Purification and characterization of NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) from porcine brain cortex synaptosomes Filip Kukulski and Michal Komoszyn ´ ski Department of Biochemistry, Institute of General and Molecular Biology, N. Copernicus University, Torun ´ , Poland We purified to homogeneity and characterized NTPDase1 and NTPDase2 from porcine brain cortex synaptosomes. SDS/PAGE and immunoblotting with antibodies specific to these enzymes revealed a molecular mass estimated at 72 kDa for NTPDase1 and 66 for NTPDase2. Both enzymes exhibited kinetic properties typical for all members of the NTPDase family, e.g. low substrate specificity for tri- and diphosphonucleosides, divalent cations dependency and insensitivity towards ATPase inhibitors. The calculated K m value for NTPDase1 in respect to ATP as a substrate (97 l M ) was three times lower in comparison to analogous values for NTPDase2 (270 l M ). Additionally, NTPDase1 had a three times higher K cat /K m coefficient than NTPDase2 (860 and 833 lmol productÆs )1 , respectively). We have also demonstrated that in spite of differences in the affinity of ATP for both hydrolases, these enzymes have similar molecular activity. Taken together, these results indicate that NTPDase1 would terminate P2 receptor-mediated signal transmission whereas activity of NTPDase2 may contribute to decreasing high (toxic) concentrations of ATP and/or to production of another signal molecule, ADP. Keywords: central nervous system; extracellular purines; P2 receptors; signal transmission; ecto-nucleoside triphosphate disphosphohydrolase. Extracellular ATP and ADP, as well as UTP and UDP participate in biological signaling (particularly, neurotrans- mission processes in the central nervous system, CNS) by activating nucleotide P2 receptors [1,2]. Nucleotide medi- ated signal transmission is terminated by hydrolysis of pyrophosphate bonds present in the agonist structure [3,4]. In the CNS, extracellular tri- and diphosphonucleosides are degraded by three representatives of NTPDase family of enzymes (NTPDase1–3) [5–7]. NTPDases cloned from CNS cells are integral cell membrane proteins that share high amino acid sequence homology [5,7,8]. Multiple sequence alignments of these enzymes show several regions of amino acid identity, termed apyrase conserved regions (ACR) [9–11]. ACR domains are thought to play a critical role in the binding and hydrolysis of substrates as site directed mutagenesis within these domains lead to the loss of biological activity of NTPDases or changed their affinity towards ATP and ADP [11–15]. NTPDase1 differs from NTPDase2 in respect to reaction products of ATP hydrolysis and in the ratio of the rate of ATP hydrolysis to the rate of ADP hydrolysis [16–19]. NTPDase1 degrades ATP and ADP directly to AMP, whereas NTPDase2 hydrolyses ATP to ADP [17]. NTPDase3 is a functional intermediate between NTPDase1 and 2, characterized by the ATP/ADP ratio of 3 [6]. Coexpression of NTPDase1 and NTPDase2 has been observed in some nerve structures [5,20]. Hitherto obtained results strongly indicate that NTPDase1 participates in the termination of P2 receptor-mediated signal transmission [4,21,22], whereas the function of NTPDase2 remains a matter of speculation. In this work we purified two NTPDases from porcine brain cortex synaptosomes. The physicochemical and biochemical properties of homogeneous preparations of these enzymes allowed us to classify them as NTPDase1 and 2. These results may contribute to the determination of biological function fulfilled by both ecto-nucleotidases. Materials and methods Materials Analytical grade reagents purchased from Fluka, Serva, Sigma, Merck, ICN, POCH (Gliwice, Poland) were used. Pig brains were obtained directly from the slaughterhouse. Electrophoresis, Western blotting and isoelectric focusing were performed in a Mini-Protean II apparatus obtained from Bio-Rad. Qualitative and quantitative purine analysis was performed using HPLC equipment from Pharmacia LKB, UV/VIS detector from Shimadzu, Supelcosil TM LC-18-DB column purchased from Supelco (15 cm · 4.6 mm, 5 lm) and computer software CHROMA from PolLab (Warsaw, Poland). MonoQ HR 5/5 column was obtained from Pharmacia. Nitrocellulose membrane NC2 was obtained from Serva. Silver stain kit and BCIP/NBT fast tablets (blue tetrazolium and 5-bromo-4-chloro-3- indolyl phosphate) were provided by Sigma. Ringo antibodies and BGO were obtained from A. Beaudoin Correspondence to F. Kukulski, Le Centre Hospitalier Universitaire de Que ´ bec (CHUQ), Centre de recherche ´ du pavillon CHUL, 2705 boulevard Laurier, local T1-49, Que ´ bec, Canada, G1V 4G2. Fax: + 1 418 654 2765, Tel.: + 1 418 654 2772, E-mail: Filip.kukulski@crchul.ulaval.ca Abbreviations: CNS, central nervous system; BGO, 1-hydroxy- naphtalene-3,6-disulfonic acid; NEM, N-ethylmaleimide; TBA, tetrabutyloammonium hydrogen sulfate. (Received 20 March 2003, revised 29 May 2003, accepted 30 June 2003) Eur. J. Biochem. 270, 3447–3454 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03734.x (Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Quebec, Canada). Isolation of NTPDases from synaptosomes Synaptosomes were isolated and purified by the method of Jones and Matus [23]. NTPDases were extracted from membranes using 0.9% polydocanol (w/v) in 10 m M Hepes/ OH buffer pH 7.6 containing 10% glycerol (v/v), 40 m M KCl, 1 m M EDTA and 1 m M phenylmethanesulfonyl fluoride. During extraction, a constant ratio of protein/ detergent was maintained at 1 : 3 (w/w). Synaptosomes were incubated in a detergent solution for 30 min at 0 °C, and subsequently centrifuged for 60 min at 100 000 g (Beckman centrifuge, 45 Ti rotor). The resulting super- natant was used for further purification. NTPDase purification The solubilized proteins was applied to a column (5 mm · 1.5 cm) filled with Con A/Sepharose 4B and equilibrated with 20 m M start buffer Tris/HCl pH 7.6 containing 75 m M NaCl, 1 m M CaCl 2 ,1m M MgCl 2 ,10% glycerol (v/v) and 0.05% polydocanol (w/v). The column was washed with start buffer and then glycoproteins were eluted from the column with 500 m M a-methylglucoside in the start buffer. The NTPDase enriched fractions were concentrated and separated on a Toyopearl HW55-S gel (2.5 cm · 60 cm column), equilibrated with 50 m M Tris/ maleate buffer pH 7.6 containing 100 m M KCl and 0.05% polydocanol (w/v). The fractions containing ATP/ADPase activity were concentrated, dialyzed and separated on a Mono Q HR 5/5 column equilibrated with 20 m M Tris/HCl buffer pH 8.0 with 0.05% polydocanol (w/v). The proteins were eluted from the column with a following KCl gradient in the start buffer: 0–10 min 0% KCl, 11–70 min from 0 to 100% (1.5 M ) KCl, 71–80 min from 100 to 0% KCl. All above described purification procedures were performed at the flow-rate of 1 mLÆmin )1 . Electrophoresis Electrophoresis under nondenaturing and denaturing con- ditions was performed according to the procedure described by Ogita and Markert [24]. The proteins were silver stained using Sigma Silver Stain Kit according to manufacturer’s instructions. Immunoblotting Proteins separated on a 10% acrylamide SDS gel were transferred to a Serva nitrocellulose membrane NC2. Electrotransfer was performed for 14 h at 4 °C according to a procedure described by Towbin et al.[25].The nitrocellulose membrane after washing and blocking with skimmed milk was incubated for 4 h in a solution of the primary rabbit antibody Ringo (anti-ACR4), diluted 2000 times in Tris-buffered saline. After several washes, the membrane was incubated with alkaline phosphatase conju- gated goat anti-rabbit Igs at a dilution of 1 : 30 000. The bands were visualized using blue tetrazolium and 5-bromo- 4-chloro-3-indolyl phosphate (BCIP/NBT Sigma fast tablets) as the substrates, in accordance to supplier’s instructions. Isoelectric focusing of NTPDase1 and NTPDase2 under nondenaturing conditions Native isoelectric focusing was performed according to the method described by Bollag and Edelstein [26]. The separated proteins were extracted from the gel with a solution of 0.1% polydocanol (w/v) in 200 m M Hepes/OH buffer pH 7.6. NTPDase activity in extracts was determined using HPLC. NTPDase1 and NTPDase2 activity assay Aliquots of 100 lL mixture containing 50 m M Hepes/OH pH 7.6, 3 m M CaCl 2 or 8 m M EGTA, 2 m M ouabain and 2m M nucleotides was preincubated for 10 min at 37 °C. The reaction was initiated by adding 100 lLofenzyme preparation. Reaction was performed for 30 min. Then the orthophosphate, nucleotide and nucleoside formed during reaction were analysed. Determination of orthophosphate liberated in the reaction The reaction was stopped by addition of 100 lL10%SDS (w/v) with 10 m M EDTA to the sample. The liberated orthophosphate was quantitated according to Hegyvary’s method [27] modified by Komoszynski and Skalska [28]. The activity of the enzymes was calculated based on a difference of colour intensity developed for samples con- taining Ca 2+ or EGTA. Determination of nucleotide reaction products using HPLC Enzymatic reactions were performed as described above and inhibited by addition of 100 lL incubation mixture to 100 lL cold (0–4 °C) 1 M perchloric acid. The sample was cooled immediately to 0 °C and centrifuged for 2 min in a microcentrifuge. The supernatant was neutralized with 1 M KOH (4 °C), centrifuged and then lipids were removed by extraction with n-heptane (5 : 1, v/v). Samples prepared in such a manner were analysed isocratically in 100 m M KH 2 PO 4 /K 2 HPO 4 buffer pH 7.0, containing 5 m M EDTA, 2.5% methanol (v/v) and 12.5 m M TBA. The flow-rate of eluent was 1 mLÆmin )1 . The separated fractions were analysed spectrophotometrically at k ¼ 260 nm. Protein determination Protein was determined by the Bradford method [29] or with bis-cinchonic acid [30] using bovine serum albumin as a standard. Results Isolation and purification Polydocanol at a concentration of 0.9% released 70% of ATP/ADPase active protein from purified synaptic 3448 F. Kukulski and M. Komoszyn ´ ski (Eur. J. Biochem. 270) Ó FEBS 2003 membranes. Further purification was performed by affinity, size-exclusion and ion exchange chromatography. The specific activity of purified NTPDase1 corresponded to 69 and 49 lmol P i Æmin )1 Æmg )1 for ATP and ADP as substrates, respectively. NTPDase2 had specific activity of 80 lmol P i Æmin )1 Æmg )1 protein in respect to ATP and 6.7 lmol P i Æmin )1 Æmg )1 protein in respect to ADP (Table.1). Both enzymes were homogeneous in a 5% acrylamide gel under nondenaturing conditions and in a 10% acrylamide SDS gel (Figs 1,2A). Ion exchange chromatography on a MonoQ HR 5/5 column was a crucial step of purification and separation of synaptosomal NTPDases. Chromatography on this carrier allowed not only a marked purification of both proteins but also separation of NTPDase1 from NTPDase2. The NTPDase2 was eluted from the MonoQ column with 105 m M KCl (Fig. 3). It readily hydrolysed ATP to ADP and poorly ADP to AMP (ATP/ADP ratio corresponded to 11.7) (Table 1, Fig. 4B). The protein eluted from the column with 225 m M salt exhibited properties characteristic for NTPDase1. This enzyme, with a ATP/ ADP ratio of 1.4 hydrolysed both ATP and ADP to AMP (Table 1, Figs 3,4A). Apparent molecular weights of purified ecto-nucleotidases during electrophoresis in SDS and Western blotting were estimated to be 72 kDa for NTPD- ase1 and 66 kDa for NTPDase2 (Fig. 2B). Kinetic properties Synaptosomal NTPDases differed slightly by isoelectric point and pH optima (Table 2). Isoelectric points were determined to be 5.1 for NTPDase1 and 5.4 for NTPDase2, whereas the optimum pH value, was, respectively, 7.6 and 7.8 independently of tested substrates (ATP or ADP). As expected, NTPDases from porcine synaptosomes exhibited a high specificity towards pyrophosphate bonds and low substrate specificity. They hydrolysed all purine and pyrimidine nucleotides tested. NTPDase1 preferentially hydrolysed ATP and UTP, whereas NTPDase2 UTP, TTP, ITP and ATP. Both enzymes did not hydrolyse ester bonds (Table 3). The purified hydrolases were activated by divalent cations, however, EDTA did not inhibit their activity completely (Table 4). Interestingly, ATP hydrolysis by both enzymes was more effective with Mg 2+ and Mn 2+ ions, whereas ADP hydrolysis was more effective with Ca 2+ ions. In the presence of Mg 2+ and Mn 2+ cations, the ATP hydrolysis rate increased three times, whereas ADP hydrolysis in the presence of these ions increased only 0.5 times (Table 4). Ca 2+ ions increased both ATP and ADP hydrolysis by 2.5 times. The highest activation of NTPDases by calcium ions was observed at 1.5 m M [Ca 2+ ]. Cu 2+ and Ba 2+ were strong inhibitors of the analysed enzymes, whereas the presence of monovalent ions in the incubation mixture did not affect the rate of ATP and ADP hydrolysis (Table 4). ATP and ADP were hydrolysed by synaptosomal NTPDases in accordance with Michaelis–Menten kinetics. Table 1. Purification of NTPDase1 and NTPDase2 from porcine brain cortex synaptosomes. Purification step Total protein (mg) Total activity (lmolÆP i Æmin )1 ) Specific activity (lmolÆP i Æmin )1 Æ mg of protien )1 ) ATP/ADP (ratio) Purification (-fold)ATP ADP ATP ADP Synaptosomes 240 42 15 0.18 0.06 2.8 1.0 MonoQ–NTPDase1 0.04 2.6 1.8 69.4 49 1.4 777 a MonoQ–NTPDase 0.004 0.35 0.03 79.6 6.7 12 455 b a In respect to ADP as substrate. b In respect to ATP as substrate. Fig. 1. PAGE of NTPDase1 and NTPDase2 during different steps of purification. Theproteinswereseparatedona5%acrylamidegel containing 0.4% Triton X-100 and then silver stained. An amount of protein (1–30 lg per well) was loaded. (A) Lane 1, polidocanol extract; lane 2, NTPDase2 after chromatography on MonoQ column; lane 3, NTPDase1 after chromatography on MonoQ column; lane 4, NTPDase-enriched fraction after affinity chromatography on Con-A Sepharose column; lane 5, NTPDases after molecular filtration. (B) NTPDase activity in acrylamide gel was assayed in 50 mm Hepes pH 7.4 containing 10 m M Ca 2+ and 1 m M ATP or ADP. In the spots containing NTPDase activity white precipitates of calcium phosphate were formed. Lanes 6 and 7, NTPDase activity with ADP and ATP as substrates, respectively. Ó FEBS 2003 NTPDase1 and NTPDase2 from porcine synaptosomes (Eur. J. Biochem. 270) 3449 NTPDase1 had a similar K m for ATP and ADP (97 ± 0.09 l M and 95 ± 0.13 l M , respectively; Table 2). Affinity of ADP to NTPDase2 (K m ¼ 5.5 ± 0.07 l M )was more than 20 times lower than the affinity of ATP to this enzyme (K m ¼ 270 ± 1.2 l M ) (Table 2). Despite the dif- ferences in affinity of ATP of NTPDase1 and NTPDase2, both enzymes possessed a similar molecular activity of 860 and 833 lmol productÆs )1 , respectively. Simultaneously, NTPDase1 characterized by a three times higher K cat /K m coefficient (9 · 10 6 M )1 Æs )1 ) in comparison with the ana- logous coefficient for NTPDase2 (3 · 10 6 )(Table2). The activity of analysed enzymes was inhibited by sodium azide, 8-butyl-thioATP and suramin, all known to inhibit NTPDases [3,31–33]. ATPase inhibitors, e.g. oua- bain, oligomycin and sodium orthovanadate, did not change the rate of ATP and ADP hydrolysis. NTPDase inhibition by azide was noncompetitive (data not shown). NTPDase1 was more sensitive to azide than NTPDase2 (Table 5). Synaptosomal NTPDases were also noncom- petitively inhibited by suramin, an antagonist of P2 receptors and simultaneously an inhibitor of ecto-ATPases [2,31]. This compound decreased activity of analysed hydrolases to a similar extent (Table 5). The strongest inhibitor of both enzymes was BGO (Table 5), that was shown previously to inhibit ATP/ADPase of bovine spleen fractions [33]. This compound decreased the activity of synaptosomal NTPDases competitively (data not shown). Purified ecto-hydrolases of pork brain synaptosomes in contrast to ATP-diphosphohydrolase from rat brain cortex synaptosomes [34], were insensitive to -SH group reagents, i.e., NEM and p-chlorohydroksymercuric benzoic acid (data not shown). Fig. 2. SDS/PAGE and Western blot of NTPDase1 and NTPDase2 separated by ion-exchange chromatography on MonoQ column. NTP- Dase1 and 2 obtained after ion-exchange chromatography were elec- trophorized through 10% SDS/acrylamide gel and transferred onto a nitrocellulose sheet. (A) An amount of 10 ng of protein was applied per well and the proteins were silver stained. Apparent molecular masses for NTPDase1 and NTPDase2 were estimated to be 72 and 66 kDa, respectively. Lane 1, molecular mass markers; lane 2, NTPDase1; lane 3, NTPDase2. (B) Nitrocellulose sheet was incubated with primary rabbit antibodies (RINGO) and then the bands were visualized using alkaline phosphatase conjugated anti-rabbit Igs and BCIP/NBT Sigma fast tablets as described. Lane, 4, molecular mass markers; lane 5, NTPDase1; lane 6, NTPDase2. Fig. 3. Chromatography of NTPDase1 and NTPDase2 on MonoQ column. 3450 F. Kukulski and M. Komoszyn ´ ski (Eur. J. Biochem. 270) Ó FEBS 2003 Discussion The presence of NTPDase1 and NTPDase2 in membranes of brain cells was confirmed previously by analysis of a cDNA library and Northern blot [5,7,10]. In the present work, we have demonstrated the presence of both enzymes in the synaptosomes of pig brain cortex. NTPDase1 and 2 were purified to homogeneity and revealed kinetic proper- ties typical for representatives of this group of enzymes, i.e., low substrate specificity for tri- and diphosphonucleosides, activation by divalent cations, and sensitivity to inhibitors [3,8]. In various brain structures, receptors activated by ATP, ADP, UTP and UDP occur simultaneously [2,35]. The investigated enzymes hydrolysed effectively, not only ATP, but also UTP and CTP, suggesting that they may partici- pate in interruption of the signal transmitted by nucleotides other than adenine. We observed the high activation of ATP hydrolysis (over 300%) by Mg 2+ and Mn 2+ ions and low activation of ADP hydrolysis by the same ions (less than 48%). In contrast, calcium ions stimulated the hydrolysis of both substrates in equal degrees (200%). The majority of the enzymes hydrolysing ATP prefer Mg 2+ –ATP complex as a substrate [3]. NTPDase2 derived from chicken gizzard smooth muscle was activated the most strongly by Mg 2+ [36], whereas NTPDase1 from human placenta most efficiently hydrolysed ATP in the presence of Ca 2+ [37]. Our results indicate that the complexes Mg 2+ –ATP or Mn 2+ –ATP are preferred as substrate by synaptosomal NTPDases, while in the case of ADP, the Ca 2+ –ADP complexes are hydrolysed most effectively. Results of previous examinations have indicated that NTPDases show a high similarity in structure [4–6,10]. All inhibitors used in our experiments changed the activity of NTPDase1 and NTPDase2 to a similar extent. This confirms that both purified NTPDases belong to the same group of enzymes. So far, the metabolic function fulfilled by NTPDase2 in the synapse has not been explained. Homogeneity of the obtained preparations allowed a precise analysis and comparison of the kinetic properties of both enzymes. The results of these experiments may help to define potential roles of NTPDase1 and NTPDase2 in the neurotransmis- sion mediated by nucleotides. We found that ATP has three times higher affinity to NTPDase1 than to NTPDase2. At the same time, both enzymes have similar molecular activity. This indicates that under high substrate concen- tration (close to V max for NTPDase2), the active sites of both enzymes are fully saturated by ATP. Under such conditions, both NTPDase1 and NTPDase2 will hydrolyse ATP with a similar velocity. However, NTPDase1 has a three times higher K cat /K m coefficient than NTPDase2. This Fig. 4. Products of ATP hydrolysis in the reaction catalysed by purified NTPDases. Reactions were carried out in the presence of 1 mm ATP. Products of ATP degradation were analysed using HPLC RP. (A) NTPDase1 hydrolysed ATP directly to AMP, (B) NTPDase2 hydro- lysed ATP to ADP; (—–) control (– – –) after 10 min (- - - -) after 20 min. Table 2. Physicochemical properties of purified NTPDases. Woolf–Augustinsson–Hofstee plot and GRAPHPAD PRISM softwarewereusedtoevaluate K m and V max withATPandADPconcentrationfrom0.01to2m M . Molecular activity and K cat /K m coefficient were calculated for ATP as a substrate. Values are expressed as mean ± SEM of three separate experiments, each conducted in triplicate. Properties NTPDase1 NTPDase2 Optimum pH 7.6 7.8 Isoelectric point 5.1 5.4 K m values K m(ATP) ¼ 97 ± 0.09 l M K m(ATP) ¼ 270 ± 1.2 l M K m(ADP) ¼ 95 ± 0.13 l M – Molecular activity 860 lmol of product per s 833 lmol of product per s K cat /K m coefficient 0.9 · 10 7 M )1 Æs )1 0.3 · 10 7 M )1 Æs )1 Apparent molecular mass 72 kDa 66 kDa Ó FEBS 2003 NTPDase1 and NTPDase2 from porcine synaptosomes (Eur. J. Biochem. 270) 3451 in turn suggests that when the ATP concentration in the extracellular space is low (close to V max for NTPDase1), ATP will be hydrolysed three times faster by NTPDase1 than by NTPDase2. Previously obtained results imply that the function of NTPDase1 in the neurotransmission processes is the removal of agonists (by conversion) from the P2 receptors [4]. ATP concentration in synapses, even after stimulation of neurons, does not exceed 20 l M [38]. Thus, under physiological conditions ATP would be predominantly hydrolysed by NTPDase1, having a three times higher K m for ATP than NTPDase2. Moreover, NTPDase1 has similar K m values with respect to ATP and ADP and the product of ATP and ADP hydrolysis is AMP – the compound with a very low affinity for P2 receptors. The above results confirm the participation of NTPDase1 in the scavenging of P2 receptor agonists. However, in some situations, e.g., during an epilepsy seizure or due to hypoxia or ischemia, the extracellular ATP concentration increases reaching milimolar values [39–41]. High mili- molar ATP concentrations are cytotoxic, thus, there is a requirement for their rapid degradation [40,41]. With the increase of ATP concentration to millimolar levels the catalytic activity of NTPDase2 rises distinctly and attains, due to a similar molecular activity, a value similar to that of NTPDase1. It indicates that NTPDase2 could be responsible for the removal of high cytotoxic ATP concentrations from the synaptic cleft. On the other hand, it should be remembered that even at low ATP concentrations, NTPDase2 will be active and as a result of its activity another signal molecule, ADP, will be formed. Then, the amount of ADP would increase with the increase of ATP concentration in the synapse. Moreover, in nerve cells, a colocalization between P2Y 2 receptors (stimulated by ATP) and P2Y 1 receptors (stimulated by ADP) has been observed [2] . The latter activated by ADP can participate in the inhibition of releasing of neurotransmitters, including those involved in metabolism stimulation [42,43]. As a consequence, the extent of injury caused by anoxia, may be limited. In summary, NTPDase2 activity could contribute to a lowering of high toxic concentrations of ATP on the one hand and to production of another signal molecule, ADP, on the other. The latter function seems possible, as there is no evidence that ADP is secreted from the cell by exocytosis Table 3. Substrate specificity of NTPDase1 and NTPDase2 from por- cine brain cortex synaptosomes. Reactions were carried out for 20 min inthepresenceof1m M substrates and liberated orthophosphate was measured as described in Materials and methods. The specific activity obtained with ATP as a substrate corresponds to 67.1 and 78.4 lmol P i Æmin )1 Æmg protein )1 for NTPDase1 and 2, respectively. NTPDase1 hydrolysed tri- and diphosphonucleosides with a similar velocity, whereas NTPDase2 preferred triphosphonucleosides as substrates. Both enzymes did not hydrolyse AMP and pNPP. Substrate (1 m M ) NTPDase1 activity NTPDase2 activity ATP 1.00 1.00 UTP 0.94 1.11 GTP 0.91 0.98 CTP 0.87 0.91 TTP 0.82 1.06 ITP 0.78 1.02 ADP 0.71 0.08 UDP 0.56 0.12 GDP 0.61 0.05 CDP 0.42 0.00 TDP 0.44 0.00 AMP 0.00 0.00 pNPP 0.00 0.00 Table 4. Effect of divalent cations on NTPDase1 and NTPDase2 activity. The influence of divalent cations on NTPDase1 and 2 activity was determined in the presence of 1.5 m M ions and 1 m M ATP or ADP as substrates. Na + /K + ions were used to exclude contamination of Na + /K + - ATPase, and they did not affect activity of examined enzymes. 5 m M EDTA inhibited activity of synaptosomal NTPDases, however, not completely. Results are average ± SEM of three different experiments, each carried out three times. Cation (1.5 m M ) NTPDase1 activity (lmol P i Æmin )1 Æmg protein )1 ) NTPDase2 activity (lmol P i Æmin )1 Æmg protein )1 ) ATP ADP ATP ADP Control 25.6 ± 1.2 18.7 ± 1.5 34.4 ± 2.4 2.6 ± 0.1 Ca 2+ 69.4 ± 2.1 50.1 ± 2.1 81.7 ± 3.2 6.6 ± 1.4 Mg 2+ 94.7 ± 4.6 30.5 ± 1.3 121.8 ± 5.7 3.2 ± 0.1 Mn 2+ 84.2 ± 1.8 25.4 ± 0.7 115.9 ± 2.2 3.1 ± 0.1 Zn 2+ 19.4 ± 3.0 0.00 11.7 ± 3.0 0.00 Ba 2+ 4.1 ± 0.7 0.00 8.3 ± 2.6 0.00 Cu 2+ 1.3 ± 0.1 0.00 5.5 ± 1.0 0.00 100 m M Na + /4 m M K + 26.1 ± 1.1 18.1 ± 1.3 34.1 ± 0.9 2.5 ± 0.9 5m M EDTA 10.8 ± 0.9 7.3 ± 0.7 12.7 ± 1.7 0.8 ± 0.3 Table 5. Inhibition of NTPDase1 and NTPDase2 by BGO, suramin and sodium azide. Inhibition constants were estimated using Dixon plots. Inhibitor NTPDase1 NTPDase2 BGO K i(ATP) ¼ 103 ± 1.2 l M K i(ATP) ¼ 150 ± 2.6 l M K i(ADP) ¼ 70 ± 0.9 l M Suramin K i(ATP) ¼ 1.8 ± 0.6 m M K i(ATP) ¼ 2.1 ± 0.03 m M K i(ADP) ¼ 1.7 ± 0.04 m M Sodium azide K i(ATP) ¼ 12 ± 0.6 m M K i(ATP) ¼ 45 ± 1.2 m M K i(ADP) ¼ 5 ± 0.7 m M 3452 F. 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Purification and characterization of NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) from porcine brain cortex synaptosomes Filip Kukulski and Michal. specificity of NTPDase1 and NTPDase2 from por- cine brain cortex synaptosomes. Reactions were carried out for 20 min inthepresenceof1m M substrates and liberated