The hydrolysis of extracellular adenine nucleotides by arterial smooth muscle cells. Regulation of adenosine production at the cell surface

The extracellular reaction sequence ATP----ADP----AMP----adenosine participates in regulating the time course of cellular response during crisis or signaling events, such as thrombus formation or neurotransmission. We have investigated the whole time course of hydrolysis of ATP to adenosine by recirculating adenine nucleotide substrates over smooth muscle cells attached to polystyrene beads. Kinetic parameters were estimated for each reaction by fitting observed time courses to models of the pathway. In spite of the inhibition of 5'-nucleotidase by ADP, adenosine was produced very rapidly by smooth muscle cells. Comparisons of the apparent Km values of ADPase and 5'-nucleotidase (determined from experiments in which each substrate was used as the initial substrate with Km values observed when each substrate was supplied from the upstream reaction) suggest that the local concentrations of substrate supplied from the preceding reactions are very much higher than those in the bulk phase. This enhancement of efficiency overcomes the effect of the feed-forward inhibition to give rise to very rapid adenosine production from ADP or ATP. These observations are in marked contrast to our previous findings with endothelial cells (Gordon, E. L., Pearson, J. D., and Slakey, L. L. (1986) J. Biol. Chem. 261, 15496-15504), on which feed-forward inhibition causes a profound lag in adenosine production from adenine nucleotides and on which there are no apparent surface effects on substrate delivery.     

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg²⁺.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group.     

Effect of subchronic treatment with mercury chloride on NTPDase, 5′-nucleotidase and acetylcholinesterase from cerebral cortex of rats

The aim of the present investigation was to evaluate the effect of a subchronic treatment (30 days/30 doses) with subcutaneous injections (0.1 mg/kg) of HgCl₂ on NTPDase (E.C. 3.6.1.5), 5′-nucleotidase (E.C. 3.1.3.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in brain from adult rats. NTPDase and 5′-nucleotidase were measured in cortical synaptosomal fraction and AChE was measured in the homogenate of cerebral cortex and hippocampus. After the subchronic treatment (30 days), NTPDase activity was enhanced approximately 35% (p < 0.05) with ATP and ADP as substrates and no difference was observed in 5′-nucleotidase activity (AMP hydrolysis). In addition, AChE activity was enhanced in the cerebral cortex (22%, p < 0.05) and hippocampus (26%, p < 0.05) after the subchronic treatment. Mercury deposited in brain was measured by cold vapor (atomic absorption spectrometry) and no difference between the control and the subchronically treated group was observed. Here we showed for the first time that exposure to low levels of Hg²⁺, which resembles occupational exposure to low levels of mercury, caused a marked increase in NTPDase and AChE activities. The relationship of these alterations with the neurotoxicity of inorganic mercury deserves further studies.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5′-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na⁺, K⁺ and Rb⁺ changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5′-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.     

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium

This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM     

Properties and subcellular localization of adenosine diphosphatase in arterial smooth muscle cells in culture

The properties and subcellular localization of adenosine diphosphatase (ADPase) activity in smooth muscle cells cultured from pig aortas have been investigated. The pH optimum of ADPase activity was 7.3 and the apparent K_m for ADP was 10.3 μM. ADPase activity was inhibited completely by EDTA and was restored by the addition of divalent cations. The enzyme activity was not inhibited by 2-glycerophosphate, a substrate for non-specific phosphatases, nor by levamisole, a specific inhibitor of alkaline phosphatase. Smooth muscle cells were homogenized and a post-nuclear supernatant was applied to a sucrose density gradient in a Beaufay automatic zonal rotor. The distribution of ADPase activity in the density gradient was similar to that of 5′-nucleotidase activity, a marker enzyme for the plasma membrane, and distinct from the distributions of the marker enzymes for the other organelles. When the cells were homogenized in the presence of digitonin, an agent which binds to cholesterol and increases the equilibrium density of the plasma membrane, the modal equilibrium densities of ADPase activity and of 5′-nucleotidase activity were increased to similar extents, thus confirming the plasma membrane localization of ADPase activity.     

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.Abbreviations DCCD dicyclohexylcarboiimide - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Pi inorganic phosphate Enzymes ATP diphosphohydrolase, Apyrase (EC 3.6.1.5) - ATPase ATP phosphohydrolase (EC 3.6.1.3) 5-nucleotidase (EC 3.1.3.5) Hexokinase (EC 2.7.1.1) Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) Adenylate kinase (EC 2.7.4.3) Inorganic pyrophosphatase (EC 3.6.1.1) - ATP pyrophosphohydrolase (EC 3.6.1.8) - LDH lactate dehydrogenase (EC 1.1.1.27) - SDH succinate dehydrogenase (EC 1.3.1.6) - ACHE acethylcholinesterase (EC 3.1.1.7) - G-6-Pase glucose-6-phosphatase (EC 3.1.3.9) - NADPH cytoehrome c oxidoreductase (NCR) (EC 1.6.2.4)     

Elevated specific activity of 5′-nucleotidase in a spinal cord myelin fraction from shiverer mice comparison with other myelin-associated enzymes and myelin proteins

Meylin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5′-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2′,3′-cyclic nucleotide-3′-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiveres, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hhypothetically, the 5′-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.     

Solubilization of rat heart sarcolemma 5′-nucleotidase by phosphatidylinositol-specific phospholipase C

The influence of a phosphatidylinositol-specific phospholipase C treatment on rat heart sarcolemmal 5′-nucleotidase was investigated. Upon complete hydrolysis of all phosphatidylinositol in the sarcolemma, 75% of 5′-nucleotidase activity was found in the solubilized form. The insolubilized enzyme after this treatment has the same K_m for AMP as the untreated, sarcolemmal-bound enzyme (0.04 mM), whereas the solubilized enzyme has a 40-fold increase in K_m for AMP (0.16 mM). Other sarcolemmal-bound enzymes were not affected by the same treatment. Hence, the specific involvement of phosphatidylinositol in the binding of 5′-nucleotidase to the sarcolemma of the rat heart is clearly demonstrated.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Adenosine triphosphate hydrolysis in rat dental tissues

Summary The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: -glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde.By comparing the enzymatic stainings obtained with the various substrates and at the different pH: s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5-nucleotidase, inorganic pyrophosphatase, 3:5-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.     

The Arrhenius plot behaviour of rat liver 5′-nucleotidase in different lipid environments

The plasma membrane enzyme 5′-nucleotidase (EC 3.1.3.5) was prepared from rat liver as a complex with sphingomyelin or in detergent-solubilized forms. Each preparation exhibited a K_m and Arrhenius break temperature indistinguishable from that of 5′-nucleotidase in plasma membranes. Measurement of fatty acid profiles, cholesterol and phospholipid content however showed a very wide variation between these preparations. We conclude that the biphasic nature of the Arrhenius plot of 5′-nucleotidase may be a property of the enzyme rather than its lipid environment.     

Effects of protein-modifying reagents on an isoenzyme of potato apyrase

Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on V_max. and K_m suggest different pK values for the amino acid residues at the ATP and ADP sites.     

Energy Metabolism Response to Low-Temperature and Frozen Conditions in Psychrobacter cryohalolentis

Studies of cold-active enzymes have provided basic information on the molecular and biochemical properties of psychrophiles; however, the physiological strategies that compensate for low-temperature metabolism remain poorly understood. We investigated the cellular pools of ATP and ADP in Psychrobacter cryohalolentis K5 incubated at eight temperatures between 22°C and −80°C. Cellular ATP and ADP concentrations increased with decreasing temperature, and the most significant increases were observed in cells that were incubated as frozen suspensions (<−5°C). Respiratory uncoupling significantly decreased this temperature-dependent response, indicating that the proton motive force was required for energy adaptation to frozen conditions. Since ATP and ADP are key substrates in metabolic and energy conservation reactions, increasing their concentrations may provide a strategy for offsetting the kinetic temperature effect, thereby maintaining reaction rates at low temperature. The adenylate levels increased significantly <1 h after freezing and also when the cells were osmotically shocked to simulate the elevated solute concentrations encountered in the liquid fraction of the ice. Together, these data demonstrate that a substantial change in cellular energy metabolism is required for the cell to adapt to the low temperature and water activity conditions encountered during freezing. This physiological response may represent a critical biochemical compensation mechanism at low temperature, have relevance to cellular survival during freezing, and be important for the persistence of microorganisms in icy environments.     

Early Temporal Changes in Ecto-Nucleotidase Activity after Cortical Stab Injury in Rat

During a variety of insults to the brain adenine nucleotides are released in large quantities from damaged cells, triggering multiple cellular responses to injury. Here, we evaluated changes in extracellular ATP, ADP and AMP hydrolysis at different times (0–24 hours) after unilateral cortical stab injury (CSI) in adult rats. Results demonstrated that 24 hours following CSI, ATP and ADP hydrolyzing activities were not significantly altered in injured cortex. Based on calculated V _ATP/V _ADP ratio it was concluded that ATP/ADP hydrolysis was primarily catalyzed by NTPDase1 enzyme form. In contrast, AMP hydrolysis, catalyzed by 5’-nucleotidase, was significantly reduced at least 4 hours following CSI. Kinetic analysis and Lineweaver-Burk transformation of the enzyme velocities obtained over the range of AMP concentrations (0.05–1.50 mM) revealed that inhibition of 5’-nucleotidase activity after CSI was of the uncompetitive type. Taken together our data suggest that injured tissue has reduced potential for extracellular metabolism of adenine nucleotides in early stages after CSI.     

Mathematical modeling of intracellular transport processes and the creatine kinase systems: a probability approach

A probability approach was used to describe mitochondrial respiration in the presence of substrates, ATP, ADP, Cr and PCr. Respiring mitochondria were considered as a three-component system, including: 1) oxidative phosphorylation reactions which provide stable ATP and ADP concentrations in the mitochondrial matrix; 2) adenine nucleotide translocase provides exchange transfer of matrix adenine nucleotides for those from outside, supplied from medium and by creatine kinase; 3) creatine kinase, starting these reactions when activated by the substrates from medium. The specific feature of this system is close proximity of creatine kinase and translocase molecules. This results in high probability of direct activations of translocase by creatine kinase-derived ADP or ATP without their leak into the medium. In turn, the activated translocase with the same high probability directly provides creatine kinase with matrix-derived ATP or ADP. The catalytic complexes of creatine kinase formed with ATP from matrix together with those formed from medium ATP provide activation of the forward creatine kinase reaction coupled to translocase activation. Simultaneously the catalytic complexes of creatine kinase formed with ADP from matrix together with those formed from medium ADP provide activation of the reverse creatine kinase reaction coupled to translocase activation. The considered probabilities were arranged into a mathermatical model. The model satisfactorily simulates the available experimental data by several groups of investigators. The results allow to consider the observed kinetic and thermodynamic iriegularities in behavior of structurally bound creatine kinase as a direct consequence of its tight coupling to translocase.     

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).     

Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

The ribose-modified chromophoric and fluorescent analog of ATP 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5′-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635–647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293–297). In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties. Their visible absorption and fluorescent properties were found to be quite similar. Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity. TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively. TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems. The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.     

A P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.