Interaction of metal ions with nucleic acids. Interaction of copper(II) with adenosine and its derivatives.

The interaction of copper(II) with adenosine, 2'-deoxyadenosine, 1-methyladenosine, 7-deazaadenosine and AMP was studied by spectroscopic and magnetochemical methods. In non-aqueous medium, copper(II) interacts with adenosine and AMP at N-7 and N-1, and with 1-methyladenosine at N-7 and N-3. The copper ion is not bound to the NH2 group. In aqueous solution, copper(II) interacts both with N-7 and N-1 of adenosine, and in AMP additionally with the phosphate group. The interaction of copper(II) with the heterocyclic part, but not withthe phosphate group, is dependent on the extent of protonation of the molecular. A crystalline AMP-copper(II) complex [Cu(C10H12N5O7P).(H2O)2] was obtained; the phosphate group and probably N-7 are involved in the complex formation.     

A Comparative study of Fe(II) and Fe(III) interactions with DNA duplex: major and minor grooves bindings

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.     

Respective role of each of the purine N-6 amino groups of 5'-O-triphosphoryladenylyl(2'----5')adenylyl(2----5')adenosine in binding to and activation of RNase L

We have synthesized a series of 2-5A (ppp5'-A2'p5'A2'p5'A) analogs in which each adenosine residue has been sequentially replaced by inosine: viz., ppp5'I2'p5'A2'p5'A, ppp5'A2'p5'I2'p5'A, and ppp5'A2'p5'A2'p5'I. These transformations enabled us to delineate the role of each of the three purine N-6 amino groups of 2-5A in determining oligonucleotide binding to and activation of the 2-5A-dependent endoribonuclease, RNase L. With the RNase L activity of both mouse L cells and human Daudi lymphoblastoid cells, we found that the N-6 amino group of the first adenosine nucleotide residue (from the 5'-terminus) is of crucial importance in determining binding to the endonuclease; however, removal of the N-6 amino moieties of the second or third adenosine nucleotide residues resulted in only a minimal decrease in binding to the endonuclease. On the other hand, conversion of the third adenosine residue to inosine effected a dramatic (10,000-fold compared to 2-5A) loss in ability to activate the nuclease; however, execution of the same N-6 amino group conversion at either the first or second adenosine residue did not cause a major change in nuclease activation ability when the accompanying decreased endonuclease binding was considered. These results clearly demonstrate that the N-6 amino group of the first adenosine residue of 2-5A is critical in RNase L binding whereas the N-6 amino function of the third adenosine residue of 2-5A is crucial for the activation of RNase L.     

Interaction of purine nucleotides with cobalt-hexammine, cobalt-pentammine and cobalt-tetrammine cations. Evidence for the rigidity of adenosine and flexibility of guanosine and deoxyguanosine sugar conformations.

The interaction of adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP) and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) with the [Co(NH3)6]3+, [Co(NH3)5Cl]2+ and [Co(NH3)4Cl2]+ cations has been investigated in aqueous solution with metal/nucleotide ratios (r) of 1/2, 1 and 2 at neutral pH. The solid complexes have been isolated and characterized by FT-IR and 1H-NMR spectroscopy. The complexes are polymeric in nature both in the crystalline solid and aqueous solution. The binding of the cobalt-hexammine cation is indirectly (via NH3) through the N-7 and the PO3(2-) groups of the AMP and via O-6, N-7 and the PO3(2-) of the GMP and dGMP anions (outer-sphere). The cobalt-pentammine and cobalt-tetrammine bindings are through the phosphate groups (inner-sphere) and the N-7 site (outer-sphere) of these nucleotide anions. The ribose moiety shows C2'-endo/anti conformation, in the free AMP and GMP anions as well as in the cobalt-ammine-AMP complexes, whereas a mixture of teh C2'-endo/anti and C3'-endo/anti sugar puckers were observed for the Co(NH3)6-GMP, Co(NH3)5-GMP and a C3'-endo/anti conformer for the Co(NH3)4-GMP complexes. The deoxyribose showed an O4'-endo/anti conformation for the free dGMP anion and a C3'-endo/anti for the Co(NH3)6-dGMP, Co(NH3)5-dGMP and Co(NH3)4-dGMP complexes.     

An FTIR Spectroscopic Study of Calf-thymus DNA Complexation with Al(III) and Ga(III) Cations

Abstract

The interaction of calf-thymus DNA with trivalent Al and Ga cations, in aqueous solution at pH =6–7 with cation/DNA(P) (P=phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4 and 1/2 was characterized by Fourier Transform infrared (FTIR) difference spectroscopy.

Spectroscopic results show the formation of several types of cation-DNA complexes. At low metal ion concentration (r=l/80, 1/40), both cations bind mainly to the backbone PO₂ group and the guanine N-7 site of the G-C base pairs (chelation). Evidence for cation chelate formation comes from major shifting and intensity increase of the phosphate antisymmetric stretch at 1222 cm-¹ and the mainly guanine band at 1717 cm¹. The perturbations of A-T base pairs occur at high cation concentration with major helix destabilization. Evidence for cation binding to A-T bases comes from major spectral changes of the bands at 1663 and 1609 cm-¹ related mainly to the thymine and adenine in-plane vibrations. A major reduction of the B-DNA structure occurs in favor of A-DNA upon trivalent cation coordination.     

Topography of the interaction of recA protein with single-stranded deoxyoligonucleotides

recA protein, in the presence of adenosine 5'-(gamma-thio)triphosphate, formed stable complexes with single-stranded deoxyoligonucleotides between 9 and 20 residues in length but not with those 8-residues long. The binding of recA protein to a 15-mer and 20-mer completely protected the sugar-phosphate backbone of the nucleic acid from digestion by pancreatic deoxyribonuclease I and protected the 5'-terminal phosphate from cleavage by calf intestinal alkaline phosphatase. Ethylation of the phosphate backbone at any position by ethylnitrosourea blocked the binding of recA protein to the 15-mer but not to the 20-mer. Ethylation of phosphates near the ends of the 15-mer interfered less, suggesting a minimum binding site requirement. In contrast to the protection of the nucleic acid backbone, recA protein did not protect the N-7 position of guanine or the N-3 position of adenine from methylation by dimethyl sulfate, but rather enhanced the methylation of guanine. These results indicate that recA protein binds primarily to the phosphate backbone of single-stranded DNA, leaving the bases free for homologous pairing. We present a model for the organization of the presynaptic filament.     

Binding of oxovanadium ions to the major and minor grooves of DNA duplex: stability and structural models.

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses that are relative to the occupational exposure. Oxovanadium compounds also exert preventive effects against chemical carcinogenesis in animals and form complexes with DNA in vivo. This study was designed to examine the interaction of calf-thymus DNA with VO2+ and VO3 ions in aqueous solution at physiological pH, with a constant DNA concentration of 12.5 mmol/L and vanadium-DNA (phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared difference spectroscopy were used to determine the cation binding site, the binding constant, the helix stability, and DNA conformation in the oxovanadium-DNA complexes. Structural analysis showed that VO2+ binds DNA through guanine and adenine N-7 atoms and the backbone PO2 group with apparent binding constants of KG = 8.8 x 10(5) (mol/L)-1 and KA = 3.4 x 10(5) (mol/L)-1. The VO3 shows weaker binding through thymine, adenine, and guanine bases, with K = 1.9 x 10(4) (mol/L)-1 and no interaction with the backbone phosphate group. A partial B-to-A DNA transition occurred upon VO-DNA complexation, while DNA remains in the B-family structure in the VO3 complexes.     

Structure-affinity relationships of the affinity of 2-pyrazolyl adenosine analogues for the adenosine A2A receptor

The structure-affinity relationships of two novel 2-substituted adenosine series containing a substituted pyrazole attached at the N-1 or C-4 position for the adenosine (ADO) A2A receptor are described. Compounds in the 2-(N-1-pyrazolyl) adenosine series IV provided the highest affinity for the ADO A2A receptor as compared to the 2-(C-4-pyrazolyl) series V. The main structural differences between the two series point to the N-1 nitrogen of series IV imparting more favorable binding interactions with the receptor than those of series V.     

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.     

2,4,6-Trinitrobenzenesulfonate labels an essential amino group near the bound phosphate at the catalytic site of mitochondrial F1-ATPase

The amino reagent 2,4,6-trinitrobenzenesulfonate (TNBS) was found to inactivate mitochondrial F₁-ATPase through covalent labeling, which was not reversed by dithiothreitol. The observed rate of inactivation was retarded by inorganic phosphate, but enhanced by prior labeling of F₁ with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1). These observations are consistent with the presence of an essential amino group near the bound inorganic phosphate at the catalytic site of F₁. A comparison of the observed protection of F₁ from NBD-C1 and 5′-p-fluorosulfonyl-benzoyladenosine (FSBA) respectively by inorganic phosphate and by 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) suggests that NBD-C1 labels an essential Tyr residue in the positively charged locus for binding the polyphosphate end of ATP, and that FSBA labels an essential Tyr residue in the more hydrophobic locus for binding the adenosine moiety of ATP at the catalytic site of F₁.     

Structure of pyrimidine 5'-nucleotidase type 1. Insight into mechanism of action and inhibition during lead poisoning

Eukaryotic pyrimidine 5'-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5'-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35 A resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25 A, 2) phosphoenzyme intermediate analog at 2.30 A, 3) product-transition complex analog at 2.35 A, and 4) product complex at 2.1A resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35 A) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.     

Conformation of complexes of thiamin pyrophosphate with divalent cations as studied by nuclear magnetic resonance spectroscopy.

The binding of Ni-2+ and Mn-2+ to thiamin phosphate and thiamin pyrophosphate (thiamin-PP) has been compared with the binding of these ions to oxythiamin phosphate and oxythiamin pyrophosphate, analogues of thiamin in which the C-4 amino group has been replaced by an -OH group. The replacement of the NH2 group results in reduced basicity of N-1 of the pyrimidine ring of oxythiamine derivatives. The effects of pD, ligand concentration, and temperature on the binding of metal ions to N-1 have been studied by observing the metal ion-induced shifting and broadening of the C-6-H signal of these compounds. The results indicate the following: (a) the metal ion is held near N-1, resulting in a "folded" conformation, because of a favorable bonding interaction between N-1 and the metal ion rather than for general conformational reasons alone; and (b) the amount of "folded" conformation present in the different pyrophosphate complexes at neutral pH follows the order: Ni-2+-thiamin-PP greater than Mn-2+-thiamin-PP greater than Mn-2+-oxythiamin-PP and Ni-2+-oxythiamin-PP It is concluded that the strength of the metal ion-pyrimidine interaction in the "folded" conformation depends strongly both on the coordination affinity of the metal ion and on the basicity of N-1. Since the interaction of the phosphate-bound metal ion with the pyrimidine ring in the Mg-2+-thiamin-PP complex is probably weaker than the corresponding interaction in the Mn-2+-thiamin-PP complex, these results predict that the Mg-2+-thiamin-PP complex in solution, at neutral pH, exists predominantly in an "unfolded" conformation.     

Interaction of adenylic acid with alkaline earth metal ions in the crystalline solid and aqueous solution. Evidence for the sugar C'2-endo/anti, C'3-endo/anti and C'4-exon/anti conformational changes

The reaction of adenosine 5'-monophosphoric acid (H2-AMP) with the alkaline earth metal ions has been investigated in aqueous solution at neutral pH. The solid salts of Mg-AMP.5H2O, Ca-AMP.6H2O, Sr-AMP.7H2O and Ba-AMP.7H2O were isolated and characterized by Fourier transform infrared, 1H-NMR spectroscopy and X-ray powder diffraction measurements. Spectroscopic and other evidence showed that the Sr-AMP.7H2O and Ba-AMP.7H2O are isomorphous, whereas the Mg-AMP.5H2O and Ca-AMP.6H2O are not similar. The Mg2+ binding is through the N-7 (inner-sphere) and the phosphate group (outer-sphere via H2O), while the Ca2+ binds to the phosphate group (inner-sphere) and to the base N-7 site (outer-sphere through H2O). The Sr2+ and Ba2+ bind to H2O molecules, H-bonding to the N-7, N-1 and the phosphate group (outer-sphere). In aqueous solution, an equilibrium between the inner- and outer-sphere metal ion bindings can be established. The sugar moiety exhibited C'2-endo/anti conformation, in the free H2-AMP acid and the magnesium salt, C'3-endo/anti in the calcium salt and unusual C'4-exo/anti, in the strontium and barium salts.     

The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.     

Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate.

2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively. Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP. In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained. The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase. All compounds under study were found to be activators of the enzyme. The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect. All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase. The inhibition was competitive with respect to cyclic AMP in all cases. All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme. These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.     

"Single Addition" substrates for the synthesis of specific oligoribonucleotides with polynucleotide phosphorylase. Synthesis of 2'-(alpha-methoxyethy) nucleoside 5'-diphosphates.

A number of synthetic methods for the preparation of the 2-O-(alpha-methoxyethyl) derivatives of the 5-diphosphates of adenosine, cytidine, guanosine, and uridine have been studied in order to provide nucleotide substrates that can be applied to the synthesis of specific oligoribonucleotides using polynucleotide phosphorylase. The reaction of nucleoside 5-diphosphates with methyl vinyl ether for a limited time produces low yields of the corresponding 2-O-(alpha-methoxyethyl) derivatives because the rate of methoxyethylation of the 3-hydroxyl groups. A study of the rates of acidic hydrolysis of alpha-methoxyethyl groups in the 2 and 3 positions of nucleosides and nucleotides has been made, and the results obtained form the basis of a more efficient method for the synthesis of the blocked nucleoside diphosphates. The method involves the reaction of nucleoside 5-diphosphates with methyl vinyl ether to give the corresponding 2,3-di-O-(alpha-methoxyethyl)nucleoside 5-diphosphates, and exploits the fact that, in the acidic hydrolysis of these derivatives, the rate of removal of the 3-methoxyethyl group is about twice that of the group in the 2 position. Alternative syntheses were based on the phosphorylation of methoxyethylated nucleosides and nucleotides. The derivatives, 2-O- and 2,3-di-O-(alpha-methoxyethyl)uridine, were prepared by the methoxyethylation of 3,5-di-O-acetyluridine and 5-O-acetyluridine followed by removal of the acetyl groups. The corresponding guanosine derivatives were made by the synthetic routes: (i) guanosine leads to O-2,O-3,O-5,N-2-tetrabenzoylguanosine leads to 2-N-benzoylguanosine leads to O3-acetyl-N-2,O5-dibenzoylguanosine leads to 2-O-(alpha-methoxyethyl)guanosine, and (ii) 2,3-O-isopropylideneguanosine leads to N-2,O5-diacetyl-2,3-O-isopropylideneguanosine leads to N-2,O-5-diacetylguanosine leads to 2,3-di-O-(alpha-methoxyethyl)guanosine. These methoxyethylated nucleosides were converted to the corresponding 5-phosphates by reaction with cyanoethyl phosphate and dicyclohexylcarbodiimide, and then to the corresponding 5-diphosphates by subsequent reaction with 1,1-carbonyldiimidazole and inorganic phosphate.     

Effects of the allosteric modulator SCH-202676 on adenosine and P2Y receptors

The G protein-coupled receptor allosteric modulator SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine), which affects a wide range of structurally unrelated G protein-coupled receptors, has highly divergent effects on purine receptors. SCH-202676 inhibited radioligand binding to human adenosine A(1), A(2A), and A(3) receptors (IC(50) = 0.5-0.8 microM) and affected dissociation kinetics, but at the human P2Y(1) nucleotide receptor it had no effect. SCH-202676 (10 microM) selectively accelerated agonist dissociation at adenosine A(3) receptors and either slowed (adenosine A(1) receptors) or accelerated (adenosine A(2A) receptors) antagonist dissociation. Thus, SCH-202676 differentially modulated A(1), A(2A), and A(3) receptors as well as agonist- and antagonist-occupied receptors.     

13C magnetic resonance investigation of mercury (II) binding to nucleosides and thiolated nucleosides in dimethyl sulfoxide.

Natural abundance, proton-decoupled 13C magnetic resonance spectroscopy is shown to be a useful technique for identifying the mercury (II) binding sites on nucleosides and especially thiolated nucleosides. Measurements made on dimethyl sulfoxide-d6 solutions, 0.5 M in nucleoside and 0.15 M in mercury, reveal that both CH3 HgCl and HgCl2 bind principally to the sulfur atoms of s6 Guo and s8 Guo. The 13C NMR spectra of the unthiolated nucleosides in the presence of excess (4:1) mercury reveal that HgCl2 binds to N-3 of cytidine, to more than one site on adenosine and guanosine, but not strongly to uridine. Excess HgCl2 shifts the thiocarbonyl carbon atoms in s6 Guo and s8 Guo approx. 16 ppm upfield compared to the free nucleosides, and there is evidence for additional coordination to N-7 of s6 Guo. Binding to the ribose hydroxyl groups is clearly ruled out. At least in these instances, 13C NMR proves to be useful for assigning the mercury (II) binding sites, complementing the results of proton magnetic resonance studies. Proton NMR data for the binding of CH3 HgCl and HgCl2 to s6 Guo and s8 Guo are also presented.     

Interaction between adenosine A 2B-receptors and alpha2-adrenoceptors on the modulation of noradrenaline release in the rat vas deferens: possible involvement of a group 2 adenylyl cyclase isoform

In the prostatic portion of rat vas deferens, activation of adenosine A 2B-receptors, beta2-adrenoceptors, adenylyl cyclase or protein kinase A caused a facilitation of noradrenaline release. Blockade of alpha2-adrenoceptors with yohimbine (1 microM) attenuated the facilitation mediated by adenosine A 2B-receptors and by direct activation of adenylyl cyclase with forskolin but not that mediated by beta2-adrenoceptors or by direct activation of protein kinase A with 8-bromoadenosine-3',5'-cyclicAMP. The adenosine A 2B- and the beta2-adrenoceptor-mediated facilitation was prevented by the adenylyl cyclase inhibitors, 2',5'-dideoxy-adenosine (3 microM) and 9-cyclopentyladenine (100 microM), at concentrations that also attenuated the release enhancing effect of forskolin, but were not changed by the phospholipase C inhibitor 1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122, 1 microM). Facilitation of noradrenaline release mediated by adenosine A 2B-receptors was also attenuated by activation of protein kinase C with the phorbol ester 12-myristate 13-acetate (1 microM) and by inhibition of Gbetagamma subunits with an anti-betagamma peptide; facilitation mediated by beta2-adrenoceptors was mainly attenuated by the calmodulin inhibitor calmidazolium (10 microM) and by the calmodulin kinase II inhibitor (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzene-sulfonamide phosphate (KN-93, 5 microM). The results suggest that adenosine A 2B- but not beta2-adrenoceptor-mediated facilitation of noradrenaline release is enhanced by an ongoing activation of alpha2-adrenoceptors. They further suggest that adenosine A 2B-receptors and beta2-adrenoceptors are coupled to distinct adenylyl cyclase isoforms what may explain the different influence of alpha2-adrenoceptor signalling pathway on the facilitatory effects mediated by the two adenylyl cyclase coupled receptors.     

The Adenosine Receptors Present on the Plasma Membrane of Chromaffin Cells Are of the A2b Subtype

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.