Dependent ecto-ATPase Research Articles

Trypanosoma cruzi: Effects of heat shock on ecto-ATPase activity

In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.

CrATP interferes in the promastigote-macrophage interaction inLeishmania amazonensisinfection

Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.

CrATP as a new inhibitor of ecto-ATPases of trypanosomatids

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite-host cell interaction.

This study describes the possible role of Mg(2+)-dependent ecto-ATPase activity on the Trypanosoma cruzi-host cell interaction. Mg(2+)-dependent ecto-ATPase activity is observed on the cell body and flagellar membranes of the parasite and is about 20 times greater in trypomastigotes, as compared with epimastigotes. Suramin (a competitive antagonist of P2 receptors) and the impermeant agent 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS), both inhibitors of ecto-ATPases, strongly inhibited ATPase activity and the adhesion and internalization of both evolutive forms by mouse resident macrophages. Suramin inhibited the growth of epimastigotes, suggesting a direct participation of ecto-ATPase activity in this process. To overcome the presence of suramin in the culture medium during the time of growth, Mg(2+) ecto-ATPase activity was enhanced 4-fold, as compared with control parasites. The over-expression in enzyme activity was followed by a dramatic increase in the adhesion of epimastigotes to resident macrophages above the level observed for non-treated parasites.

Ectonucleotide diphosphohydrolase activity in Crithidia deanei.

In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.

Characterization of an ecto-ATPase of Tritrichomonas foetus

In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl 2 to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46 mM MgCl 2. The ecto-ATPase activity was also stimulated by MnCl 2 and CaCl 2, but not by SrCl 2. The Mg 2+-dependent ATPase presents two apparent K m values for Mg-ATP 2− ( K m1=0.03 mM and K m2=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg 2+-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A 1, ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60–70% of the Mg 2+-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg 2+-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4′-diisothiocyanostylbene-2′,2′-disulfonic acid (DIDS) as well as suramin, an antagonist of P 2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg 2+-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50 mM d-galactose. Since previous results showed that d-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg 2+-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.

Ectonucleotide Diphosphohydrolase Activities in Hemocytes of Larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 ± 0.94 nmol of Pi/h × 106 cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 ± 1.74 nmol of Pi/h × 106 cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP−4 and Mg-ATP2− were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.

Ectonucleotide Diphosphohydrolase Activities in Entamoeba histolytica

In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol Pi/h/105 cells). Interestingly, in the presence of 5 mM MgCl2 an ecto-ATPase activity of 300 nmol Pi/h/105 cells was observed. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl2. Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2 but not by SrCl2, ZnCl2, or FeCl3. In fact, FeCl3 inhibited both Mg2+-dependent and Mg2+-independent ecto-ATPase activities. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.4 and 8.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5′-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg2+ (basal activity), the apparent Km for ATP4− was 0.053 ± 0.008 mM, whereas at saturating MgCl2 concentrations, the corresponding apparent Km for Mg-ATP2− for Mg2+-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM ± 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4′-4′-diisothiocyanostylbenzene-2′-2′-disulfonic acid. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg2+-dependent ecto-ATPase but not the Mg2+-independent ATPase activity.

ATP reception and chemosensory adaptation in Tetrahymena thermophila.

Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog &bgr;- &ggr; -methylene ATP are both effective depolarizing chemorepellents in Tetrahymena thermophila. Chemorepellent behavior consists of repeated bouts of backward swimming (avoidance reactions) that can easily be quantified to provide a convenient bioassay for purinergic reception studies. Chemosensory adaptation occurs following prolonged exposure (10 min) to the repellents, and cells regain normal swimming behavior. Adaptation is specific since cells that are behaviorally adapted to either ATP or &bgr;- &ggr; -methylene ATP still retain full responsiveness to the chemorepellents GTP and lysozyme. However, cross adaptation occurs between ATP and &bgr;- &ggr; -methylene ATP, suggesting that they involve the same receptor. Behavioral sensitivity to both ATP and &bgr;- &ggr; -methylene ATP is increased by the addition of Na+, but addition of either Ca2+ or Mg2+ dramatically decreases the response to ATP. These ionic effects are correlated with in vivo ATP hydrolysis, suggesting that divalent ions decrease purinergic sensitivity by activating a Ca2+- or Mg2+-dependent ecto-ATPase to hydrolyze the ATP signal. In vivo [32P]ATP binding studies and Scatchard analysis suggest that the behavioral adaptation is due to a decrease in the number of surface binding sites, as represented by decreased Bmax values. All these changes are reversible (de-adaptation) after 12 min in a repellent-free buffer. Electrophysiological analysis showed that both &bgr;- &ggr; -methylene ATP (10 micromol l-1) and ATP (500 micromol l-1) elicited sustained, reversible depolarizations while GTP (10 micromol l-1) produced a transient depolarization, suggesting that the chemosensory response pathways for ATP and GTP reception may differ. There may be separate ATP and GTP receptors since ATP and GTP responses do not cross-adapt and 'cold' (unlabeled) GTP is not a good inhibitor of [32P]ATP binding. These results suggests that T. thermophila possess high-affinity surface receptors for ATP that are down-regulated during chemosensory adaptation. These ATP receptors may act as chemorepellent receptors to enable T. thermophila to recognize recently lysed cells and avoid a possibly deleterious situation. This is the simplest eukaryotic organism to show an electrophysiological response to external ATP.

Differential degradation of extracellular adenine nucleotides by folliculated oocytes of Xenopus laevis.

The degradation of extracellular ATP and ADP by Xenopus oocytes was studied to investigate whether one or two ecto-enzymes are responsible for breakdown of both nucleotides. At a concentration of 100 microM, the half-life of ATP and ADP was 33 and 40 min, respectively. Degradation of ATP caused an initial fast and then a sustained accumulation of ADP in the buffer, while the concentration of AMP in the buffer increased slowly, but progressively, in a relatively linear manner. The rates of degradation of ATP and ADP were similar at pH levels between 7 and 10, but the velocity of breakdown of ATP was significantly higher than that of ADP at pH of 5-6. In divalent cation-free buffer, the addition of 0.1 mM of Ca2+, but not equimolar Mg2+, significantly potentiated the degradation of ATP by oocytes while, in the case of ADP, each of these divalent cations were able to potentiate its degradation. The rate of hydrolysis of ATP and its kinetic constants were not significantly different in the presence or absence of ADP (50 microM). In conclusion, differences in pH- and cation-dependency, and absence of inhibitory effect of ADP on degradation of ATP suggest that degradation of ATP and ADP by oocytes is provided by separate enzymes, namely Ca2+/Mg(2+)-dependent ecto-ATPase and ecto-ADPase, rather than by one ecto-enzyme.

Insulin-Stimulated Phosphorylation of Recombinant pp120/HA4, an Endogenous Substrate of the Insulin Receptor Tyrosine Kinase

Insulin binding to the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 20,000. Analysis of the deduced amino acid sequence of rat liver pp120/HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr488 and Tyr513), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser503). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe488 and Ala503) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr488 is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser513 may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg(2+)-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg(2+)-dependent ecto-ATPase activity.

Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown.

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (Pi) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg(2+)-dependent and Ca2+/Mg(2+)-independent dephosphorylation of ATP. Ca2+/Mg(2+)-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg(2+)-independent ATPase activity was active over a range of 0.1-30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0-9.0), but increased in a relatively linear manner with: (1) time of reaction (0-90 min), (2) number of cells (1-20 oocytes), and (3) temperature (10-37 degrees C). Ecto-ATPase activity was unaffected by ouabain (100 microM), sodium azide (100 microM), and oligomycin (5 micrograms/ml) (as inhibitors of endo-ATPases) and beta-glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP>>alpha, beta-methylene ATP, beta, gamma-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido)napthalene-1,3,5-trisul phonic acid), 100 microM] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg(2+)-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and characterization of a parotid Ca(2+)-dependent ecto-ATPase.

Parotid acini were isolated and tested to further establish the presence of ecto-ATPase in the intact cells. Inhibitors were used to determine if the inhibitor profile of the ATPase was similar to that of a Ca(2+)-ATPase from parotid membranes identified previously as an ecto-ATPase. The Ca(2+)-ATPase of intact cells was insensitive to oligomycin (10 micrograms/ml), N-ethylmaleimide (NEM) (0.1 mM), ruthenium red (0.1 mM), sodium azide (1 mM), and was inhibited approximately 22% by sodium orthovanadate (Na3VO4) (1 mM). This profile was similar to the Ca(2+)-ATPase of intact cells. Trifluoperazine (TFP) (0.1 mM) inhibited the enzyme in intact cells by approximately 32%. The nucleotide substrate specificity of the enzyme also reflected very closely the pattern seen in isolated membranes.

Membrane ecto-ATPase on epidermal Langerhans cells.

Epidermal Langerhans cells (LC) possess a plasma membrane Mg(++)- or Ca(++)-dependent ecto-ATPase (mATPase), the function of which is not known. On the other hand, extracellular ATP (ATPo) has been shown to cause a rapid increase in cell-membrane permeability for selected cells, thus inducing the entry of exogenous substances, leading eventually to cell death. ATPo has also been shown to be a mediator of T cell-mediated cytotoxicity. We have observed human LC to be resistant to the permeabilizing effects of ATPo when compared to cultured human keratinocytes and J774 macrophages. In addition, our preliminary evidence in vitro suggests that mATPase on LC may provide protection against the lytic effects of ATPo. Finally, we propose that this latter mechanism may also operate in vivo.

The involvement of ecto-ATPase activity in the phosphorylation of intracellular proteins by the addition of extracellular [32P]ATP in PC12 cells

Abstract We have investigated the possibility that ecto-phosphorylation by extracellular ATP may play a role in the development of PC12 cells. To test this model and to identify putative target membrane proteins, intact PC12 cells were radiolabeled by the addition of 20 μM [γ- 32 P]ATP. An analysis of the labeled proteins revealed that a 57 kDa protein was the most abundant phosphorylated protein even within time periods as short as 3 min and continued to be labeled over and above the level of other proteins. This protein was identified as tyrosine hydroxylase by immunoprecipitation with antiserum to tyrosine hydroxylase. When intact cells were incubated with either [γ- 32 P]ATP or 32 P i of comparable specific radioactivity, the overall protein labeling pattern and the degree of phosphorylation of tyrosine hydroxylase were similar. There were no discrete proteins that were labeled by [γ- 32 P]ATP and not by 32 P i that would provide evidence for ecto-kinase activity in PC12 cells. Also, the addition of nonradioactive P i reduced the incorporation of radioactivity into the protein from extracellular [γ- 32 P]ATP. These results suggested that the phosphorylation of tyrosine hydroxylase by extracellular [γ- 32 P]ATP required the initial hydrolysis of ATP and the subsequent incorporation of the 32 P i into the intracellular ATP pool. To support this interpretation, we have demonstrated directly the presence of ecto-ATPase activity in intact PC12 cells by measuring the hydrolysis of extracellular [γ- 32 P]ATP. Nearly 50% of the total ATP added (20 μM) was hydrolyzed within 10 min under conditions identical to those used to demonstrate intracellular protein phosphorylation. PC12 cells express both a Ca 2+ -dependent ecto-ATPase activity and a Mg 2+ -dependent ecto-ATPase activity. In addition, extracellular ATP is degraded enzymatically not only to ADP, but sequentially to adenosine. Our results also point out the difficulties inherent in attempts to identify ecto-kinase activity in cells that also contain ecto-ATPase activities.

The Kinetic Properties of the Ecto-ATPase of Human Peripheral Blood Lymphocytes and of Chronic Lymphatic Leukemia Cells

This study examines whether the activity of the Mg2+-dependent ecto-ATPase of the surface membrane of the human lymphocyte is changed in chronic lymphocytic B-cell leukemia (CLL-B) and may be an indicator of malignant transformation. The ecto-ATPase activities of preparations consisting predominantly of T or B cells were compared to each other and to the ecto-ATPase of the CLL peripheral blood lymphocytes (PBL). The specific activities and kinetic constants of the ecto-ATPase of the cell preparations were determined with [gamma-32P] adenosine triphosphate (ATP) as substrate. B-enriched lymphocytes had nearly fourfold greater specific activity and apparent Vmax than T-enriched lymphocytes, while the Km values of both cell types showed no significant difference. The specific activities and kinetic constants of the ecto-ATPase of the CLL PBL were significantly higher than the corresponding values of PBL or of B-enriched lymphocytes. Judging from the kinetic constants the ecto-ATPase of the CLL-B lymphocyte appears to be an enzyme that is distinctly different from that of the normal B cell. On the basis of the kinetic properties, the ecto-ATPase of the B cell appears to be identical with that of the T cell. The differences in the maximal velocities of the hydrolysis of ATP by B and T cells are likely due to a greater number of enzymatic sites on the B cell.

Extracellular ATP, ecto-ATPase and calcium influx in Dictyostelium discoideum cells

There is evidence that a protein kinase is present on the surface of Dictyostelium discoideum cells [l-3] and is capable of phosphorylating certain plasma membrane proteins [3]. Such phosphorylation may be involved in membrane permeability and transport, cell movement, and the activation of cyclic AMP receptors which occurs during early differentiation [4]. External ATP induces cell aggregation, decreases the chemotactic sensitivity of cells to a cyclic AMP gradient and inhibits intercellular contact sites outside the aggregation center [ 1,5]. We found that ATP (0.1-0.8 PM produced at 1.5 X lo7 cells/ml) is present extracellularly in cell suspensions. This reflected a steady state concentration since a Mg”-dependent ecto-ATPase was continually hydrolyzing extracellular ATP. The ectoATPase was inhibited by suramin and I-ethyl-3(3-dimethyl-aminopropyl)carbodiimide (EDAC), but not by oligomycin or ouabain. Ecto-ADPase, -AMPase and nucleotide diphosphokinase activities were also detected. Calcium influx was inhibited by suramin, l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC), KCN, dinitrophenol, carbonyl-mchlorophenylhydrazone (CCCP), adenyl imidodiphosphate, adenyl methylene diphosphate, and by adding hexokinase or apyrase to split extracellular ATP. Addition of ATP (optimum level 10e6 M) to cell suspensions stimulated calcium influx and partially overcame the KCN-induced inhibition of this influx. Glucose uptake was not affected by suramin or apyrase.

Neuroblasts-glia interaction: The effect of Co-cultivation upon ecto-ATPase activity of neuroblastoma and glioma cells

The effect of cell contact and cell medium upon the ecto-enzymes, Mg 2+- and Ca 2+-dependent ATPase and 5′-nucleotidase were studied in nervous system cells in tissue culture. Conditions were worked out for co-culture and rseparation of glioblastoma and neuroblastoma cells so that the effects upon each of the co-cultured cell lines after interaction of these cells could be reliably determined. Co-cultivation of mouse neuroblastoma and glioma cell lines markedly enhanced Mg 2+- and Ca 2+-dependent ecto-ATPase activity. Evidence was obtained which indicates that increase in ecto-ATPase of co-cultured neuro- and glioblastoma cells occurs in both cell types. Ecto-ATPase was 500% of the original level in clonal line NN astroblasts after co-culture with M1 neuroblasts. This activity decreased over 50 transfers during the period of about a year. Increase in ecto-ATPase and morphological differentiation of M1 neuroblastoma cells after co-culture with NN astroblasts could also be brought about simply by treatment with the medium from NN cell cultures. Co-cultivation of neuroblastoma and glioma cells does not change significantly the specific activity of ecto-5′-nucleotidase.