58-64-0

Usage

Uses

1. Cellular Processes:
Adenosine-5'-diphosphate is used as an energy source and precursor for ATP in various cellular processes, including respiration, biosynthetic reactions, motility, and cell division.
2. Endothelium-Dependent Vascular Response Studies:
Adenosine 5′-diphosphate (5′-ADP or ADP) is used as a test compound for studying the endothelium-dependent vascular response in salt-sensitive (DS) and salt-resistant Dahl rats (DR). This application helps researchers understand the role of ADP in vascular health and its potential implications in various health conditions.
3. P2-Purinergic Receptor Subtype Studies:
ADP is used to study different P2-purinergic receptor subtypes on canine vascular smooth muscle and endothelium. This research contributes to the understanding of the mechanisms of action of ADP and its potential therapeutic applications.
4. Agricultural Uses:
In agriculture, Adenosine diphosphate (ADP) acts as a source of energy in biochemical reactions, playing a vital role in the growth and development of plants.

Biological Activity

adenosine-5'-diphosphate is an agonist of purinergic receptors.purinergic receptors, also known as purinoceptors, are a family of plasma membrane molecules that are found in almost all mammalian tissues. within the field of purinergic signalling, these receptors have been implicated in learning and memory, locomotor and feeding behavior, and sleep. more specifically, purinergic receptors are involved in several cellular functions, such as proliferation and migration of neural stem cells, vascular reactivity, apoptosis and cytokine secretion.

Biochem/physiol Actions

Adenosine 5′-diphosphate induces human platelet aggregation and non-competitively blocks the stimulated human platelet adenylate cyclase.

in vitro

adenosine 5'-diphosphate (adp) is an adenine nucleotide having two phosphate groups esterified to the sugar moiety at the 5’ position. adp is formed through dephosphorylation of adenosine 5’-triphosphate (atp) by atpases and can be converted back to atp by atp synthases. adp can also be metabolized to adenosine 5’-monophosphate (amp) and 2’-deoxyadenosine 5’-diphosphate (dadp). adp can modulate several receptors, such as activating certain purinergic receptors and inhibiting others, inhibiting rat ecto-5’nucleotidase (ki = 0.91 nm), as well as regulating the phosphorylation status of amp-activated protein kinase [1, 2].

Enzyme inhibitor

This adenine nucleotide (FWfree-acid = 504.16 g/mol; CAS 58-64-0; Molar Absorptivity = 15,400 M–1cm–1, l = 259 nm) is a product in ATPdependent transphosphorylases, phosphohydrolases, and molecular motors; as such, ADP often inhibits these enzymes. Enzymatic Phosphorylation: ADP is a substrate for adenylate kinase (Reaction: ADP2– + MeADP " MeATP2– + AMP) and other enzymes that stabilize ATP concentrations in prokaryotes [e.g., acetate kinase (Reaction: MgADP + Acetyl-phosphate ! MeATP2– + Acetate)] and eukaryotes [e.g., pyruvate kinase (Reaction: MgADP + Phosphoenolpyruvate ! MgATP2– + Pyruvate), creatine kinase (Reaction: MgADP + Creatine-phosphate ! MgATP2– + Creatine), arginine kinase (Reaction: MgADP + Arginine-phosphate ! MgATP2– + Arginine), and nuclecleotide diphosphate kinase (Reaction: ADP2– + MgGTP2– " MgATP2– + GDP2–)]. ATP Synthase: ADP is a primary substrate for the FOF1 ATP synthase (Reaction: MgADP + Pi + High Chemiosmotic Gradient Energization State ! MgATP2– + Low Chemiosmotic Gradient Energization State). ADP can also become entrapped within a catalytic site of the rotary motor, when proton motive is low, absent, or uncoupled, and its inhibitory action under such conditions is believed to prevent wasteful hydrolysis of ATP (Reaction: MgATP2– + H2O ? MgADP + Pi). Metal Ion Binding Properties: As a polyanion, ADP not only binds physiologic divalent cations Mg2+ and Ca2+, but also forms reversible complexes with Mn2+ and Co2+. For reversible complexation of ADP2– with a metal ion Me2+, (Reaction: ADP2– + Me2+ ! MeADP), Kformation = [MeADP]/[ADP2–]free[Me2+]free, indicating that [MeADP]/[ADP2– ]free = Kformation ′ [Me2+]free. In many cases, metal-free ADP is not a substrate and instead acts as a revesible inhibitor. Good experimental design therefore demands rigorous control of free metal ion concentration to control the ratio of metal-bound and metal-free forms. When exposed to Cr(III) at elevated temperature, ADP also forms ligand exchange-inert complexes with Cr3+. Platelet Aggregation: ADP is also a well-known activator of platelet aggregation, as mediated by the ADP receptors P2Y1, P2Y12 and P2X1. Upon conversion to adenosine by ecto-ADPases, platelet activation is inhibited by means of adenosine receptors. Target(s): Hydrogenomonas facilis ribulosediphosphate (RuDP) carboxylase and NADH-, ATP-dependent CO2 fixation; platelet (Na+/K+)-ATPase; hydrogen-ion transport in chloroplasts; pyruvate dehydrogenase kinase; 5-oxo-L-prolinase, or L-pyroglutamate hydrolase; a-NADHdependent reductase, rat liver microsomes; nitrogenase; Trypanosoma cruzi hexokinase; maize leaf acetyl-coenzyme A carboxylase; rat brain mitochondrial calcium-efflux; sarcoplasmic reticulum Ca2+ ATPase; Na+-Na+ exchange mediated by (Na+/K+)ATPase reconstituted into liposomes; nitrate and nitrite assimilation in Zea mays under dark conditions; PGE1-activated platelet adenylate cyclase in rats and rabbits; mitochondrial F1-ATPase, inactive complex formed upon binding ADP at a catalytic site; ATP-sensitive K+ channels, frog skeletal muscle; human 5-phosphoribosyl-1pyrophosphate synthetase; Crithidia fasciculata glutathionylspermidine synthetase; myosin V ATPase; cystic fibrosis transmembrane conductance regulator (ABC transporter) via its adenylate kinase activity; V type ATPase/synthase.

IC 50

67 nm for p2x2/3

Purification Methods

It is characterised by conversion to the acridine salt by addition of alcoholic acridine (1.1g in 50mL), filtering off the yellow salt and recrystallising from H2O. The salt has m 215o(dec), max 259nm ( 15,400) in 2O. [Baddiley & Todd J Chem Soc 648 1947, 582 1949, cf LePage Biochemical Preparations 1 1 1949, Martell & Schwarzenbach Helv Chim Acta 39 653 1956]. [Beilstein 26 III/IV 2369.]

references

1. azran, s.,frster, d.,danino, o., et al. highly efficient biocompatible neuroprotectants with dual activity as antioxidants and p2y receptor agonists. j. med. chem. 56(12), 4938-4952 (2013).2. jarvis, m.f.,bianchi, b.,uchic, j.t., et al. [3h]a-317491, a novel high-affinity non-nucleotide antagonist that specifically labels human p2x2/3 and p2x3 receptors. journal of pharmacology and experimental therapeutics 310(1), 407-416 (2004).

Upstream product

• 606-67-7 disodium adenosine-5' triphosphate 
• 56-65-5 ATP 
• 7732-18-5 water 
• 1062-98-2 adenosine 5'-tetraphosphate 
• 58-85-5 biotin 
• 61-19-8 5'-adenosine monophosphate 
• 506-68-3 bromocyane 
• 59286-20-3 Adenosine 5'<(S)α-thio>diphosphate 
• 81347-80-0 adenosin 5'-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl diphosphate) 
• 73116-39-9 γ-(18)O4-ATP 
• 76947-02-9 tris(tetra-n-butylammonium) hydrogen pyrophosphate 
• 5135-30-8 5'-tosyladenosine 
• 35094-46-3 adenosine-5'-O-(3-thio)triphosphate 
• 58-98-0 UDP 

Downstream Products

• 2564-35-4 2'-deoxyguanosine 5'-triphosphate 
• 5959-90-0 diadenosine 5^',5^'''-P¹P³-triphosphate 
• 5959-90-0 Diadenosine triphosphate 
• 61-19-8 5'-adenosine monophosphate 
• 56-65-5 ATP 
• 64060-84-0 C₁₀H₁₃N₅O₁₀P₂ 
• 23600-16-0 8-bromo-ADP 
• 58-61-7 adenosine 
• 86119-84-8 phosphoric acid 
• 60-92-4 cAMP 
• 3615-55-2 D-ribose 5-phosphate 
• 73-24-5 7H-purin-6-ylamine 
• 86-04-4 diphosphoric acid mono-inosin-5'-yl ester 
• 1927-31-7 dATP 

Relevant academic research and scientific papers

Thermodynamic analysis of F1-ATPase rotary catalysis using high-speed imaging

F1-ATPase (F1) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high-speed imaging of F1 rotation at various temperatures using the total internal reflection dark-field (TIRDF) illumination system, which allows resolution of the F1 catalytic reaction into elementary reaction steps with a high temporal resolution of 72 μs. At a high concentration of ATP, F1 rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature-sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate (Pi). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, Pi release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, ΔG? = 53-56 kJ mol-1, the contributions of the enthalpy (ΔH?), and entropy (ΔS?) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both ΔH? and ΔS?. The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.

Molecular identification of N-acetylaspartylglutamate synthase and β-citrylglutamate synthase

The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and L-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and β-citrylglutamate.

Kinetic mechanism and rate-limiting steps of focal adhesion kinase-1

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH2, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values kcat (0.052 ± 0.001 s-1), KMgATP (1.2 ± 0.1 μM), KiMgATP (1.3 ± 0.2 μM), KFAK-tide (5.6 ± 0.4 μM), and K iFAK-tide (6.1 ± 1.1 μM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the βγ-bridge:β-nonbridge positional oxygen exchange of [γ-18O4]ATP in the presence of 1 mM [γ- 18O4]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of kx/kcat = 0.14 ± 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on kcat/KFAK-tide, while kcat and kcat/KMgATP were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on kcat/Km for MgATP but not on kcat/K m for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (kcat) is rate-limited by both reversible phosphoryl group transfer (kforward ≈ 0.2 s-1 and kreverse ≈ 0.04 s-1) and a slow step (kconf ≈ 0.1 s-1) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.

Coupling of proteolysis to ATP hydrolysis upon Escherichia coli Lon protease functioning: I. Kinetic aspects of ATP hydrolysis

Some aspects of the Escherichia coli Lon protease ATPase function were studied around the optimum pH value. It was revealed that in the absence of the protein substrate the maximum ATPase activity of the enzyme is observed at an equimolar ratio of ATP and Mg2+ ions in the area of their millimolar concentrations. Free components of the substrate complex (ATP-Mg)2- inhibit the enzyme ATPase activity. It is hypothesized that the effector activity of free Mg2+ ions is caused by the formation of the "ADP-Mg-form" of ATPase centers. It was shown that the activation of ATP hydrolysis in the presence of the protein substrate is accompanied by an increase in the affinity of the (ATP-Mg)2- complex to the enzyme, by an elimination of the inhibiting action of free Mg2+ ions without altering the efficiency of catalysis of ATP hydrolysis (based on the kcat value), and by a change in the type of inhibition of ATP hydrolysis by the (ADP-Mg)- complex (without changing the Ki value). Interaction of the Lon protease protein substrate with the enzyme area located outside the peptide hydrolase center was demonstrated by a direct experiment.

The ATPase activities of sulfonylurea receptor 2A and sulfonylurea receptor 2B are influenced by the C-terminal 42 amino acids

Unusually among ATP-binding cassette proteins, the sulfonylurea receptor (SUR) acts as a channel regulator. ATP-sensitive potassium channels are octameric complexes composed of four pore-forming Kir6.2 subunits and four regulatory SUR subunits. Two different genes encode SUR1 (ABCC8) and SUR2 (ABCC9), with the latter being differentially spliced to give SUR2A and SUR2B, which differ only in their C-terminal 42 amino acids. ATP-sensitive potassium channels containing these different SUR2 isoforms are differentially modulated by MgATP, with Kir6.2/SUR2B being activated more than Kir6.2/SUR2A. We show here that purified SUR2B has a lower ATPase activity and a 10-fold lower K m for MgATP than SUR2A. Similarly, the isolated nucleotide-binding domain (NBD) 2 of SUR2B was less active than that of SUR2A. We further found that the NBDs of SUR2B interact, and that the activity of full-length SUR cannot be predicted from that of either the isolated NBDs or NBD mixtures. Notably, deletion of the last 42 amino acids from NBD2 of SUR2 resulted in ATPase activity resembling that of NBD2 of SUR2A rather than that of NBD2 of SUR2B: this might indicate that these amino acids are responsible for the lower ATPase activity of SUR2B and the isolated NBD2 of SUR2B. We suggest that the lower ATPase activity of SUR2B may result in enhanced duration of the MgADP-bound state, leading to channel activation.

Zn(II) enhances nucleotide binding and dephosphorylation in the presence of a poly(ethylene imine) dendrimer

The interaction of a second-generation poly(ethylene imine) dendrimer (L) with the nucleotides AMP, ADP and ATP was analysed by means of potentiometric titrations (0.1 M Me4NCl, 298.1 K), in the absence and in the presence of Zn2+, to perform speciation of the systems and determination of complex stability constants. Protonated forms of L interact with anionic forms of the nucleotides giving rise to stable anion complexes. In the presence of Zn2+, ternary complexes (ion-pair complexes) become the main species in solution, the presence of each partner enhancing the binding of the other one (positive cooperativity effect). The stability constants determined by the same method and under the same experimental conditions for the formation of PO43-, P2O7 4- and P3O105- complexes with L showed a strict similarity between the binding of AMP, ADP and ATP and their inorganic counterparts, indicating that the main interaction of nucleotides with protonated ligand forms takes places through their inorganic tails. Dephosphorylation of ATP to form ADP and phosphate was monitored by means of 31P NMR spectra at pH 3 and 9 in the presence of L and of its Zn 2+ complexes. The rate of ATP dephosphorylation is enhanced by about 2 times at pH 3 and 5 times at pH 9 in the presence of L and by about 4 times at pH 3 and 6 times at pH 9 in the presence of its metal complex, relative to the uncatalysed process, the dephosphorylation reactions being much faster in the acidic media where successive cleavage of ADP to give AMP and phosphate was also observed. Depending on the pH, the observed dephosphorylation enhancements have been interpreted in terms of formation of phosphoramidate intermediates, formation of ATP-Zn2+ coordinative bonds and positive allosteric effects due to metal ion coordination.

A zinc(II)-based receptor for ATP binding and hydrolysis

A protonated Zn(II) complex with a terpyridine-containing pentaamine macrocycle catalyses ATP hydrolysis in the presence of a second metal ion, which acts as cofactor assisting the phosphoryl transfer from ATP to an amine group of the receptor. The Royal Society of Chemistry 2005.

A general framework for inhibitor resistance in protein kinases

Protein kinases control virtually every aspect of normal and pathological cell physiology and are considered ideal targets for drug discovery. Most kinase inhibitors target the ATP binding site and interact with residue of a hinge loop connecting the small and large lobes of the kinase scaffold. Resistance to kinase inhibitors emerges during clinical treatment or as a result of in vitro selection approaches. Mutations conferring resistance to ATP site inhibitors often affect residues that line the ATP binding site and therefore contribute to selective inhibitor binding. Here, we show that mutations at two specific positions in the hinge loop, distinct from the previously characterized "gatekeeper," have general adverse effects on inhibitor sensitivity in six distantly related kinases, usually without consequences on kinase activity. Our results uncover a unifying mechanism of inhibitor resistance of protein kinases that might have widespread significance for drug target validation and clinical practice.

Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase: Product inhibition, cooperativity, and magnesium activation

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mM tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min-1, the Km for ATP by 0.104 ± 0.024 mM, and the Ki for ADP by 0.037 ± 0.007 mM. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2+] in response to changes in available light.

Magnesium coordination controls the molecular switch function of DNA mismatch repair protein MutS

The DNA mismatch repair protein MutS acts as a molecular switch. It toggles between ADP and ATP states and is regulated by mismatched DNA. This is analogous to G-protein switches and the regulation of their "on" and "off" states by guanine exchange factors. Although GDP release in monomeric GTPases is accelerated by guanine exchange factorinduced removal of magnesium from the catalytic site, we found that release of ADP from MutS is not influenced by the metal ion in this manner. Rather, ADP release is induced by the binding of mismatched DNA at the opposite end of the protein, a long-range allosteric response resembling the mechanism of activation of heterotrimeric GTPases. Magnesium influences switching in MutS by inducing faster and tighter ATP binding, allowing rapid downstream responses. MutS mutants with decreased affinity for the metal ion are impaired in fast switching and in vivo mismatch repair. Thus, the G-proteins and MutS conceptually employ the same efficient use of the high energy cofactor: slow hydrolysis in the absence of a signal and fast conversion to the active state when required.