Stainless steel electrospray probe: a dead end for phosphorylated organic compounds?

A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs.

The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47-50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.

Nucleoside diphosphate kinase (NDPK/NM23) and the waltz with multiple partners: possible consequences in tumor metastasis.

Tumor metastasis is responsible for a high degree of mortality in cancer patients. One of the genes involved in tumor metastasis is NM23. At present, eight human isoforms, transcribed from different NM23 genes, have been detected. The gene products have been identified as nucleoside diphosphate kinases (NDPKs), most of which catalyse the transfer of the gamma-phosphate of a (deoxy)nucleoside triphosphate to a (deoxy)nucleoside diphosphate. However, the function of NDPK isoforms involved in tumor metastasis cannot be explained on the basis of their phosphotransferase activity alone. At present, several other properties, like transcriptional regulation and protein kinase activity, have been assigned to these proteins. Moreover, it has also been shown that NDPKs interact with several other proteins, and binding partners of NDPKs are identified at an increasing rate. Accumulating evidence indicates that protein-protein interactions modulate the molecular action of NDPKs. In this review we provide a brief overview of how NDPKs are correlated with cancer, and discuss when and how the activities assigned to NDPKs may affect metastasis, with special emphasis on the role of protein-NDPK interactions in this process.

Short capillary ion-pair high-performance liquid chromatography coupled to electrospray (tandem) mass spectrometry for the simultaneous analysis of nucleoside mono-, di- and triphosphates.

A liquid chromatography/mass spectrometry (LC/MS) method for the analysis of complex mixtures of nucleoside mono-, di- and triphosphates has been developed. A short capillary column (35mm x 0.3mm i.d.) was operated under ion-pair high-performance liquid chromatography conditions and hyphenated to (negative) electrospray (tandem) mass spectrometry. As such, the separation of 12 nucleotides was performed by a binary gradient elution using CH(3)OH/H(2)O and N,N-dimethylhexylamine (N,N-DMHA) as ion-pairing agent. The influence of different N,N-DMHA concentrations on the chromatographic and mass spectrometric performance was evaluated to achieve optimal LC/MS conditions. In addition it was demonstrated that a controlled admission of ammonium dihydrogen phosphate (NH(4)H(2)PO(4)) improved both chromatographic performance and mass spectrometric detection. Because the system was hyphenated to an orthogonal designed electrospray interface (Z-spraytrade mark), long acquisition times were possible without loss of sensitivity.

Structural basis of allotypes of ecto-nucleotide pyrophosphatase/phosphodiesterase (plasma cell membrane glycoprotein PC-1) in the mouse and rat, and analysis of allele-specific xenogeneic antibodies.

Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) have been implicated in bone calcification, type II diabetes, control of purinergic signalling and tumour invasion. The gene for the plasma cell membrane glycoprotein PC-1 in the mouse (Enpp1) has been known since 1970 to exist in two allelic forms, but their structural basis was heretofore unknown. We show that the Enpp1a and Enpp1b alleles differ by only two amino acids, at positions 650 and 679 in the C-terminal nuclease-like domain. Histidine 650 but not arginine 679 forms an essential part of the Enpp1a epitope recognized by monoclonal antibody IR-518. Sequences of LEW and LOU rats and the rat glioma cell line C6 differ from that of the mouse by about 60 amino acids. The LOU and C6 cell line sequences differ by only three amino acids, but differ from the LEW sequence by 10 amino acids. All three rat strains possess the mouse Enpp1b allele at positions 650 and 679. Despite numerous other differences from the mouse, rats immunized with Enpp1a mouse cells have generated monoclonal antibodies specific for the Enpp1a allele, suggesting that amino acids 650 and 679 may be particularly immunogenic. The cytoplasmic tails of the mouse and rat are highly conserved, but are significantly different from human cytoplasmic tails.

Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells.

The soluble protein fraction of tobacco bright yellow 2 cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-binding activity, detected with both a conventional binding assay and a surface plasmon resonance biosensor. A cAMP-agarose-based affinity purification procedure yielded three proteins which were identified by mass spectrometry as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and two nucleoside diphosphate kinases (NDPKs). This is the first report describing an interaction between cAMP and these proteins in higher plants. Our findings are discussed in view of the reported role of the interaction of cAMP with GAPDH and NDPK in animals and yeast. In addition, we provide a rapid method to isolate both proteins from higher plants.

Agonists of the P2Y(AC)-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma.

We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the P2Y(AC)-receptor with EC(50) values of 250 +/- 37 pM and 1 +/- 0.5 microM, respectively. P2Y(AC)-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y(AC)-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP(3)-independent Ca(2+)-influx was observed after P2Y(AC)-receptor activation. Inhibition of this influx by Ca(2+)-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap(3)A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA(Asn19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-purinoceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-dependent, but ras- and Ca(2+)-independent cascade.

Natural inhibitors of neutrophil function in acute respiratory distress syndrome.

OBJECTIVE: Neutrophils play a key role in the physiopathogenesis of acute lung injury in general and acute respiratory distress syndrome (ARDS) in particular. To identify the anti-inflammatory mediators with a protective effect on lung tissue damage in ARDS, we correlated the concentration of the Clara cell 16-kD protein (CC16; an inhibitor of neutrophil chemotaxis), angiogenin (an inhibitor of degranulation), and the total radical oxygen neutralizing activity with the amount of elastase (a marker of neutrophil activation) and with the Pao2/Fio2 ratio, which is inversely related to lung injury. SETTING: University hospital. PATIENTS: Patients with ARDS (n = 12) and patients at risk for developing ARDS (n = 14). INTERVENTIONS: Patients underwent bronchoalveolar lavage 12 hrs after diagnosis of ARDS or at-risk status. MEASUREMENTS AND MAIN RESULTS: The amount of CC16 and radical oxygen neutralizing activity was not significantly different in patients with or at risk for ARDS. In contrast, the amount (mean +/- sem) of angiogenin in the bronchoalveolar lavage of ARDS patients (45 +/- 14 ng/mL, n = 12) was increased 11-fold (p <.05) compared with patients at risk for ARDS (4 +/- 1 ng/mL, n = 14). In patients with ARDS, the amount of protein and angiogenin in bronchoalveolar lavage increased with decreasing concentration of CC16 (p <.05). In addition, CC16 correlated with the Pao2/Fio2 ratio (p <.05) and inversely with the amount of elastase (p <.05) and thus may be regarded as a reliable protective agent for lung injury. CONCLUSION: A high concentration of CC16, a natural inhibitor of neutrophil function, decreases neutrophil-mediated lung damage of patients with ARDS. Strategies to increase natural anti-inflammatory agents, and thus influence the disruption of the balance between natural inflammatory and anti-inflammatory or protective factors, could be useful to modulate the tissue destruction and the course of ARDS.

P2Y(AC)(-)-receptor agonists enhance the proliferation of rat C6 glioma cells through activation of the p42/44 mitogen-activated protein kinase.

1. Extracellularly added P(1),P(3)-di(adenosine-5') triphosphate (Ap(3)A), P(1),P(4)-di(adenosine-5') tetraphosphate (Ap(4)A), ATP, ADP, AMP and adenosine are growth inhibitory for rat C6 glioma cells. Analysis of nucleotide hydrolysis and the use of nucleotidase inhibitors demonstrated that the latter inhibition is due to hydrolysis of the nucleotides to adenosine. 2. Agonists of the P2Y(AC)(-)-receptor enhance the growth of C6 cells if their hydrolysis to adenosine is inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). In these conditions, the potency to stimulate cell growth parallels the ranking of the receptor agonists, i.e. 2-methylthioadenosine-5'-diphosphate (2MeSADP)>Ap(3)A>Ap(4)A. ATP and ADP are still hydrolysed in the presence of PPADS and have no proliferative effect on C6 cells. 3. The enhanced growth is due to a P2Y(AC)(-)-receptor-mediated activation of p42/44 mitogen-activated protein kinase (MAPK) as shown by immunoblotting and protein kinase assays for active MAPK and the use of the MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. 4. The UTP-induced enhancement of the growth of C6 cells is due to activation of MAPK by a PPADS sensitive nucleotide receptor. 5. In conclusion, the effect of nucleotides on the growth of C6 cells is determined by ecto-nucleotidases and by activation of nucleotide receptors. Hydrolysis of nucleotides to adenosine induces growth inhibition while inhibition of the hydrolysis of agonists of the P2Y(AC)(-)-receptor enhances cell growth by activation of MAPK.

Protein tyrosine kinase-dependent regulation of adenylate cyclase and phosphatidylinositol 3-kinase activates the expression of glial fibrillary acidic protein upon induction of differentiation in rat c6 glioma.

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.

Establishment of serum-free pre-colony forming unit assays for differentiation of primitive hematopoietic progenitors: serum induces early macrophage differentiation and inhibits early erythroid differentiation of CD34++CD38- cells.

In this report we show that serum has differentiation-inducing effects on primitive hematopoietic progenitor cells with the CD34++CD38- immunophenotype. Using the pre-colony forming unit (pre-CFU) assay as a model for early myelopoiesis, we compared the effects of serum-containing and serum-free media and evaluated different cytokine cocktails [interleukin (IL)-1, IL-3, IL-6, kit ligand with and without the Flt3/Flk2 ligand (FL)]. In this assay, pre-CFUs are defined as cells unable to form colonies when plated directly in semi-solid assays, but which can differentiate into CFUs when cultured in liquid medium containing early-acting cytokines. In one of the investigated serum-free media, the average myeloid expansion in liquid medium reached up to more than 50% of that obtained in serum-containing medium. In addition, our experiments revealed differences in the clonogenic output between cells cultured in serum-free medium and those cultured in serum-containing medium, demonstrating that serum has a monocyte differentiation-inducing effect on primitive hematopoietic progenitors. Also in serum-free medium, higher proportions of erythroid progenitors were generated. These differentiation-inducing effects of serum further emphasize the need for serum-free culture protocols for hematopoietic graft engineering. Addition of FL to the culture media ameliorated cellular expansion and resulted in a decrease in the proportion of erythroid and granulocyte progenitors and an increase in the proportion of monocyte progenitors. In conclusion, this study shows that good serum-free conditions are available for differentiation assays with primitive hematopoietic progenitors and demonstrates that serum and FL have biasing effects on the initial phase of hematopoietic differentiation, favoring the monocyte lineage.

Phosphatidylinositol 3-kinases in tumor progression.

Many cellular processes have been identified in which phosphatidylinositol 3-kinase has a key regulatory function. As an oncogene, it is also involved in the development of cancer. The transformation and progression of normal cells towards an advanced stage tumor and/or towards metastatic lesions involves a complex series of events, including genetic alterations, leading to aberrant cell cycle progression, altered adhesion and motility characteristics, inhibition of apoptosis and induction of angiogenesis. This review highlights the processes involved in the pathogenesis of cancer in which phosphatidylinositol 3-kinase is involved and provides an overview of the possible mechanisms by which the enzyme exerts its oncogenic action.

Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.

Identification of the tumor metastasis suppressor Nm23-H1/Nm23-R1 as a constituent of the centrosome.

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Nucleoside diphosphate kinase beta (Nm23-R1/NDPKbeta) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma.

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKbeta and Nm23-R2/NDPKalpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

Reperfusion injury after focal myocardial ischaemia: polymorphonuclear leukocyte activation and its clinical implications.

The only way to rescue ischaemic tissue is to re-instate the oxygen supply to the tissue. However reperfusion of the ischaemic area not only oxygenates the tissue but also initiates a cascade of processes, which may in some cases result in temporary dysfunction of the myocardium. In order to devise protective measures, it is essential to understand the mechanisms and the triggers of this reperfusion phenomenon. In this review we will mainly focus on the inflammatory response caused by reperfusion. We will cover the different steps of polymorphonuclear leukocyte activation and will briefly discuss the molecular biology of the receptors involved. The currently used pharmacological medications in acute cardiology will be reviewed and in particular their actions on polymorphonuclear leukocyte activation, adhesion and degranulation. This review is a compilation of the current knowledge in the field and the therapeutic progress in the prevention of reperfusion injury made today.

Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.

1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures.

Purification of rat C6 glioma plasma membranes by affinity partitioning.

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.

Isolation of plasma membranes from rat C6 glioma cells cultivated on microcarriers.

We have studied the feasibility of rat C6 glioma cell cultivation on microcarrier beads and the isolation of their plasma membranes from the beads. Cells were cultivated on Cytodex-1 microcarrier beads and the plasma membranes were subsequently isolated from confluent cell monolayers on the beads. This approach yielded approximately 4 x 10(6) cells/ml in a 1 L spinner vessel. Enzymatic assays indicated an 18-fold enrichment of plasma membranes isolated from the beads with minor contamination by other cell organelles. Assay for IGF-I receptor binding capacity revealed that 70% of the total receptor binding capacity could be recovered in the plasma membrane fraction isolated from the beads as compared with the receptor binding capacity of intact cells, demonstrating the functional integrity of the isolated membranes. Electron microscopy and immunofluorescence analysis indicated that the isolated plasma membranes were highly homogeneous with the majority exposing the cytoplasmic surface. Our procedure of C6 glioma cell cultivation on microcarriers and subsequent plasma membrane isolation, provides large quantities of homogeneous and metabolically active membranes which can be used to study receptor-mediated effects on cell proliferation and differentiation.

An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma.

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.