Ligand Binding Properties of Bovine Liver Adenosine Kinase

Abstract

Bovine liver adenosine kinase displays a characteristic intrinsic fluorescence due to 3 tryptophans/mol. This fluorescence is very sensitive to ligand binding and was used to characterize the ligand binding sites of the enzyme. ADP or ATP showed a monophasic saturation curve consistent with the existence of one binding site. In contrast, adenosine and AMP gave biphasic saturation curves suggesting the existence of at least two binding sites with high and low affinity. These binding sites were further characterized by studying the complexation of adenosine kinase with O-(N-methylanthraniloyl)adenosine nucleoside or nucleotide analogues.     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

Exogenous adenosine activates A2A adenosine receptor to inhibit RANKL-induced osteoclastogenesis via AP-1 pathway to facilitate bone repair

Background

Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms.

Methods and results

The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses.

Conclusions

These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.

     

Modes of Binding of 2′-AMP to RNase T1 A Computer Modeling Study

Abstract

The modes of binding of adenosine 2′-monophosphate (2′-AMP) to the enzyme ribonuclease (RNase) T₁ were determined by computer modelling studies. The phosphate moiety of 2′-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites - (1) The primary base binding site where the guanine of 2′-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3′-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2′-AMP to RNase T₁ were found to be of nearly the same energy implying that in solution 2′-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T₁ - 2′-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T₁ catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2′-AMP and 2′-GMP with RNase T₁ reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme - 2′-GMP complex.     

Interaction of Adenosine and Guanine Derivatives in the Rat Hippocampus: Effects on Cyclic AMP Levels and on the Binding of Adenosine Analogues and GMP

Guanine nucleotides (GN) have been implicated in many intracellular mechanisms. Extracellular actions, probably as glutamate receptor antagonists, have also been recently attributed to these compounds. GN may have a neuroprotective role by inhibiting excitotoxic events evoked by glutamate. Effects of extracellular GN on adenosine-evoked cellular responses have also been reported. However, the exact mechanism of such interaction is not known. In the present study, we showed that GN potentiated adenosine-induced cAMP accumulation in slices of hippocampus from young rats. However, neither GMP nor the metabotropic glutamate receptor agonist, 1S,3R-ACPD, inhibited the binding of the adenosine receptor agonist [³H]NECA (when binding to adenosine A2 receptors), or the binding of the adenosine A2a receptor agonist [³H]CGS 21680 in hippocampal membrane preparations. GppNHp, probably by interacting with G-proteins, decreased [³H]CGS 21680 binding. [³H]GMP binding was assayed in order to evaluate the GN sites which are not G-proteins. [³H]GMP binding was inhibited by GMP and GppNHp, but not by 1S,3R-ACPD. The interaction of endogenous adenosine with the GMP-binding sites was determined by incubating membranes in the presence or absence of adenosine deaminase (ADA). NECA, CADO, CGS 21680 and CPA (only at the highest concentration used) increased GMP binding in the presence of ADA. However, in the absence of ADA, the control levels of GMP binding were as high as in the presence of added ADA plus adenosine agonists, indicating that endogenous adenosine modulates the binding of GMP. If this site has a neuroprotective role, adenosine may be increasing its neuromodulator and proposed protective action.     

Strategically Functionalized Adenosines: Agonists for Adenosine Receptors

Abstract

The syntheses of three classes of adenosine analogues involving cyclosubstitution at the 6-position and functionalization at the 2-position are reported. The target molecules synthesized are stable with respect to hydrolytic deamination by mammalian adenosine deaminase, and, because of major structural changes at the 2- and 6-positions, these compounds are expected to be poor phosphorylation substrates for the kinases. Adenosine receptor binding data reveal that several of the compounds synthesized show excellent A₁ receptor affinity and A₂/A₁ selectivity.     

Adenosine and 2-Chloroadenosine Deaza-Analogues as Adenosine Receptor Agonists1

Abstract

Several deaza-analogues of adenosine and 2-chloro-adenosine have been examined for their adenosine receptor affinity. It was found that the relative contribution of the nitrogen atoms of the purine moiety to binding at A₁ rat brain adenosine receptor, follows the order N⁷ > N³ > N¹. The affinity of the adenosine analogues for the adenosine rat brain receptor was besides compared with their activity as inhibitors of platelet aggregation. A synthesis of 2-chloro-1-deazaadenosine by two alternative routes starting from 7-nitroimidazo[4,5-b]pyridine-4-oxide is also reported.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

Molecular Modelling of the Antagonist Binding Site on the Adenosine A,Receptor

Abstract

With the aid of molecular modelling both adenosine and adenosine A, receptor antagonists belonging to various chemical classes were compared. A model for the antagonist binding site was developed. As a consequence 1H-imidazo[4, 5-c]-quinolin-4-amines were synthesized, constituting a novel class of potent non-xanthine adenosine receptor antagonists.     

Unilateral Entorhinal Cortex Lesion - An Animal Model For Cognitive Impairment in Human Disease: Effects on Adenosine Receptors and Second Messengers

Abstract

Entorhinal cortex pathology has been demonstrated in several neuropsychiatric diseases. Decreased binding to adenosine A1 receptors and adenylate cyclase in the dentate gyrus after entorhinal cortex lesion indicates impaired adenosinergic neuromodulation in these diseases.     

Potent and Selective Ligands for Adenosine Binding Sites

Abstract

A number of selective ligands for the different binding sites of adenosine have been synthesized and tested in several pharmacological models. The aim of these synthetic efforts is both to improve the knowledge of structure-activity relationships in the adenosine-related biological systems and to develop drugs from some of these molecules.     

Synthesis of Triple Helix Forming Oligonucleotides Containing 2′-Deoxyformycin A

Abstract

N⁷-Benzoyl-2′-deoxyformycin A (5) was prepared from formycin A and incorporated into the triple helix forming oligonucleotide PRE2ap at CG inversion sites. The modified oligonucleotide containing three substitutions of 2′-deoxyformycin A displayed a 10-fold increase in binding affinity as compared to its unmodified counterpart. This provided a method to accommodate CG inversion sites within target sites for antiparallel triple helix formation.     

[3H]Nitrobenzylthioinosine Binding as a Probe for the Study of Adenosine Uptake Sites in Brain

Abstract: The binding of the potent adenosine uptake inhibitor [³H]nitrobenzylthioinosine ([³H]NBI) to brain membrane fractions was investigated. Reversible, saturable, specific, high-affinity binding was demonstrated in both rat and human brain. The K_d in both was 0.15 nM with B_max values of 140–200 fmol/mg protein. Linear Scatchard plots were routinely obtained, indicating a homogeneous population of binding sites in brain. The highest density of binding sites was found in the caudate and hypothalamus in both species. The binding site was heat labile and trypsin sensitive. Binding was also decreased by incubation of the membranes in 0.05% Triton X-100 and by treatment with dithiothreitol and iodoacetamide. Of the numerous salt and metal ions tested, only copper and zinc had significant effects on [³H]NBI binding. The inhibitory potencies of copper and zinc were IC₅₀= 160 μM and 6 mM, respectively. Subcellular distribution studies revealed a high percentage of the [³H]NBI binding sites on synaptosomes, indicating that these sites were present in the synaptic region. A study of the tissue distribution of the [³H]NBI sites revealed very high densities of binding in erythrocyte, lung, and testis, with much lower binding densities in brain, kidney, liver, muscle, and heart. The binding affinity in the former group was approximately 1.5 nM, whereas that in the latter group was 0.15 nM, suggesting two types of binding sites. The pharmacologic profile of [³H]NBI binding was consistent with its function as the adenosine transport site, distinct from the adenosine receptor, since thiopurines were very potent inhibitors of binding whereas adenosine receptor ligands, such as cyclohexyladenosine and 2-chloroadenosine, were three to four orders of magnitude less potent. [³H]NBI binding in brain should provide a useful probe for the study of adenosine transport in the brain.     

Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I

BackgroundCardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI_135–209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI_146–158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI_1–37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury.MethodsSoluble fragments of cTnI containing its N- and C-terminal tails, cTnI_1–77 and cTnI_135–209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail.ResultscTnI_135–209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases.ConclusionsBoth N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies.General significancecTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury.     

L-Glutamate Receptor Heterogeneity: Labeling of Distinct Receptor Sub-Types Using Radioligand Binding Techniques

Abstract

This study demonstrates (1) that L-[³H]glutamate labels 3 distinct binding sites (types A1, A2 and A4) in isolated rat brain membranes and (2) that only the N-methyl-aspartate (A1) and quisqualate (A2) receptor classes are associated with the postsynaptic density (PSD). L-[³H]glutamate bound to PSDs with K_d 339 nM and B_max 6.1 pmol/mg protein. These sites were resolved into 2 distinct sub-types on the basis of inhibition studies. N-Methyl-aspartate maximally inhibited 57% of PSD-located L-glutamate binding sites (the A1 site) and quisqualate 43% (the A2 site); the effects of both substances were additive. The ligand selectivities of these 2 sites indicated their identity with the N-methyl-D-aspartate and quisqualate receptor classes defined electrophysiologically. The Cl^?-dependent population of L-glutamate binding sites (the A4 site) which predominates in synaptic membranes was absent from PSDs.     

Adenosine Receptor Classification: Quo Vadimus?

Abstract

Considerable progress has been made in our understanding of the diversity of adenosine receptors during the last decade, with the cloning of the orphan receptors RDC7 and RDC8 (1), and their subsequent characterisation as canine A₁ and A₂ receptors respectively (2,3), in the late 1980s. The principal objective of this review is to produce an integrated view of adenosine receptor classification, using the important observations from studies of molecular biology, receptor binding characteristics and functional pharmacology.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Survey of Nonxanthine Derivatives as Adenosine Receptor Ligands

Abstract

The binding affinities at rat A₁, A_2a, and A₃ adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [³H]PIA or [³H]CGS 21680 in rat brain membranes or [¹²⁵I]AB-MECA in CHO cells stably transfected with rat A₃ receptors. Several new classes of adenosine antagonists (e. g. 5- oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11- hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidin-one, folic acid, and cytochalasin H and J bound to A₃ receptors selectively. Moreover, cytochalasin A, which bound to A₁ adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A₁ receptor binding.

     

Distribution of the T4D Bacteriophage Binding Sites on the Escherichia Coli B Surface

Abstract

A study of the distribution of the T4D bacteriophage binding sites on the Escherichia coli B bacterial surface has shown that: (1) the number of binding sites per unit surface area is larger during growth period than during the division period, (2) the density of the binding sites on one-half of the bacterial cell is larger than the density of binding sites on the other half; (3) in newly-divided bacteria, the maximal binding site density is situated at one pole; (4) as bacteria grow, this maximum shifts to the middle of the cell; (5) when the septum is established, the middle of the cell becomes very poor in phage binding sites activity, and (6) phage adsorbs in clusters or in groups following curved lines around the bacterial cell.