Molecular Recognition of Adenosine Deaminase: 15N NMR Studies

The elucidation of the molecular recognition of adenosine deaminase (ADA), the interpretation of the catalytic mechanism, and the design of novel inhibitors are based mostly on data obtained for the crystalline state of the enzyme. To obtain evidence for molecular recognition of the physiologically relevant soluble enzyme, we studied its interactions with the in situ formed inhibitor, 6-OH-purine riboside (HDPR), by 1D-¹⁵N- and 2D-(¹H-¹⁵N)- NMR using the labeled primary inhibitor [¹⁵N₄]-PR. We synthesized both [¹⁵N₄]-PR and an [¹⁵N₄]-HDPR model, from relatively inexpensive ¹⁵N sources. The [¹⁵N₄]-HDPR model was used to simulate H-bonding and possible Zn²⁺-coordination of HDPR with ADA. We also explored possible ionic interactions between PR and ADA by ¹⁵N-NMR monitored pH-titrations of [¹⁵N₄]-PR. Finally, we investigated the [¹⁵N₄]-PR-ADA 1:1 complex by 2D-(¹H-¹⁵N) NMR. We found that HDPR recognition determinants in ADA do not include any ionic-interactions. HDPR N1 H is an H-bond acceptor, and not an H-bond donor. Despite the proximity of N7 to the Zn²⁺-ion, no coordination occurs; instead, N7 is an H-bond acceptor. We found an overall agreement between the crystallographic data for the crystallized ADA:HDPR complex and the ¹⁵N-NMR signals for the corresponding soluble complex. This finding justifies the use of ADA's crystallographic data for the design of novel inhibitors.     

15N-NMR studies of Methanobacterium thermoautotrophicum: comparison of assimilation of different nitrogen sources

Methanobacterium thermoautotrophicum can utilize glutamine and urea as well as ammonia as the sole nitrogen source during growth on H2 and CO2. High-field 15N-NMR has been used to compare the assimilation of these different nitrogen sources by this organism. The 15N-NMR spectra of extracts of cells grown in media containing [delta-15N]glutamine as the nitrogen source show that the glutamine amide nitrogen is rapidly converted to glutamate. The 15N-NMR spectra of cell extracts from cells grown on [15N]urea show a marked increase in the labeling of the alpha-NH2 of glutamate concurrent with a decrease in the urea resonance. These two nitrogen sources do not show the metabolic shift to alanine as the major resonance in stationary phase as is seen with 15NH4Cl. This behavior is discussed in terms of the enzymes of nitrogen metabolism.     

A 15N-NMR study of isolated brain in portacaval-shunted rats after acute hyperammonemia.

Acute hyperammonemia was induced by 15NH4+ infusion in portacaval-shunted (PCS) and control rats to investigate its effects on cerebral metabolism of glutamine, glutamate and gamma-aminobutyrate. Cerebral 15N-metabolites were observed by 15N-NMR spectroscopy in the ex vivo brain, removed in toto at the end of infusion. Key 15N-metabolites in the brain and liver were quantitated and their specific activities measured by NMR and biochemical assays in perchloric acid extracts of the freeze-clamped organs. In the ex vivo brain, [gamma-15N]glutamine, present at tissue concentrations of 3-5 mumol/g with 15N enrichment of 36-48%, was observable within 6-13 min of data acquisition. [alpha-15N]glutamine/glutamate, each present at 0.5-1 mumol/g (approx. 10% enrichment), were observed in 27 min. The results demonstrate the feasibility of observing these cerebral metabolites by 15N-NMR within a physiological time scale. In a rat pretreated with glutamine synthetase inhibitor, L-methionine DL-sulfoximine, cerebral [15N]gamma-aminobutyrate was observed after 910 min. In PCS rats, decreased 15NH4+ removal in the liver was accompanied by formation of approx. 2-fold higher concentration of cerebral [gamma-15N]glutamine relative to that in weight-matched controls. The result suggests that increased diffusion of blood-borne 15NH3 into the brain led to increased [gamma-15N]glutamine synthesis in astrocytes as well as ammonia-mediated inhibition of glutaminase.     

Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

Direct detection of N-H[...]N hydrogen bonds in biomolecules by NMR spectroscopy

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active (15)N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson-Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N-H donor and N acceptor moieties. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor in a single experiment. The size of the scalar couplings can be related to H-bond geometries and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight < or = approximately 25 kDa. A C15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure.     

1H-15N-NMR studies of bacteriorhodopsin Halobacterium halobium. Conformational dynamics of the four-helical bundle.

Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1-71), B1 (residues 1-155) and BP2 (residues 163-231) were investigated. By this procedure, all observed 1H-15N cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross-peaks from four (C, D, E and F) helical bundles (79-189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.     

A 15N NMR study on D-lysine metabolism in Neurospora crassa

The metabolic relationship of D-lysine, L-lysine, and L-pipecolic acid has been investigated in Neurospora crassa. Kinetic experiments show that radioactivity from D-lysine is efficiently incorporated into L-pipecolic acid and that this metabolite is converted to L-lysine. The alpha-amino group from D-[alpha-15N]lysine is lost in the course of its conversion to L-pipecolic acid and is trapped by pyruvate and alpha-keto glutarate to give L-[alpha-15N]alanine and L-[alpha-15N]glutamic acid. These amino acids are devoid of any label, however, when D-[epsilon-15N]lysine is applied to the fungus. As determined by mass and 15N NMR spectrometry the label from D-[epsilon-15N]lysine migrate via L-pipecolic acid into the alpha position of L-lysine, i.e. D-[epsilon-15N]lysine is converted to L-[alpha-15N]lysine. L-Pipecolic acid functions as an intermediate in this conversion.     

Stabilization of the non-canonical adenine-adeninium base pair by N(7) coordination of Zn(II)

A new zinc (II) compound with 9-ethyladenine (9-EtA) of formula [Zn(9-EtA-N7)Cl(3)](9-EtAH) has been synthesized and characterized by X-ray diffraction. Its X-structure consists of an Zn(II) anionic complex and 9-ethyladeninium as counteranion. The Zn(II) complex shows a distorted tetrahedral geometry in which three Cl and an 9-EtA coordinates through N(7) position are the ligands. An indirect chelation via intramolecular H-bond between N(6)H and an Cl ligand is present in the complex. The network of [Zn(9-EtA-N7)Cl(3)](9-EtAH) shows interesting features. Thus, self-association of coordinated adenine-adeninium takes place by H-bonding of N(6)-H...N(1) and N(6)-H...N(7), leading to a polymeric ribbon-like 1D supramolecular arrangement. Ab initio calculations have been applied in order to study the stability of the adenine-adeninium interaction due to the coordination of the Zn(II) to the N(7) position and to compare experimental and theoretical structural data.     

Two-dimensional NMR studies of staphylococcal nuclease: evidence for conformational heterogeneity from hydrogen-1, carbon-13, and nitrogen-15 spin system assignments of the aromatic amino acids in the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined 1H, 13C, and 15N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3',5'-bisphosphate at pH 5.1 (H2O) or pH 5.5 (2H2O). Samples of recombinant nuclease were labeled with 13C or 15N as appropriate to individual NMR experiments: uniformly with 15N (all sites to greater than 95%), uniformly with 13C (all sites to 26%), selectively with 13C (single amino acids uniformly labeled to 26%), or selectively with 15N (single amino acids uniformly labeled to greater than 95%). NMR data used in the analysis included single-bond and multiple-bond 1H-13C and multiple-bond 1H-15N correlations, 1H-13C single-bond correlation with Hartmann-Hahn relay (1H[13C]SBC-HH), and 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE). The aromatic protons of the spin systems were identified from 1H[13C]SBC-HH data, and the nonprotonated aromatic ring carbons were identified from 1H-13C multiple-bond correlations. Sequence-specific assignments were made on the basis of observed NOE relay connectivities between assigned 1H alpha-13C alpha or 1H beta-13C beta direct cross peaks in the aliphatic region [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88-101] and 1H delta-13C delta direct cross peaks in the aromatic region of the 1H[13C]SBC-NOE spectrum. The His121 1H delta 2 resonance, which has an unusual upfield shift (at 4.3 ppm in the aliphatic region), was assigned from 1H[13C]SBC, 1H[13C]MBC, and 1H[15N]MBC data. Evidence for local structural heterogeneity in the ternary complex was provided by doubled peaks assigned to His46, one tyrosine, and one phenylalanine. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3',5'-bisphosphate [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555; Loll, P. J., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183-201]. The unusual chemical shift of His121 1H delta 2 is consistent with ring current calculations from either X-ray structure.     

Crystal-state structural analysis of two gamma-lactam-restricted analogs of Pro-Leu-Gly-NH2

The crystal structures of two analogs of Pro-Leu-Gly-NH2 (1), containing a gamma-lactam conformational constraint in place of the -Leu-Gly- sequences, are described. The highly biologically active (S,R)-diastereomer 2a is semi-extended at the C-terminus, with the N-terminal Pro residue in the unusual "C5" conformation [psi 1 = -0.8(15) degrees] stabilized by a (peptide)N-H...N(amino) intramolecular H-bond [the N(3)...N(4) separation is 2.687(11)A]. Conversely, the N,N'-isopropylidene aminal trihydrate of the (S,S)-diastereomer 2b, compound 3, adopts a beta-bend conformation at the C-terminus, as already reported for 1. However, the backbone torsion angles [phi 2 = 57.4(4), psi 2 = -129.9(3) degrees; psi 3 = -92.3(4), phi 3 = 6.4(5) degrees] lie close to the values expected for the corner residues of an ideal type-II' beta-bend. A weak intramolecular 4----1 H-bond is seen between the Gly carboxyamide anti-NH and Pro C = O groups. In the newly formed 2,2,3,4-tetraalkyl-5-oxo-imidazolidin-1-yl moiety the psi 1 torsion angle is 12.9(4) degrees and the intramolecular N(3)...N(4) separation is 2.321(4)A.     

Resolution, molecular structure and biological activities of the D- and L-enantiomers of potent anti-implantation agent, DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran.

Compound 1 (DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran, CDRI 85/287) a potent anti-estrogen and anti-implantation agent has been successfully resolved into its pure D- and L-enantiomers. Biological studies showed L-enantiomer to be the active form, exhibiting a fivefold higher receptor affinity for the rat uterine cytosolic estrogen receptor, 100% contraceptive efficacy at 1.3 mg/kg dose in single day schedule and 89% inhibition of estradiol induced increase of uterine weight at its contraceptive dose. The absolute stereochemistry determined by X-ray crystallographic analysis showed that the L-enantiomer has 2R configuration at its asymmetric centre.     

A nuclear-magnetic-resonance study of the binding of novel N-hydroxybenzenesulphonamide carbonic anhydrase inhibitors to native and cadmium-111-substituted carbonic anhydrase

Various ring- and nitrogen-substituted benzenesulphonamides have been prepared and tested as potential inhibitors of carbonic anhydrase. N-Methoxysulphonamides showed no inhibitory activity, as predicted by the classic work of Krebs on N-substituted inhibitors. By contrast, N-hydroxysulphonamides proved to be very effective inhibitors of carbonic anhydrase. Using 111Cd-NMR it has been possible to analyse the molecular interaction of 4-fluoro-N-hydroxybenzenesulphon[15N]amide, with 111Cd-substituted bovine carbonic anhydrase. A large cadmium-111:nitrogen-15 spin-coupling shows that this inhibitor is directly bound to the metal via its nitrogen rather than through an oxygen atom. The mode of this binding is similar to that for the unsubstituted sulphonamide inhibitor, 4-fluorobenzenesulphon[15N]amide. The 111Cd-chemical shift of the signal for the inhibited enzyme shows that the N-hydroxysulphonamide is bound as its anion. From the relative intensities of free and complexed enzyme signals it can be deduced that the cadmium enzyme complex with the N-hydroxysulphonamide has a longer life-time than that formed with the unsubstituted sulphonamide. By contrast, native zinc-containing bovine carbonic anhydrase shows similar I50 values with both of these sulphonamides. Attempts to monitor the binding using 15N-NMR were unsuccessful, possibly due to a very long relaxation time for the nitrogen nucleus in the N-hydroxysulphonamide when bound to the enzyme leading to loss of the 15N signal.     

15N- and 1H-NMR investigations of the active-site amino acids in semisynthetic RNase S' and RNase A

Extensive 15N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively 15N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The 15N resonances of the side chains of lysine-7 (N epsilon), glutamine-11 (N gamma), and histidine-12 (N pi, tau) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and 5'AMP. The analysis of the 15N-1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the N tau signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange. For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 1H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8-6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.     

15N-CIDNP investigations during tryptophan, N-acetyl-L-tryptophan, and melatonin nitration with reactive nitrogen species

Tryptophan and melatonin are nitrated by peroxynitrite; tryptophan residues in proteins are susceptible to attack by reactive nitrogen species. Nitrated tryptophan might therefore be used as a biomarker for the involvement of reactive species derived from nitrogen oxide in a variety of pathophysiological conditions. The radical character of the tryptophan (Trp) and N-acetyl-L-tryptophan (N-AcTrp) nitration with peroxynitrite is shown using (15)N-CIDNP. During the decay of peroxynitrite-(15)N in the presence of Trp at pH 5 in the probe of a (15)N-NMR spectrometer, the (15)N-NMR signals of various nitrated tryptophans ((15)NO(2)-Trp) show emission (E). The effects are built up in radical pairs [Trp( radical), 15NO2 ](F) formed by diffusive encounters of radicals 15NO2 and Trp( radical) generated during decay of peroxynitrite-(15)N in the presence of Trp. Similar (15)N-CIDNP effects are observed during reaction of Trp and/or N-AcTrp using the nitrating systems H(15)NO(3), H(15)NO(4) and H(2)O(2)/15NO2 /HRP, which are also built up in radical pairs [Trp, 15NO2 ](F). During nitration of melatonin (Mel) with peroxynitrite-(15)N and H(15)NO(4), the (15)N-NMR signal of 4-nitromelatonin (4-(15)NO(2)-Mel) shows emission arising from radical pairs [Mel, 15NO2 ](F) which are formed in an analogous manner.     

Characterization of peptidoglycan stem lengths by solid-state 13C and 15N NMR

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.     

Comparison of inhibitory activity of isomeric triazolopyridine derivatives towards adenosine receptor subtypes or do similar structures reveal similar bioactivities?

The synthesis of an array of 8-amino-2-aryl-[1,2,4]triazolo[1,5-a]pyridine-6-carboxyl amide derivatives is described for the first time. A subset of 20 derivatives were compared to their isomeric 5-amino-2-aryl-[1,2,4]triazolo[1,5-a]pyridine-7-carboxyl amide counterparts with regard to their potential to inhibit the human adenosine 2a (hA2a) receptor and their selectivity against the human adenosine 1 (hA1) receptor. Based on the analysis of H-bond donor/acceptor capabilities of the isomeric triazolopyridine pairs it can be concluded that the H-bond donor strength of the free amino functionality is the main determinant for hA2a inhibitory activity and hA1 selectivity.     

Zinc-substituted Desulfovibrio gigas desulforedoxins: resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.     

L-[3H]Adenosine, a New Metabolically Stable Enantiomeric Probe for Adenosine Transport Systems in Rat Brain Synaptoneurosomes

The stereoenantimers D-[3H]adenosine and L-[3H]adenosine were used to study adenosine accumulation in rat cerebral cortical synaptoneurosomes. L-Adenosine very weakly inhibited rat brain adenosine deaminase (ADA) activity with a Ki value of 385 microM. It did not inhibit rat brain adenosine kinase (AK) activity, nor was it utilized as a substrate for either ADA or AK. The rate constants (fmol/mg of protein/s) for L-[3H]adenosine accumulation measured in assays where transport was stopped either with inhibitor-stop centrifugation or with rapid filtration methods were 82 +/- 14 and 75 +/- 10, respectively. Using the filtration method, the rates of L-[3H]adenosine accumulation were not significantly different from the value of 105 +/- 15 fmol/mg of protein/s measured for D-[3H]adenosine transport. Unlabeled D-adenosine and nitrobenzylthiolnosine, both at a concentration of 100 microM, reduced the levels and rates of L-[3H]adenosine accumulation by greater than 44%. These findings suggest that L-adenosine, a metabolically stable enantiomeric analog, and the naturally occurring D-adenosine are both taken up by rat brain synaptoneurosomes by similar processes, and as such L-adenosine may represent an important new probe with which adenosine uptake may be studied.     

Effective switch-on fluorescence sensing of zinc(II) ion by 8-aminoquinolino-beta-cyclodextrin/adamantaneacetic acid system in water

A water-soluble 8-aminoquinolino-beta-cyclodextrin/1-adamantaneacetic acid (1/ADA) system is prepared in situ and exhibits a unique switch-on fluorescence response to Zn(2+) over other common metal ions. Spectrophotometric studies demonstrate that this system can strongly coordinate Zn(2+) through a cyclodextrin/substrate/metal triple recognition mode, and the resulting 1/ADA/Zn(2+) ternary complex emits the blue-green fluorescence (lambda=490nm) that can be easily distinguished by eyes in aqueous solution. Significantly, the switch-on fluorescence response of 1/ADA to Zn(2+) is barely affected by various metal ions except Cu(2+). As a result, this system can behave as an efficient supramolecular fluorescence sensor for Zn(2+) in water.     

Adenosine modulation of D-[3H]aspartate release in cultured retina cells exposed to oxidative stress

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.