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.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.     

Demonstration of a novel ecto-enzyme on human erythrocytes, capable of degrading ADP and of inhibiting ADP-induced platelet aggregation

The role of ADP as an important inducer of platelet aggregation is generally accepted. Therefore it has been postulated by many authors that the enzymatic removal of extracellular ADP from the circulation is essential to avoid platelet aggregation and thrombus formation. Here we show that erythrocytes essentially contribute to the clearance of ADP. The removal of ADP from suspensions of washed human erythrocytes was due to at least two different activities. One activity, which had already been observed by earlier workers, was identified as adenylate kinase, on the basis of the reaction products and the inhibition by adenosine(5')pentaphospho(5')adenosine (Ap5A). This enzyme was not associated with the cells and was always detectable in cell-free supernatants, indicating that the enzyme had leaked from the cells into the extracellular medium. In contrast, the second activity, which is described here for the first time, was tightly bound to the cells. The activity was not inhibited by Ap5A. The main product of the reaction was AMP, and enzyme activity depended on the presence of divalent cations. The Michaelis constant was about 28 mumol/l. This activity seemed to be an ecto-ADPase. Studies with various inhibitors revealed that degradation of ADP was not due to a non-specific phosphatase. Besides the well known ADPase on the endothelium, the ecto-activity on erythrocytes may play an important part in destroying pro-aggregatory ADP.     

Purification and characterisation of bovine spleen ADPase.

ADPase has been purified from bovine spleen. Electrophoresis revealed a minor contaminent in the preparation that may represent ADPase that has been decreased in size by limited proteolysis during extraction and purification. The native enzyme behaves on SDS/PAGE as a 100-kDa protein but ADPase is a glycoprotein and so its electrophoretic behaviour may be anomalous. The apparent molecular mass is decreased to 70 kDa after removal of carbohydrate by treatment with a glycosidase. The use of a cross-linking reagent followed by electrophoresis suggests that the enzyme is composed of a single subunit. The specific activity of the purified material was 115 U/mg protein. The enzyme catalyses the hydrolysis of nucleoside di- and tri-phosphates but nucleoside monophosphates are not acted upon. The Km for ADP (approx. 10-15 microM) is unaffected by the state of purification of the enzyme, but catalytic activity of the purified material is stimulated by inclusion of detergent in the assay system and by calcium ions. Maximum activity is seen at pH 8.0-8.5 with ADP as substrate but the optimum shifts to 7.5-8.0 for ATP hydrolysis.     

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 rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 mM MgCl2, and the apparent Km was 20 microM. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, beta-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation parallel ouabain-sensitive (Na+-K+)ATPase and 5'-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.     

Phospholipase C axis is the preferential pathway leading to PKC activation following PTH or PTHrP stimulation in human term placenta

In the present report we describe an NTPDase 1 (ATP diphosphohydrolase; ecto-apyrase; EC 3.6.1.5) in rat hippocampal slices. The effect of glutamate on the ATPase and ADPase activities in rat hippocampal slices of different ages was also studied since adenosine, the final product of an enzymatic chain that includes NTPDase 1 and 5'-nucleotidase, can act upon A1 receptors in turn decreasing the release of glutamate. Hippocampal slices from 7, 14, 20-23 and 60 day-old rats were prepared and ATPase and ADPase activities were measured. The parallelism of ATPase and ADPase activities in all parameters tested indicated the presence of an ATP diphosphohydrolase. In addition, a Chevillard plot indicated that ATP and ADP are hydrolyzed at the same active site on the enzyme. ATPase activity was significantly activated by glutamate in 20-23 and 60 day-old rats, but ADPase activity was not activated. These results could indicate distinct behavior of the ATPase and ADPase activities of NTPDase 1 in relation to glutamate or the simultaneous action of the ecto-ATPase. Activation of ATPase activity by glutamate may constitute an important role in this developmental period, possibly protecting against the neurotoxicity induced by ATP, as well as producing high levels of ADP, by increasing adenosine production, a neuroprotective compound.     

Substitution of His59 converts CD39 apyrase into an ADPase in a quaternary structure dependent manner

The two transmembrane domains of CD39 ecto-apyrase regulate the formation of fully active homotetramers. We show that mutations in apyrase conserved region 1 (ACR1) have two dramatically different sets of effects determined by whether they occur in intact tetramers or in disrupted tetramers or monomers. In intact tetramers, substitution of H59 in the rat brain CD39 ACR1 with G or S abolishes more than 90% of the ATPase activity but less than 50% of the ADPase activity, converting the enzyme into an ADPase with relative ADP:ATP hydrolysis rates of 6:1 or 8:1, respectively. In contrast, the same substitutions in tetramers lacking either transmembrane domain, in monomers lacking both transmembrane domains, or in detergent-solubilized full-length monomers have no effect on ATPase activity and increase ADPase activity approximately 2-fold, resulting in equal ATPase and ADPase activities. N61R substitution has a much smaller effect on the ADPase:ATPase ratio in both cases. While the data for truncated and monomeric constructs are consistent with the proposed role of ACR1 as the beta-phosphate binding domain by analogy with the actin/hsp70/hexokinase superfamily, the finding that H59 substitutions in full-length CD39 primarily diminish the ATP hydrolysis rate suggests that ACR1 may play a different role in intact tetramers. We propose that CD39 uses different ATPase and ADPase mechanisms in different quaternary structure contexts, and that H59 in ACR1 plays a central role specifically in ATP hydrolysis in intact tetramers.     

ATPase and Adpase activities in synovial membrane of equine metacarpophalangeal joint

ATPase and ADPase activities capable of hydrolyzing nucleoside di- and triphosphates in the presence of Ca2+ are present in synovial membrane of metacarpophalangeal joint mainly associated to membrane fractions. These hydrolytic activities have been considered involved in the inflammatory process where ATP and ADP are inflammatory mediators while adenosine counteracts this effect. Both, subcellular localization and kinetic properties of these nucleotidase activities, suggest that could correspond to single enzyme called ATP-diphosphohydrolase or apyrase. The comparison of the activity on ATP-Ca and ADP-Ca from normal and pathological equine synovial membrane did not show significant differences either in the subcellular fraction distribution or in the enrichment of each subcellular fraction. Neither differences on 5'-nucleotidase activity present in the microsomal fraction were observed.     

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.     

A putative adenosine kinase family protein possesses adenosine diphosphatase activity

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as “adenosine-specific kinase” family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the “adenosine-specific kinase” family and suggests a need to reexamine its functional annotation.     

Characterization of ATP and ADP hydrolysis activity in rat gastric mucosa

The degradation of nucleotides is catalyzed by the family of enzymes called nucleoside triphosphate diphosphohydrolases (NTPDases). The aim of this work was to demonstrate the presence of NTPDase in the rat gastric mucosa. The enzyme was found to hydrolyze ATP and ADP at an optimum pH of 8.0 in the presence of Mg2+ and Ca2+. The inhibitors ouabain (0.01-1 mM), N-ethylmaleimide (0.01-4 mM), levamisole (0.10-0.2 mM) and Ap5A (0.03 mM) had no effect on NTPDase 1 activity. Sodium azide (0.03-30 mM), at high concentrations (>0.1 mM), caused a parallel hydrolysis inhibition of ATP and ADP. Suramin (50-300 microM) inhibited ATP and ADP hydrolysis at all concentrations tested. Orthovanadate slightly inhibited (15%) Mg2+ and Ca2+ ATP/ADPase at 100 microM. Lanthanum decreased Mg2+ and Ca2+ ATP/ADPase activities. The presence of NTPDase as ecto-enzyme in the gastric mucosa may have an important role in the extracellular metabolism of nucleotides, suggesting that this enzyme plays a role in the control of acid and pepsin secretion, mucus production, and contractility of the stomach.     

Salivary apyrase of Aedes aegypti: characterization and secretory fate

Salivary gland homogenates of female adult Aedes aegypti hydrolyzed ATP and ADP thereby defining an apyrase activity. Activity is divalent cation dependent with an optimum pH of 9.0. ATPase and ADPase activities could not be dissociated thus suggesting the presence of a true apyrase enzyme. Apyrase activity is low on the day of emergence but increases to 160 mU per pair of glands on the second day. The site at which mosquitoes probed into warm polyacrylamide gels retains apyrase activity, confirming the secretory fate of this activity.     

Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.     

Characterization and importance of the dimer interface of human calcium-activated nucleotidase

Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical as the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Tissue distribution of adenosine diphosphatase activity in the rat

The specific activity of adenosine diphosphatase (ADPase) was determined in rat tissue homogenates using a specific radioassay. 14 different tissues were sampled and activity was found in most homogenates examined. The levels ranged from very little activity in the pancreas to high levels in the duodenum and ileum. High levels of ADPase activity found in the gastrointestinal tract could be partially attributed to non-specific alkaline phosphatase. It is concluded that ADPase activity is widely distributed in rat tissues.     

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.     

The equilibrium constant and the reversibility of the reaction catalysed by nicotinamide-adenine dinucleotide kinase from pigeon liver.

The reversibility of the NAD+ kinase reaction was established, and the kinetic parameters of the rate equation in the reverse direction were determined. The equilibrium constant of the reaction was determined by using the purified pigeon liver enzyme and radioactively labelled nicotinamide nucleotides. The relationship between kinetic parameters of the forward and reverse reactions is in reasonable agreement with the measured equilibrium constant. As expected from the proposed mechanism of action, the enzyme does not catalyse isotope exchange between NAD+ and NADP+ in the absence of ADP and ATP. Although homogeneous as judged by polyacrylamide-gel electrophoresis, the enzyme preparation exhibits ADP/ATP isotope-exchange activity which could not be separated from NAD+ kinase activity, but kinetic evidence suggests that this is probably due to a contaminant.     

Multiple ecto-nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression

The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface-located enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K(m)-values of 283 microM and 243 microM, respectively. Using PCR we identified the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-specific alkaline phosphatase and ecto-5'-nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5'-nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto-nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto-nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.