Inhibitory Effect Of ADP Research Articles

A new fluorescence method for monitoring PNK activity in vitro, natural compounds screening and intracellular imaging

Polynucleotide kinase (PNK) is critical for nucleic acid metabolism, as its overexpression can increase ROS production and activate the inflammation pathway, leading to chronic inflammation. In this study, we proposed a new fluorescence method for monitoring PNK in vitro, natural compounds screening and intracellular imaging. Quantitative detection depicted that the limit of detection (LOD) of PNK is 0.0021 U/mL in the linear range of 0.01∼10 U/mL. Methodological evaluation showed that both of the selectivity and the inhibitory effect of ADP and EDTA on PNK activity with satisfactory results. Fluorescence imaging of inflammatory cells demonstrated the reliability of platform for capturing PNK change in situ regulated by inhibitor of natural compound NS17 isolated from Kadsura heterolita. Mechanism study showed that the inhibitor NS17 can reduce inflammation by scavenging intracellular ROS, killing inflammatory cells and down-regulating COX-2 expression, the processes of which are related with PNK activity. In summary, above results suggest that this new platform could monitor PNK activity in situ and screen inflammation inhibitors targeted on PNK.

Inhibitory Effects of Adenine Nucleotides on Brain Mitochondrial Permeability Transition

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca(2+) and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca(2+) × mg protein(-1). ADP or ATP also prevented Ca(2+)-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K(i)) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca(2+)-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.

Adiponectin inhibits tissue factor expression and enhances tissue factor pathway inhibitor expression in human endothelial cells

Tissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-alpha (TNF-alpha)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HUVECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-alpha-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-alpha, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp on TNF-alpha-induced TF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IkappaB-alpha was decreased by Adp, but phosphorylation of p44/42 MAPK, SAPK/JNK, and p38 MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor-kappaB (NF-kappaB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-alpha-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY294002, suggesting that Akt activation might inhibit TF expression induced by TNF-alpha. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-kappaB through stabilization of IkappaB-alpha and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

The role of lysine 185 in the kir6.2 subunit of the ATP-sensitive channel in channel inhibition by ATP.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, forms ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on Kir6.2. 2. Previous studies have shown that mutation of the lysine residue at position 185 (K185) in the C-terminus of Kir6.2 to glutamine, decreased the channel sensitivity to ATP without affecting the single-channel conductance or the intrinsic channel kinetics. This mutation also impaired 8-azido[32P]-ATP binding to Kir6.2. 3. To determine if K185 interacts directly with ATP, we made a range of mutations at this position, and examined the effect on the channel ATP sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 4. Substitution of K185 by a positively charged amino acid (arginine) had no substantial effect on the sensitivity of the channel to ATP. Mutation to a negatively charged residue markedly decreased the channel ATP sensitivity: the Ki for ATP inhibition increased from 85 microM to >30 mM when arginine was replaced with aspartic acid. Substitution of neutral residues had intermediate effects. 5. The inhibitory effects of ADP, ITP and GTP were also reduced when K185 was mutated to glutamine or glutamate. 6. The results indicate that a positively charged amino acid at position 185 is required for high-affinity ATP binding to Kir6.2. Our results demonstrate that ATP does not interact with the side-chain of K185. It remains unclear whether ATP interacts with the backbone of this residue, or whether its mutation influences ATP binding allosterically.

Differential degradation of extracellular adenine nucleotides by folliculated oocytes of Xenopus laevis.

The degradation of extracellular ATP and ADP by Xenopus oocytes was studied to investigate whether one or two ecto-enzymes are responsible for breakdown of both nucleotides. At a concentration of 100 microM, the half-life of ATP and ADP was 33 and 40 min, respectively. Degradation of ATP caused an initial fast and then a sustained accumulation of ADP in the buffer, while the concentration of AMP in the buffer increased slowly, but progressively, in a relatively linear manner. The rates of degradation of ATP and ADP were similar at pH levels between 7 and 10, but the velocity of breakdown of ATP was significantly higher than that of ADP at pH of 5-6. In divalent cation-free buffer, the addition of 0.1 mM of Ca2+, but not equimolar Mg2+, significantly potentiated the degradation of ATP by oocytes while, in the case of ADP, each of these divalent cations were able to potentiate its degradation. The rate of hydrolysis of ATP and its kinetic constants were not significantly different in the presence or absence of ADP (50 microM). In conclusion, differences in pH- and cation-dependency, and absence of inhibitory effect of ADP on degradation of ATP suggest that degradation of ATP and ADP by oocytes is provided by separate enzymes, namely Ca2+/Mg(2+)-dependent ecto-ATPase and ecto-ADPase, rather than by one ecto-enzyme.

Effect of inorganic phosphate and ADP on the myofilament sliding induced by laser flash photolysis of caged ATP

Using the technique of laser flash photolysis of caged ATP, we have suggested that, under nearly isometric conditions, the unitary distance of myofilament sliding per ATP molecule (myosin head powerstroke) is about 10 nm. To give further information about the mechanism of myofilament sliding, we studied the effect of inorganic phosphate (P i) and ADP on the photoreleased ATP-induced shortening of single glycerinated muscle fibers under very small external loads. Both the velocity and the distance of the myofilament sliding induced by 150 μM ATP increased by P i (20 mM), and decreased by ADP (0.4 mM). On the other hand, P i and ADP showed no significant effect on the myofilament sliding induced by 100 and 75 μM ATP. The potentiating effect of P i on the myofilament sliding with 150 μM ATP can be explained as being due to the increase in population of AM · ADP · P i with corresponding decrease in population of AM · ADP, and also the increase in population of M · ADP · P i and M · ATP. Meanwhile, the inhibitory effect of ADP can be simply accounted for to be due to an accumulation of AM · ADP that already finished their force generating process. The ineffectiveness of P i and ADP on the myofilament sliding with 100 and 75 μM ATP is consistent with the view that it is caused by almost synchronized single myosin head powerstrokes.

ADP inhibition of the plasmalemma ATPase from Jerusalem artichoke tubers (Hèlianthus tuberosus L.)

The inhibitory effect of ADP on the plasma membrane ATPase from the parenchyma cells of Jerusalem artichoke (Helianthiis tuberosus L.) tubers was investigated in both physiological states (dormant or non-dormant). To begin with, several sources of ADP were employed, as many commercial ADP contain a variable and non-negligible amount of vanadate the inhibitory effect of which on plasmalemma ATPase is well known. By using vanadium-free ADP, it was possible to show that the plasma membrane ATPase was inhibited by ADP. Similar levels of inhibition were measured in dormant and non-dormant samples, showing that endogenous ADP is not responsible for the physiological regulation of ATPase activity in dormant and non-dormant materials.

Nucleoside triphosphate pyrophosphatase of rabbit matrix vesicles, a mechanism for the generation of inorganic pyrophosphate in epiphyseal cartilage

Inorganic pyrophosphate (PP i) may be important in the regulation of mineralisation but its origin in epiphyseal cartilage is ill-defined. Nucleoside triphosphate pyrophosphatase is one potential source, as this enzyme catalyses the formation of PP i from nucleoside triphosphates. This enzyme has been identified in matrix vesicles derived from rabbit epiphyseal cartilage and a method developed to measure the activity using ATP as substrate in intact matrix vesicles under relatively physiological conditions. The enzyme had a high affinity for ATP ( K m < 10 μM) and was also active towards GTP, CTP and UTP. Disruption of the matrix vesicle membrane by sonication failed to alter the activity. Treatment of sonicated matrix vesicles with Triton X-100 increased the activity which may indicate a direct effect of the detergent on the enzyme. Activity towards ATP was inhibited substantially by ADP and AMP and by another potential substrate β,γ-methyleneadenosine 5′-triphosphate. Dichloromethylene bisphosphonate, an analogue of the product PP i, inhibited the activity to a lesser extent. Two other potential substrates, NADP + and thymidine 5′-monophosphate p-nitrophenyl ester were only weakly inhibitory as was 1-hydroxyethylidene 1,1-bisphosphonate. These results imply that nucleoside triphosphates are the substrates in vivo and the inhibitory effects of ADP and AMP suggest mechanisms whereby this activity could be regulated.

Correlation between thylakoid protein phosphorylation and molecular organization of the photosynthetic apparatus in a dynamic system

In-vitro thylakoid protein phosphorylation has been studied in synchronized cells of Scenedesmus obliquus at the 8- and 16-h of the life cycle, stages which are characterized by the maximum and minimum photosynthetic activities, respectively. The stage of maximum photosynthetic activity (8-h) is characterized by the highest protein phosphorylation in vitro and in vivo, by the largest proportion of the heavy subfraction of thylakoids, and by maximum oligomerization of the light-harvesting chlorophyll a/b-protein complex, altogether creating the highest energy charge of the thylakoid membranes. Protein phosphorylation in vitro decreases the amount of the heavy subfraction and increases the amount of oligomerization of the antenna of photosystem I (PSI) (increase of chlorophyll b in the light fraction). Concomittantly, PSII units become smaller (longer time for the rise in fluorescence induction) and photosynthetic efficiency increases (decrease of fluorescence yield). In-vivo protein phosphorylation is controlled mainly endogenously during the 8-h of the life cycle but is exogenously modulated by light to optimize the photosynthetic activity by redistribution of pigment-protein complexes. In-vitro protein phosphorylation seems to restore partially the conditions prevalent in vivo and lost during the preparation of membranes. The effect is greater in 16-h cells which have less-stable membranes. The regulatory mechanism between membrane stabilization and oligomerization on the one hand and redistribution of the light-harvesting chlorophyll a/b-protein complex from PSII to PSI on the other hand remains unexplained. We have confirmed that the mechanism of protein phosphorylation is regulated via plastohydroquinone, but experiments with the plastohydroquinone analogue 2,3,5,6-tetramethyl-p-benzoquinone demonstrated that plastohydroquinone is not solely responsible for the differences in protein phosphorylation of 8- and 16-h thylakoids. The inhibitory effect of ADP and the distinct rates of kinase reaction indicate that the adenylate energy charge and changes in the organization of the photosynthetic apparatus also contribute to the observed differences in protein phosphorylation. Phosphorylation in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea indicated that the 32-kDa phosphoprotein and the herbicide-binding QB protein may be the same. These experiments also indicated that 3-(3',4'-dichlorophenyl)-1,1-dimethylurea-binding reduces kinase activity directly and not only by inhibiting electron transport.

Regulation of C4 Photosynthesis A Role for Pyruvate in Regulating Pyruvate,Pi Dikinase Activity in vivo

Results are presented in support of the view that pyruvate levels are critical in the light-dark regulation of pyruvate,PI dikinase in vivo. In an in vitro system containing purified Zea mays pyruvate,PI dikinase, increasing pyruvate reduced both the initial rate and the final extent of ADP-mediated inactivation of the enzyme. Pyruvate also reversed the inhibitory effect of ADP on the initial rate of PI-mediated activation of pyruvate,P*i dikinase. With intact Zea mays leaves, inactivation of pyruvate,PI dikinase in darkened leaves was prevented by anaerobic conditions or reduced temperature; both treatments resulted in elevated steady-state levels of pyruvate. Added pyruvate was shown to substantially increase the level of active pyruvate,PI dikinase in chloroplasts incubated with PI in the dark but not in the light. The likely physiological significance of these results is discussed.

Kinetic and thermodynamic properties of beef heart mitochondrial ATPase: effect of co-solvent systems.

The effects of glycerol and methanol upon beef heart mitochondrial ATPase (F1) were studied. Glycerol was found to be a potent reversible inhibitor of the F1-catalyzed hydrolysis of ATP and ITP. The inhibition of ATP hydrolysis was linear with respect to glycerol concentrations, while that of ITP was not. From the temperature dependence of Vmax for F1-catalyzed ATP and ITP hydrolysis in glycerol or methanol solutions, the energy of activation and the enthalpy of activation were calculated. The inhibitory effect of ADP on F1 hydrolytic activity was studied in three solvent systems (totally aqueous, 20% methanol, and 20% glycerol). Compared to the aqueous system, methanol decreased the potency of ADP as an inhibitor, and glycerol enhanced the potency.

ADP is a potent inhibitor of human platelet plasma membrane adenylate cyclase.

The potent inducer of platelet aggregation, ADP, inhibits both basal and stimulated cyclic AMP production in intact platelets1,2. However, several attempts have failed to detect an effect of the nucleotide on adenylate cyclase activity in broken cell preparations2,3. This paradox has led to suggestions that ADP may act on unidentified sites before the adenylate cyclase step, such as a nucleoside diphosphate kinase4 and a Ca2+ transport system5,6. It is difficult to demonstrate the effect of physiologically effective concentrations (0.1–5 µM) of ADP on a crude membrane preparation of adenylate cyclase because of the presence of nucleoside phosphohydrolase activities which may degrade either the substrate ATP or the putative effector ADP to products (such as ADP, AMP and adenosine) displaying diverse effects. A substrate regenerating system which converts ADP to ATP obviously provides no solution. The contamination by adenine nucleotides and catecholamines in widely used crude lysate preparations aggravates the problem7–9. Using a purified human platelet plasma membrane preparation, and the non-hydrolysable ATP analogue APP(NH)P as substrate, we have now demonstrated direct inhibition of adenylate cyclase by ADP in the physiologically effective concentration range. The inhibitory effect of ADP is immediate in onset and is equal to that produced by adrenaline; it is shared by ADP-βS (and 2′-deoxy ADP to some extent) but not by AP(CH2)P or other adenine nucleotides. The Ki, for ADP is ∼ 0.3 µM and maximal inhibition is observed at 2 µM

Localization of inhibition by adenosine diphosphate of phosphoglycerate-dependent oxygen evolution in a reconstituted chloroplast system.

ADP was shown to inhibit phosphoglycerate-dependent O2 evolution in a simplified reconstituted chloroplast system containing 3-phosphoglycerate kinase and triose phosphate dehydrogenase. Rates of O2 evolution in the simplified system are comparable with those obtained by using stromal protein rather than purified enzymes. ADP does not inhibit O2 evolution with glycerate 1,3-biphosphate as substrate nor does it inhibit triose phosphate dehydrogenase. The inhibitory effect of ADP is attributed to an increase in the rate of conversion of glycerate biphosphate into phosphoglycerate. The results are discussed in terms of control by ADP of phosphoglycerate-dependent oxygen evolution.

Energy-dependent control of the latency of mitochondrial enzymes.

1 At neutral pH, the temperature-dependent release of glutamate-oxaloacetate aminotrasferase, glutamate-oxaloacetate aminotransferase, glutamate dehydrogenase, malate dehydrogenase, malate dehydrogenase and β-hydroxybutyrate dehydrogenase from mitochondria in an in vitro system, is prevented as a function of the concentration of ATP, oxidizable substrates and ADP. On the other hand, orthophosphate causes the release of mitochondrial enzymes (optimum at pH 6.5). This effect of Pi is completely inhibited by ADP but not by ATP. UTP and UDP partially replace ADP for this activity. The inhibitory effect of ADP is insensitive to either substrate or rutamycin. 2 In absence of added phosphate and at acid pH values (optimum at pH 6.0), ATP or oxidizable substrates cause marked release of latent mitochondrial enzymes. ATP shows magnesium requirement for this activity. Inhibitors of electron transport and phosphoryl transfer reactions completely prevent these effects of ATP and substrates. Inhibitors of mitochondrial alkali metal cation uptake are inactive. Rutamycin and dinitrophenol prevent effects of ATP in freshly prepared but not in aged mitochondria. 3 Chromatographically pure phosphatidylinositol, phosphatidylcholine and phosphatidyl-ethanolamine, but not saturated phospholiopids, prevent phosphate from stimuating leakage of latent enzymes from mitochondria. 4 Release of mitochondrial enzymes effected by oxidizable substrates, ATP, ADP and orthophosphate, but not phospholipids, seems to be associated with energy-dependent mitochondrial swelling. 5 It is concluded that the temperature-dependent release of latent enzymes from mitochondria may depend on structural transformations of the mitochondrial membrane controlled in part by the oxidative phosphorylation mechanism.