Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence

Ecto-3′-nucleotidase/nuclease (3′NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3′mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3′-nucleotidase activity (La3′-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3′-nucleotidase inhibitor and approach the possible involvement of ecto-3′-nucleotidase in cellular adhesion. La3′-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3′-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu²⁺ ions. Interestingly, ecto-3′-nucleotidase activity is 60-fold higher than that of ecto-5′-nucleotidase in L. amazonensis. Additionally, ecto-3′-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage–parasite attachment/invasion was increased by 400% in the presence of adenosine 3′-monophosphate (3′AMP); however, this effect was reverted by TTM treatment. We believe that La3′-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.     

Giardia duodenalis: Biochemical characterization of an ecto-5′-nucleotidase activity

In this work, we biochemically characterized the ecto-5′-nucleotidase activity present on the surface of the living trophozoites of Giardia duodenalis. Two sequences of the 5′-nucleotidase family protein were identified in the Giardia genome. Anti-mouse CD73 showed a high reaction with the cell surface of parasites. At pH 7.2, intact cells were able to hydrolyze 5′-AMP at a rate of 10.66 ± 0.92 nmol Pi/h/10⁷ cells. AMP is the best substrate for this enzyme, and the optimum pH lies in the acidic range. No divalent cations had an effect on the ecto-5′-nucleotidase activity, and the same was seen for NaF, an acid phosphatase inhibitor. Ammonium molybdate, a potent inhibitor of nucleotidases, inhibited the enzyme activity in a dose-dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. The results indicate the existence of an ecto-5′-nucleotidase that could play a role in the salvage of purines.     

Stimulation of an α1-adrenergic receptor downregulates ecto-5′ nucleotidase activity on the apical membrane of RPE cells

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5′ nucleotidase (CD73). This study identifies ecto-5′ nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5′AMP dephosphorylation in the posterior rat eye. The ecto-5′ nucleotidase inhibitor αβmADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5′ nucleotidase. The production of phosphate from 5′AMP by ARPE-19 cells was inhibited by αβmADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5′AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the α1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37°C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5′AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5′ nucleotidase, with activity on the apical membrane, and stimulation of α-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time.     

Biomass production and fatty acid profile of a Scenedesmus rubescens-like microalga

This investigation examined the effects of nitrogen–phosphate combined deficiency on the biomass yield, fatty acid methyl esters (FAME) production and composition from Scenedesmus rubescens-like microalga. A 15-day indoor culture was performed as a 3 × 3 factorial design (NaNO₃ levels: 3, 10 and 20 mM; KH₂PO₄ levels: 20, 50 and 150 μM). The algae grown under medium nitrogen concentration (10 mM) and high phosphate concentration (150 μM) reached the highest biomass (1223.5 ± 152.5 mg/L). Both nitrogen and phosphate had a significant influence on the FAME yield (P < 0.05 and P < 0.0001, respectively). The FAME yield from algae grown under low nitrogen (3 mM) and phosphate concentration (20 μM) increased throughout the experiment and the highest FAME yield (42.2 ± 2.5% of AFDW) as well as C16 and C18 content (95.8 ± 1.6% of AFDW) was achieved under these conditions. Algae grown under medium nitrogen concentration (10 mM) and low phosphate concentration (20 μM) had the highest FAME productivity (426.0 mg/L ± 135.0 mg/L). Thus, the lower nitrogen concentration (3 mM–10 mM) and low phosphate concentration (20 μM) would be an optimal combination tested to produce the most FAME from S. rubescens-like algae.     

Regional localization of human ecto-5′nucleotidase to chromosome 6q14–q21

Summary Human and mouse hybrids that contain fragments of human chromosome 6 as translocations were analysed for expression of ecto-5nucleotidase enzymic activity measured by the conversion of AMP to adenosine and for antigenicity recognized by a monoclonal antibody specific for the human isozyme. Both methods allow a regional assignment of ecto-5nucleotidase to 6q14–q21.     

Phosphorus uptake and translocation in salt-stressed melon plants

Melon seedlings (Cucumis melo L. cv.Galia) were grown hydroponically to study the effect of salinity (80 mmol/LNaCl) on phosphate (Pi) uptake and translocation at two levels of Pi (25 μmol/L and 1 mmol/L). Net uptake rates of Pi were determined by depletionof the medium and by plant content. Salinity decreased Pi uptake at low Pi (high affinity uptake mechanism), 25 μmol/L, although no specific competitive inhibition of Pi uptake by Cl⁻ was observed. When plants were grown with high Pi (1 mmol/L), the uptake of Pi through the low affinity system was increased by 80 mmol/L NaCl. Salinity also reduced the phosphorus flux, as Pi, through the xylem. It is hypothesised that high levels of NaCl decrease the mobility of Pi stored in vacuoles, and as a result, inhibit export from this storage compartment to other parts of the plant.     

A new histo-and cytochemical method for demonstration of cyclic 3′, 5′-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat

Summary Cyclic 3, 5-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo-and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5 nucleotidase activity instead of an exogenous 5 nucleotidase source (snake venom). Cyclic GMP and cyclic AMP were used as substrates. When cyclic GMP was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase.To confirm the propriety of our new cytochemical method, the localization of 5 nucleotidase was also studied utilizing 5 AMP or 5 GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5 nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments. After inhibition of the intrinsic 5 nucleotidase activity with the use of 1 mM Ni-ions or 10 mM NaF no demonstration of cyclic nucleotide PDEase activity was possible; the existence of intrinsic 5 nucleotidase activity is necessary for the release of free phosphateions from 5 AMP (5 GMP), which are a prerequisite for the histochemical reaction. For comparison, some sections were incubated with the conventional cyclic nucleotide PDEase incubation medium containing snake venom from Ophiophagus hannah. With this conventional method, morphological preservation was extremely poor, and moreover, the reaction itself was weaker than that with the presently described method.The authors wish to dedicate the paper to Professor Dr.Dr.h.c. A. Oksche, Justus Liebig University Gießen     

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).     

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES,CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH₂PO₄) or Pi‐sufficient (100 μM NaH₂PO₄) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH₂PO₄ decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The K_m value of Pi uptake rate was decreased by incubation with 1 μM NaH₂PO₄ and the decrease in K_m value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.     

Preferential utilization of organic and inorganic sources of phosphorus by wheat plant

On soils of low P supply organic P (Po) makes up a similar or even larger part in soil solution than inorganic P (Pi). The ability of wheat (Triticum aestivum L., cv. Star) plants to hydrolyze and absorb this Po in comparison to similar concentrations of Pi was studied. Four concentration levels of Pi and Po were obtained by extracting two soils with deionized water in a ratio of 1:1 and concentrating the resulting filtrate by freeze drying to different degrees. The concentration of Pi varied between 5 and 36 μM and Po between 3 and 22 μM. Wheat seedlings were grown in these solutions for 12 and 24 h and acid and alkaline phosphatase activity determined. The reduction of Po concentration in solution expressed on a root length basis gave the rate of Po hydrolysis and the reduction in concentration of Pi and Po gave the P inflow into the roots. No alkaline phosphatase activity was detected. The activity of wheat root acid phosphatase increased with Po concentration in solution. Phosphorus uptake was 2 to 6 fold higher from Pi than from Po at similar concentrations of both. The rate of uptake from Pi, the inflow, as well as the rate of hydrolysis of Po increased linearly with concentration but at similar concentration the inflow was 2 to 4 times higher than the rate of Po hydrolysis. Results suggest that plants can utilize Po after hydrolysis by phosphatase, but Pi is more important and preferentially used by plants; Po may be essential for plant nutrition especially in high P-fixing soils.     

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.     

Effects of Orthophosphate and Adenosine 5'-Phosphate on Threonine Synthase and Cystathionine gamma-Synthate of Lemna paucicostata Hegelm. 6746

Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine γ-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine γ-synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts.     

Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.     

Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5′-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5′nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5′-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.     

Influence of phosphate and ammonia on the growth, exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii TA1 with clover (Trifolium pratense)

The Rhizobium-legume interaction is sensitive to a number of environmental factors, among which phosphate (Pi) and ammonium availability are the most important. We investigated the effect of Pi and ammonia concentration on exopolysaccharide production and symbiosis Trifolium pratense with Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). The optimal Pi concentration in the bacterial growth medium for RtTA1 growth and exopolysaccharide production was in the range from 0.9 mM to 8.1 mM. Independently of Pi concentration, ammonium (NH4Cl) concentration above 8.1 mM in the culture medium significantly decreased EPS production, indicating a regulatory role of this nutrient on the EPS production in the RtTA1 strain. Pi availability has a beneficial effect on both partners of symbiosis. Pi concentration in the plant medium in the range from 1.7 mM to 5 mM was optimal for nodule formation, nodule occupancy and nitrogen fixation ability. Despite of T. pratense cv. Bryza tolerance on high Pi concentration, 20 mM Pi occurs to be nearly phytotoxic, which negatively affects almost all symbiotic parameters. Large amounts of starch were accumulated in the nodules formed by clover grown on medium containing high Pi concentration.     

Enzymatic characteristics of two novel Myxococcus xanthus enzymes, PdeA and PdeB, displaying 3′,5′- and 2′,3′-cAMP phosphodiesterase, and phosphatase activities

Myxococcus xanthus PdeA and PdeB, enzymes homologous to class III 3′,5′-cyclic nucleotide phosphodiesterases, hydrolyzed 3′,5′- and 2′,3′-cyclic AMP (cAMP) to adenosine, and also demonstrated phosphatase activity toward nucleoside 5′-tri-, 5′-di-, 5′- and 3′-monophosphates with highest activities for nucleoside 5′-monophosphates. The substrate specificities of PdeA and PdeB show no similarity to that of any known cNMP phosphodiesterase, nucleotidase, or phosphatase. The enzyme activities of PdeA and PdeB were stimulated by 50 μM Mn²⁺ or Co²⁺. The K_m values of PdeA and PdeB for 3′,5′-cAMP, 2′,3′-cAMP, 5′-ATP, and 5′-AMP were in the low micromolar range (1.4-12.5  μM).     

Regulation of mesophyll photosynthesis in intact wheat leaves by cytoplasmic phosphate concentrations

The effect of D-(+)-mannose, inorganic phosphate (Pi) and mannose-6-phosphate on net mesophyll CO₂ assimilation rate (A) and stomatal conductance (g_s) of wheat (Triticum aestivum L.) leaves was studied. The compounds were supplied through the transpiration stream of detached leaves from plants grown in sand in growth cabinets or glasshouses, with different concentrations of Pi (0.25, 1.0 and 4.0 mM) supplied during growth. In all cases, 10 mM D-(+)mannose caused 40–60% reduction of A within 30 min, though the time courses differed for flag leaves and the sixth leaf on the mainstem of glasshouse- and cabinet-grown plants. D-(+)Mannose had a similar effect on A in leaves having a fourfold range in total phosphate content. Effects of D-(+)mannose in reducing g_s were always slower than on A. When the CO₂ concentration in the leaf chamber was adjusted to maintain intercellular CO₂ concentration (C_i) constant as A declined after mannose supply, g_s still declined indicating that stomatal closure was not caused by changing C_i. Supplying mannose-6-phosphate at 10 and 1 mM and Pi at 5 and 10 mM concentrations caused rapid reductions in g_s and also direct reductions in A. The observed effects of mannose and Pi on assimilation are consistent with the proposed regulatory role of cytoplasmic Pi in determining mesophyll carbon assimilation that has been derived previously using leaf discs, protoplasts and chloroplasts.Abbreviations and symbols A net mesophyll CO₂-assimilation rate - C_a, C_i external (assimilation-chamber) and intercellular CO₂ concentration, respectively - g_s stomatal conductance - Man6P mannose-6-phosphate - Pi orthophosphate     

Purine catabolism in man: inhibition of 5'-phosphomonesterase activities from placental microsomes.

The 5'-phosphomonoesterase activity of 5'-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.5) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5'-nucleotidase suggest a sequential mechanism of interaction between AMP nad MgCl2, with a Km of 14 and 3 muM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhititors when AMP was the substrate, with a Ki slope of 120 muM. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. These observations suggest that 5'-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5'-nucleotidase.     

Purinergic modulation of glucose uptake into cultured rat podocytes: effect of diabetic milieu

Extracellular purines act via P1 and P2 receptors on podocytes and may influence on their function. This action may be modified under various (patho)physiological conditions leading to development of podocytopathy. Aim of study was to investigate effects of diabetic milieu, represented by high glucose concentration (HG, 30 mM glucose) on purinergic-induced changes of 2-deoxy-d-glucose (2-DG) uptake and on extracellular purines metabolism in cultured rat podocytes. Basal 2-DG uptake was 2.7-fold enhanced in HG compared to normal glucose concentration, NG (1271 ± 86 vs. 477 ± 37 nmol/h/mg protein, P < 0.001). ATP stimulated 2-DG uptake by 44 ± 4% and 29 ± 5% in NG and HG, respectively. ATP analogues, β, γ-methylene ATP and 2-methylthio ATP stimulated 2-DG uptake in range of 18–34% in NG and 16–17% in HG. Benzoylbenzoyl ATP increased 2-DG uptake about 24 ± 2% in NG however, its effect in HG reached 50 ± 1%. The antagonists of P2 receptors (suramin, reactive blue 2, PPADS) decreased basal 2-DG uptake in NG and HG; suramin and reactive blue 2 at average of 15 ± 4% in NG but in HG the effect was in following order: suramin 28 ± 3%; PPADS 20 ± 3% and RB-2 9 ± 0.9%. Extracellular adenosine concentration was higher in HG than in NG (0.48 ± 0.01 vs. 5.05 ± 0.39 μM, P < 0.05), however intracellular ATP content and extracellular ATP concentration were not affected. Neither ecto-ATPase nor ecto-5′-nucleotidase activities were affected in HG. In conclusion, diabetic milieu affects purinergic modulation of glucose transport into podocytes which may play a role in development of diabetic podocytopathy.     

Alteration of 1,2-diacylglycerol content in ischemic and reperfused heart

Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN₃ were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase.A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05–0.8 µM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates.The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.Abbreviations Ap₅A P¹ - P⁵-di(adenosine-5) Pentaphosphate - ATP-DPH ATP Diphosphohydrolase - DCCD N,N Dicyclohexycarbodiimide - Fru-P₂ase Fructose 1,6-biphosphatase - SDS Sodium Dodecyl Sulfate - TLC Thin Layer Chromatography