'-ADP, which adopt an anti), and it is the beta- rather than the alpha-phosphate group that binds to P1. 3',5'-ADP binds with the 5'-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2',5'-ADP. This inhibitor binds with either the 3' or the 5' phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2' versus 3') has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.     

A single mutation converts the nucleotide specificity of phenol sulfotransferase from PAP to AMP

Sulfotransferases (STs) catalyze all the known biological sulfonations, in which a sulfuryl group from a common sulfonate donor such as 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is transferred to a nucleophilic acceptor. In addition to PAPS, phenol sulfotransferase (PST), a member of the ST family, utilizes other nucleotides as substrates with much less catalytic efficiency [Lin, E. S., and Yang, Y. S. (2000) Biochem. Biophys. Res. Commun. 271, 818-822]. Six amino acid residues of PST have been chosen for mutagenesis studies on the basis of a model of PST and its sequence alignment with those of available cytosolic and membrane-anchored STs. Systematic analyses of the mutants reveal that Ser134 is important for the regulation of nucleotide specificity between 3'-phosphoadenosine 5'-phosphate (PAP) and adenosine 5'-monophosphate (AMP). Kinetic studies also indicate that Ser134 plays a key role in nucleotide binding (K(m)) but not in catalysis (kcat). Consequently, the catalytic efficiency (kcat/K(m)) of PST can be altered by 5 orders of magnitude with a mutation of Ser134. Moreover, the change in nucleotide specificity from PAP to AMP can be achieved by mutation of Ser134 to any of the following residues: Glu, Gln, Arg, and His. Roles of Lys44, Arg126, and Arg253, which interact directly with the 5'- and 3'-phosphate of PAP, were also investigated by mutagenesis and kinetic experiments. On the basis of these findings, we suggest that Ser134 is the key residue that enables PST to discriminate PAP from AMP.