Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin
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The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5’-triphosphates and nucleoside 5’-diphosphates with varying preference for the individual type of nucleotide. We report the cloning and functional expression of rat NTPDase3. The rat brain-derived cDNA has an open reading frame of 1590 bp encoding 529 amino acid residues, a calculated molecular mass of 59.1 kDa and predicted N- and C-terminal hydrophobic sequences. It shares 94.3% and 81.7% amino acid identity with the mouse and human NTPDase3, respectively, and is more closely related to cell surface-located than to the intracellularly located members of the enzyme family. The NTPDase3 gene is allocated to chromosome 8q32 and organized into 11 exons. Rat NTPDase3 expressed in CHO cells hydrolyzed both nucleoside triphosphates and nucleoside diphosphates with hydrolysis ratios of ATP:ADP of 5:1 and UTP:UDP of 8:1. After addition of ATP, ADP is formed as an intermediate product that is further hydrolyzed to AMP. The enzyme is preferentially activated by Ca2+ over Mg2+ and reveals an alkaline pH optimum. Immunocytochemistry confirmed expression of heterologously expressed NTPDase3 to the surface of CHO cells. PC12 cells express endogenous surface-located NTPDase3. An immunoblot analysis detects NTPDase3 in all rat brain regions investigated. An alignment of the secondary structure domains of actin conserved within the actin/HSP70/sugar kinase superfamily to those of all members of the NTPDase family reveals apparent similarity. It infers that NTPDases share the two-domain structure with members of this enzyme superfamily.
Key wordsactin apyrase ATP ectonucleotidase NTPDase PC12 cell
apyrase conserved region
Chinese hamster ovary
Dulbecco’s modified Eagle’s medium
ecto-nucleoside triphosphate diphosphohydrolase
expressed sequence tag
nerve growth factor
polymerase chain reaction
open reading frame
The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that differ regarding tissue distribution, cellular location and substrate specificity. They hydrolyze nucleoside 5′-triphosphates and nucleoside 5′-diphosphates with varying preference for the individual type of nucleotide. Depending on subtype their catalytic site may be in ectoposition facing the extracellular medium or it may be located in the lumen of intracellular organelles. Whereas the surface-located members of the protein family are thought to be involved mainly in the control of ligand availability for P2 receptors, the functional role of the intracellular members is less defined [1, 2].
The gene family has members within vertebrates, invertebrates, plants, yeast and protozoans but has not been identified in bacteria (references in [1, 3, 4, 5]). Hallmarks of all E-NTPDases are five highly conserved sequence domains (‘apyrase conserved regions,’ [3, 4, 6]) that presumably are involved in the catalytic cycle. Interestingly, E-NTPDases share two common sequence motifs with members of the actin/HSP70/sugar kinase superfamily, the actin-HSP 70-hexokinase β- and γ-phosphate binding motif [(I/L/V)X(I/L/V/C)DXG(T/S/G)(T/S/G)XX(R/K/C)] [3, 7, 8, 9], with the DXG sequence strictly conserved.
In contrast to the members of the actin/HSP70/sugar kinase superfamily, mammalian E-NTPDases are membrane- anchored proteins with one or two transmembrane domains. NTPDase5 and NTPDase6 contain a single predicted N-terminal hydrophobic domain. They are located to the endoplasmic reticulum or Golgi apparatus respectively but they can also be released in soluble form from transfected cells [10, 11, 12]. They preferentially hydrolyze nucleoside diphosphates. The two forms of NTPDase4, that differ by alternative splicing, have predicted N- and C-terminal transmembrane domains and were allocated to the Golgi apparatus (UDPase)  and to lysosomal/autophagic vacuoles (LALP70) [14, 15], respectively. The Golgi enzyme hydrolyzes a number of nucleoside 5′-di and triphosphates but not ATP and ADP. NTPDase7  is localized to intracellular organelles and hydrolyses a variety of nucleoside triphosphates with the exception of ATP. In mammals, four different surface-located subtypes of E-NTPDases have been cloned and characterized. They share a membrane topography with N- and C-terminal transmembrane domains: NTPDase1 [17, 18, 19, 20], NTPDase2 [8, 21], NTPDase3 [9, 22, 23], and most recently NTPDase8 . They all hydrolyze nucleoside tri- and diphosphates but differ significantly in catalytic properties .
Surface-located mammalian E-NTPDases have been cloned and characterized mainly from rat, human and mouse tissues. Since these enzymes vary regarding substrate preference and product pattern formation it is necessary to compare the catalytic properties of individual enzymes within the same species. We have previously cloned and characterized rat NTPDase1 and rat NTPDase2 [8, 20]. Here we report the cloning and characterization of rat NTPDase3. We analyze its functional properties and tissue distribution in brain and compare some of the key properties of primary and secondary structure to those of other members of the gene family.
Materials and methods
Rat brain cDNA library SuperScripti™, reverse transcriptase SuperScripti™II, Trizoli™ reagent, oligo(dT)-cellulose, fetal calf serum, horse serum, DMEM (Dulbecco’s modified Eagle’s medium), penicillin and streptomycin, and trypsin/EDTA solution was obtained from Invitrogen (Karlsruhe, Germany). The cell culture medium Ham’s F12 was from PAA Laboratories (Cölbe, Germany). Sawady Pwo DNA Polymerase was from PeqLab (Erlangen, Germany). Nucleoside triphosphate and diphosphate sodium salts, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), β-subunit of nerve growth factor (NGF) and proteinase K were purchased from Sigma-Aldrich (Taufkirchen, Germany). Concanavalin A (Con-A)Ysepharose, desoxycytidine 5′[α32P]-triphosphate and the enhanced chemiluminescence system were from Amersham Biosciences (Freiburg, Germany). Protease inhibitors antipain, aprotinin, benzamidine, chymostatin, leupeptin, and pepstatin were obtained form Calbiochem (Schwalbach, Germany). Restriction endonucleases were purchased from MBI Fermentas (St. Leon-Rot, Germany) or Invitrogen (Karlsruhe, Germany). BOA protein marker was from Biomol (Hamburg, Germany) and protran nitrocellulose membranes from Schleicher & Schuell (Dassel, Germany). Nucleobond AX 500 plasmid purification kit and Nucleo-Trap DNA purification kit were from Machery-Nagel (Düren, Germany). Synthetic oligonucleotides were obtained from BioSpring (Frankfurt, Germany), indocarbocyanin-3 (Cy3)-labeled anti-rabbit IgG antibody was from Dianova (Hamburg, Germany).
cDNA library screening
Total RNA from rat brain was isolated with Trizol reagent. For isolation of polyadenylated RNA from total RNA, oligo(dT)-cellulose was used according to the manufacturer’s instructions. A cDNA library was synthesized with SuperScript® II from 0.5 µg of mRNA with an oligo(dT)18 primer in accordance with the manufacturer’s instructions. Two mouse sequences from expressed sequence tag (EST) databases (GenBank accession numbers bf302156 and w46136) were used for primer design. As a probe, a 288-bp PCR fragment was amplified using forward primer 5′-CCGTCCCTGCTCCCAAGATTT-3′, reverse primer 5′-CAGGCACAGCAAGGCGATAGC-3′, and the rat brain cDNA library as a template. For library screening, electrocompetent Escherichia coli DH5α were transformed with a rat brain pCMV-SPORT 2 cDNA library amplified and plated on LuriaYBertani/ampicillin agar plates. The resulting transformants were screened by colony hybridization with the 288-bp cDNA fragment labeled with [α-32P]dCTP by PCR. Positive signal areas were amplified and rescreened for single positive colonies.
cDNA sequencing and computational sequence analysis
DNA sequencing was performed by Scientific Research and Development GmbH (Oberursel, Germany). Primer walking in both directions was employed for obtaining the complete full length sequence of the cDNA clone 18.104.22.168. The Omiga 2.0 sequence analysis program (Oxford Molecular Ltd., Oxford, UK) was used for assembling sequence fragments, translating DNA into amino acid sequences, generating hydrophobicity blots and amino acid alignment (CLUSTAL W algorithm). To align the amino acid sequences for the dendrogram, ClustalX 1.81 and for the graphic depiction BoxShade v3.31c were used. For prediction of transmembrane domains, the software TMHMM 2.0 (www.cbs.dtu.dk/services/TMHMM-2.0) was employed. For signal peptide and sorting analysis, SignalP 3.0 (www.cbs.dtu.dk/services/SingalP/) and PSORT II (http://psort.nibb.ac.jp/form2.html) were used. The DNA and deduced amino acid sequences were analyzed for similarity to known sequences with the NCBI Blast Network service (www.ncbi.nlm.nih.gov/BLAST/). Protein motif search was performed using the prosite database (www.expasy.org/prosite/). Secondary structure prediction of the amino acid sequences was performed with the SSpro tool (www.igb.uci.edu/tools/scratch/). The genomic library was screened using BLAST and the splice analysis of the genomic sequence was performed using the splice site analysis tool www.fruitfly.org/seq-tools/splice.html.
Expression of recombinant proteins
For recombinant expression, the EcoRI/NotI cDNA fragment of clone 22.214.171.124 was cloned into the EcoRI/NotI sites of pcDNA3. Chinese hamster ovary (CHO) cells were cultured as previously described . Cells were transfected by electroporation with the rat NTPDase3 pcDNA3 plasmid or with plasmids expressing rat NTPDase1  or NTPDase2  in electroporation buffer (in mM: 137 NaCl, 5 KCl, 0.7 Na2HPO4, 6 dextrose, 20 Hepes, pH 7.0) using a BTX Electrocell manipulator 600. In control experiments cells were transfected with empty vector alone. Twenty-four hours after electroporation the culture medium of transfected CHO cells was exchanged to remove dead cells and debris.
Preparation of membrane fractions
Forty-eight hours after electroporation, the culture medium was removed, cells were washed twice with isotonic buffer (in mM: 140 NaCl, 5 KCl, 0.5 EDTA, 20 MOPS, pH 7.4) and scraped from the plates in ice cold homogenization buffer (in mM: 250 sucrose, 2 EDTA, 2 iodoacetamide, 30 MOPS, pH 7.4) containing a mixture of protease inhibitors (in µg/ml: 2 chymostatin, 2 aprotinin, 1 pepstatin, 150 benzamidine, 2 antipain, 2 leupeptin). After centrifugation at 300 gav cells were resuspended in homogenization buffer, homogenized, and centrifuged for 10 min at 300 gav at 4 °C. The resulting supernatant was sonicated and subsequently centrifuged at 100,000 gav for 60 min at 4 °C, pellets were resuspended in storage buffer (in mM: 2 iodoacetamide, 25 Hepes, pH 7,4) containing the protease inhibitor mixture and 50% (v/v) glycerol, and stored at −20 °C until further processing. For preparation of membrane fractions from various brain tissues, Wistar rats obtained from Charles River Wiga (Sulzfeld, Germany) were used. Animals were anaesthetized with CO2, decapitated, the brain was dissected, homogenized in five volumes of homogenization buffer containing the protease inhibitor mixture and further processed as described above.
Measurement of nucleotidase activities
Nucleotidase activity was determined by measuring the formation of Pi liberated from nucleotides . Membrane fractions were incubated in phosphate-free solution containing 500 µM CaCl2 and 25 mM Hepes (pH 7.4), and 500 µM nucleoside tri- or diphosphates, respectively, at 37 °C. Samples were heat-inactivated for 2 min at 95 °C prior to determination of inorganic phosphate. To investigate the dependence of enzyme activity on metal ions, 50–400 µM CaCl2 or MgCl2 was added, or CaCl2 and MgCl2 were replaced by 1 mM EDTA. pH-dependency was determined using a combined buffer (25 mM Hepes and 50 mM glycine) ranging from pH 3 to 10, containing 500 µM ATP and 500 µM CaCl2 or 500 µM MgCl2. Catalytic activity of membrane fractions derived from cells transfected with the empty plasmid was subtracted from that obtained with cDNA-transfected cells. It was verified for each experimental condition applied that hydrolysis rates were constant (10–30 min). At the end of the reaction with nucleoside triphosphate it was ensured that less than 10% of the initial substrate was hydrolysed.
For determination of product formation following ATP hydrolysis (250 µM ATP, 250 µM CaCl2), aliquots were collected at various time points and subjected to HPLC analysis. Following heat inactivation, samples were centrifuged at 20,000 gav for 15 min (4 °C). ATP, ADP, AMP, and adenosine were separated by a Sepsil C18 reverse phase column (Jasco, Groβ-Umstadt, Germany) and eluted with the mobile phase, consisting of 50 mM potassium-phosphate buffer (pH 6.5), 6% methanol and 5 mM tetrabutylammonium hydrogen sulfate . The absorbance at 260 nm was continuously monitored and nucleotide concentration was determined from the area under each absorbance peak.
The anti-rat NTPDase3 antibody (N3-3i4) was raised in rabbits by direct injection of the encoding NTPDase3-pcDNA3 plasmid . Previous to Western blot analysis of membrane fractions from transfected CHO cells or mouse brain tissues, NTPDase3 was enriched using ConAY Sepharose. The 100,000 gav pellet was solubilized in ConA-buffer (in mM: 150 NaCl, 2 MgCl2, 2 CaCl2, 2 MnCl2, 20 Tris/HCl, pH 7.4) containing 0.1% Triton X-100. After an overnight incubation with ConA-Sepharose at 4 °-C, the ConA-Sepharose was washed several times with ConAbuffer containing 0.1% Triton X-100, and protein was eluted with the same buffer containing 200 mM methyl-α-d-mannopyranoside. For immunoblotting the ConA eluate was precipitated with 10% trichloroacetic acid and the pellet was resuspended in sample buffer without reducing agents. Polyacrylamide gel electrophoresis was carried out on minigels (10% acrylamide). Immunoblotting using the polyclonal NTPDase3 antibody (dilution 1:4000) was performed using the Amersham enhanced chemiluminescence system according to the manufacturer’s instructions.
Transfected CHO cells (15,000 cells per well) or nontransfected PC12 cells (15,000 cells per well) were seeded onto poly-d-lysine-coated (10 µg/ml) glass cover slips (10 mm diameter) and cultured for 2 days (CHO cells) or up to 14 days (PC12 cells) . An aliquot of 50 ng/ml of NGF was added to PC12 cells every 3–4 days. For surface labeling, the anti-rat NTPDase3 antibody (1:1000) was applied to viable cells for 15 min at 37 °C followed by repeated washing with phosphate-buffered saline (PBS, in mM: 137 NaCl, 3 KCl, 15 Na+/K+ phosphate buffer, pH 7.4) at room temperature, methanol fixation at −20 °C and application of Cy3-conjugated anti-rabbit IgG antibody together with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, 1 µg/ml). After immunolabeling, cells were mounted and investigated with an epifluorescence microscope equipped with an MCID 4 imaging analysis system (Imaging Research, St. Catherines, Canada).
Cloning and sequencing of rat NTPDase3
Relation to other members of the E-NTPDase family
The deduced amino acid sequence of the cloned rat NTPDase3 shares 94.3% and 81.7% amino acid identity, respectively, with the mouse  and human  orthologs. Compared to other rat members of the E-NTPDase family, NTPDase3 is most closely related to cell surfacelocated NTPDase2, NTPDase8, and NTPDase1 (36.9%, 36.3%, 35.1% identity, respectively). It is more distantly related to the intracellularly located rat NTPDase5 and rat NTPDase6 (17.8% and 15.2% identity, respectively). The graphic depiction of a multiple sequence alignment of 22 mammalian members of the E-NTPDase family illustrates the subdivision of the family into intracellular and surfacelocated enzymes (Figure 3). Whereas the origins of the branches for the four surface-located members (NTPDase1, NTPDase2, NTPDase3, NTPDase8) are located at close distance to each other, the intracellular members are divided into two sub-branches. One sub-branch consists of NTPDase4 and NTPDase7 that share their membrane topology with the surface-located enzymes. The other subbranch consisting of NTPDase5 and NTPDase6 is characterized by a single N-terminal hydrophobic domain, which can be cleaved resulting in the formation of a soluble enzyme .
Secondary structure prediction and relation to the actin/HSP70/sugar kinase superfamily
The similarities in the distribution of the ACRs between the members of the E-NTPDase family imply structural conservation relevant for catalytic activity. It has previously been emphasized that NTPDases share two nucleotide binding motifs with the actin/HSP70/sugar kinase superfamily [3, 8, 9]. Members of this superfamily share little overall sequence identity except for the strictly conserved DXG motifs in the consensus sequence. These motifs can be identified in ACR1 and ACR4 of all ENTPDases.
The degree of similarity in secondary structure mirrors the phylogenetic distance between sequences (comp. Figure 3). In all NTPDases, the sequences ACR1 and ACR4 are situated between two β strands. The alignment not only implies considerable structural conservation between E-NTPDases and members of the actin/HSP70/sugar kinase superfamily. It also suggests that NTPDases — in spite of their membrane anchorage — may consist of two major domains that repeat basic topology and key conserved sequence domains.
Catalytic properties of heterologously expressed rat NTPDase3
Substrate specificity of recombinant rat NTPDase3 in membrane fractions of transfected CHO cells.
Activity (% of ATPase activity)
Activity (% of ATPase activity)
20.1 ± 1.3
126.2 ± 21.9
13.3 ± 2.4
103.2 ± 11.1
25.0 ± 3.2
92.1 ± 8.8
20.2 ± 0.9
109.5 ± 20.6
14.3 ± 6.0
Antibody production and enzyme distribution
Surface staining revealed endogenous expression of NTPDase3 by the rat-derived PC12 cells (Figure 9e). Immunofluorescence was observed over the entire cell surface. The formation of small immunofluorescent dots implies a partial clustering of the protein. In addition, immunostaining was enhanced at growth cones. Application of preimmune serum either to CHO or PC12 cells yielded negative results (not shown).
Rat NTPDase3 differs significantly in its functional properties from the two other surface-located NTPDases previously isolated from rat, namely NTPDase1 and NTPDase2. Whereas rat NTPDase1 expressed in CHO cells exhibits the typical catalytic properties of an apyrase with a ratio of hydrolysis rates for ATP and ADP of 1:0.8, rat NTPDase2 has dominant ATPase activity with a ratio of 1:0.05–1:0.03 [8, 20, 35]. When expressed in CHO cells, rat NTPDase3 reveals an ATP to ADP hydrolysis ratio of 1:0.2 and thus represents a functional intermediate between the two other NTPDases. This difference is further underlined when the product formation following hydrolysis of ATP is analyzed. Whereas NTPDase1 hydrolyzes ATP directly to AMP with minimal formation of free ADP, ADP accumulates in the reaction medium following hydrolysis of ATP by NTPDase2 . NTPDase3 reveals intermediate properties. In the presence of ATP, NTPDase3 accumulates extracellular ADP which is finally converted to AMP. Rat NTPDase3 differs significantly from rat NTPDase1 and NTPDase2 regarding cation dependence. Whereas rat NTPDase1 and NTPDase2 are equally activated by Ca2+ or Mg2+ , rat NTPDase3 shows a clear preference for activation by Ca2+. In addition, ATPase and ADPase activities are differentially affected by Ca2+ or Mg2+. This differential activation remains unexplained and may depend on the difference in phosphate chain length between ATP and ADP and thus the potential coordination with the respective metal cation within the protein. Removal of divalent cations abolishes catalytic activity of all three enzymes. None of these enzymes hydrolyzes AMP. NTPDase3 shares, however, with NTPDase1 and NTPDase2 its broad substrate specificity towards purine and pyrimidine nucleoside triphosphates. It can be expected that the differences in catalytic properties between individual subtypes of NTPDases differentially affect P2 receptor signaling either by activating or inactivating P2 receptors .
The principal functional properties of rat NTPDase3 are similar to those of human NTPDase3 (HB6, ) and mouse NTPDase3 . Interestingly, the pH-dependence of ATP hydrolysis by rat NTPDase3 clearly differs from that of mouse NTPDase3. Whereas mouse NTPDase3 expresses a considerably higher activity at pH 5 than at pH 7–8 , rat NTPDase3 reveals its maximal activity at alkaline pH. Interestingly, the recently cloned NTPDase8  shares principal functional properties with NTPDase3 rather than with NTPDase1 or NTPDase2. Mouse NTPDase8 is preferentially activated by Ca2+ over Mg2+, has an ATP to ADP hydrolysis ratio of approximately 1:0.5 and accumulates ADP that is effectively further hydrolyzed to AMP . Human NTPDase3 forms a dimer  and glycosylation is essential for functional expression . The length of the ORF of rat, mouse  and human  NTPDase3 is identical (529 aa). The three enzymes share 13 cysteine residues and reveal 7 (rat, human) or 8 (mouse) potential N-glycosylation sites. The rat gene (31.0 kb) is slightly longer than the mouse gene (26.9 kb) but it shares its general organization into 11 exons of which exons 2 to 11 contain the ORF.
Rat NTPDase1 and 2 have previously been immunolocalized in the brain. NTPDase1 is associated with the endothelium of blood vessels and smooth muscle as well as with microglia [39, 40]. NTPDase2 is expressed by neural stem cells in the subventricular zone of the lateral ventricles . The immunoblot analysis identified NTPDase3 in all brain regions investigated. NTPDase3 could be detected by immunocytochemistry at the surface of viable PC12 cells. The corresponding RNA has previously been identified in PC12 cells by RT-PCR together with that of NTPDase1 and NTPDase2 . Interestingly, the hydrolysis ratio of ATP:ADP (1:0.28) and the product formation following application of ATP to viable PC12 cells is much closer to that of NTPDase3 than of NTPDase2 for which also a weak immunostaining was obtained . This suggests that NTPDase3 is the predominant ecto-nucleotidase of PC12 cells. In addition we observed an enhancement of NTPDase3 immunoreactivity at growth cones of PC12 cells suggesting that the enzyme may be preferentially associated with sites of active membrane incorporation.
The availability of all NTPDase isoforms expected from genomic analysis opens the possibility of structural and functional comparison. To date structural data for this enzyme family are not available. However, the atomic structure of a considerable number of enzymes belonging to the actin/HSP70/sugar kinase superfamily, including glycerol kinase has been derived. All these proteins are soluble, have ATP phosphotransferase or hydrolase activity, depend on divalent metal ion and tend to form oligomeric structures . Individual enzyme families lack global sequence identity. They share, however, the principal structure of two major domains (I and II) of similar folds on either side of a large cleft with an ATP binding site at the bottom of the cleft . These two domains are expected to undergo conformational changes involving movement relative to each other. Both, domain I and II are divided into subdomains (Ia, Ib, IIa, IIb). Subdomains Ia and IIa have the same basic fold with conserved secondary structure elements that share considerable similarity with those of NTPDases.
A comparison of the conserved secondary structure (Figure 5) reveals duplicate conservation of DXG motifs between β strands (ACR1 and ACR4) of NTPDases that correspond to the β- and γ-phosphate binding motifs in subdomains Ia and IIa of actin, as well as a conserved glycine in ACR5 that can be identified among all members of the actin/HSP70/sugar kinase superfamily (α3, ). This further supports the notion that E-NTPDases are members of the actin/HSP70/sugar kinase superfamily. It implies in addition that E-NTPDases, like the other members of this superfamily, consist of two major domains with one phosphate binding motif in each domain and the binding of the nucleotide in a cleft between the two opposing domains . Interestingly, some members of the E-NTPDase superfamily are entirely soluble (e.g., potato apyrase or the nucleoside triphosphatases of the protozoan parasite Toxoplasma gondii, references in ), others have one transmembrane domain and can be cleaved to form catalytically active soluble enzymes (NTPDase5, NTPDase6), and yet others are (NTPDase 1, 2, 3, 4, 8) are firmly anchored to the membrane via two transmembrane domains. The transmembrane domains of NTPDase1 were found to be important for maintaining catalytic activity and substrate specificity [6, 43], presumably by affecting tertiary and/or quarternary structure. The two transmembrane domains of NTPDase1 interact both within and between monomers and may undergo coordinated motions during the process of nucleotide binding and hydrolysis .
NTPDases differ from members of the actin/HSP70/sugar kinase superfamily by additional conserved short sequence domains (ACR2, ACR3). Differences in sequence, secondary and tertiary structure are believed to account for differences in catalytic properties between related NTPDases . The essential role of the ACRs for catalytic activity has been underpinned by a considerable number of studies using point mutations in the ACRs or ACR deletions [2, 29, 45, 46, 47, 48].
Our present study shows that rat NTPDase3 displays catalytic properties distinctly different from those of rat NTPDase1 and rat NTPDase2. Rat NTPDase3 differs from mouse NTPDase3 in its pH dependence. The enzyme is expressed in multiple brain regions and at the surface of PC12 cells. A comparison of the conserved secondary structure of actin and of NTPDases reveals apparent similarities, inferring that also basic tertiary structure may be conserved between members of the actin/HSP70/sugar kinase superfamily and NTPDases.
Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 269, A4) (to N.B., H.Z.) and from the Canadian Institutes of Health Research (to J.S.).
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