Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Release of glycosylphosphatidylinositol- (GPI-) anchored ectoenzymes from the membrane by phosphatidylinositol- (PI-) specific phospholipases may play an important role in modulating the surface expression and function of this group of proteins. To investigate how the properties of the host membrane affect anchor cleavage, porcine lymphocyte ecto-5'-nucleotidase (5'-NTase; EC 3.1.3.5) was purified, reconstituted into lipid bilayer vesicles of various lipids, and cleaved using PI-PLC from Bacillus thuringiensis (Bt-PI-PLC). Bt-PI-PLC activity was highly dependent on the chain length and unsaturation of the constituent phospholipids. Very high rates of cleavage were observed in fluid lipids with a low phase transition temperature (T(m)), in lymphocyte plasma membrane, and in a lipid mixture that formed rafts. Arrhenius plots of the rate of anchor cleavage in various lipids showed a characteristic break at the bilayer T(m), together with a discontinuity close to T(m). The activation energy for GPI anchor cleavage was substantially higher in gel phase bilayers compared to those in the liquid crystalline phase. The addition of cholesterol simultaneously abolished the phase transition and the large difference in cleavage rates observed above and below T(m). Inclusion of GM(1) and GT(1b) (components of lipid rafts) in the bilayer reduced the overall activity, but the pattern of the Arrhenius plots remained unchanged. Both gangliosides had similar effects, suggesting that bilayer surface charge has little influence on PI-PLC activity. Taken together, these results suggest that lipid fluidity and packing are the most important modulators of Bt-PI-PLC activity on GPI anchors.
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PMID:PI-specific phospholipase C cleavage of a reconstituted GPI-anchored protein: modulation by the lipid bilayer. 1180 43

We previously demonstrated that Copenhagen (COP) and DA inbred rat strains show a wide difference in a test for aerobic treadmill running that correlated positively with isolated cardiac function. The purpose of this study was to test adenosine production as a candidate intermediate phenotype that may explain part of the difference in running and cardiac performance in these genetic models for low and high aerobic capacity. Adenosine production was measured as the activity of soluble 5'-nucleotidase and membrane-bound ecto-5'-nucleotidase in the membrane pellet and supernatant fractions of left and right ventricular muscle and gracilis muscle taken from 10 DA and 10 COP rats. Ecto-5'-nucleotidase activity in the membrane pellet of hearts from both DA and COP accounted for the vast majority of the total tissue adenosine production (>90% in the left ventricle and >80% in the right ventricle). Ecto-5'-nucleotidase activity in the pellet fraction was significantly higher in the left (22.4%) and right (46.1%) ventricles of DA rats compared with COP rats, with no differences in total protein content. There were no significant differences between the strains for 5'-nucleotidase activity in the cardiac supernatant, the gracilis pellet, or the gracilis supernatant. These data support the hypothesis that an increase in cardiac adenosine production may contribute to the greater aerobic running capacity of the DA rats.
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PMID:Cardiac adenosine production in rat genetic models of low and high exercise capacity. 1206 42

Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5'-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with (3)H-labelled nucleotides and adenosine as substrates, direct evaluation of gamma-phosphate transfer from [gamma-(32)P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20 kDa surface protein was observed following incubation of Namalwa B cells with [gamma-(32)P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.
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PMID:The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells. 1209 90

Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release not only of the cotransmitters ATP and norepinephrine but also of soluble nucleotidases that break down extracellular ATP, ADP, and AMP into adenosine. In this study we show that the apparent K(m) values of the releasable enzyme activity vary depending on which of these adenine nucleotides is used as initial substrate. The K(m) value for ATP was 33.6 +/- 2.3 microM, 21.0 +/- 2.3 microM for ADP, and 10.0 +/- 1.1 microM for AMP. The ratios of the V(max) values for each enzyme reaction were 4:2:3. We have also found a different sensitivity of the metabolism of ATP and AMP by releasable nucleotidases to known nucleotidase inhibitors. Suramin inhibited the breakdown of ATP by releasable nucleotidases in a noncompetitive manner and with a K(i) value of 53 microM, but had no effect on the breakdown of AMP. The 5'-nucleotidase inhibitor alpha,beta-methylene ADP inhibited the breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-beta,gamma-dibromomethylene-D-ATP (ARL67156), an ecto-ATPase inhibitor, suppressed ATPase and AMPase activities, whereas NaN(3) (10 mM) affected neither reaction, but inhibited the ADP metabolism. Phosphatase- and phosphodiesterase inhibitors did not affect the activity of the releasable nucleotidases. This evidence suggests that the soluble nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5'-nucleotidase-like and an ecto-nucleoside triphosphate diphosphohydrolase-like activity.
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PMID:Enzyme kinetics and pharmacological characterization of nucleotidases released from the guinea pig isolated vas deferens during nerve stimulation: evidence for a soluble ecto-nucleoside triphosphate diphosphohydrolase-like ATPase and a soluble ecto-5'-nucleotidase-like AMPase. 1218 56

Polyamines were found to modulate the activity of several membrane-bound enzymes, participating in cell growth and differentiation. We have studied the effect of polyamines (spermidine, spermine and putrescine) on rat mesangial cell ectoenzymes: 5'-nucleotidase, Mg(2+)-ATPase and Ca(2+)-ATPase. Ecto-5'-nucleotidase activity was significantly increased after 48 h treatment with spermine and spermidine. Mg(2+)-ATPase was increased only after treatment with spermidine; however, Ca(2+)-ATPase was significantly increased after both spermine and spermidine treatment of mesangial cells. Culture of mesangial cells with putrescine did not change the activity of these ectoenzymes. Increased expression of mesangial cell ecto-ATPase and ecto-5'-nucleotidase after spermine and spermidine treatment could result in an increased production of adenosine, a powerful autacoid interesting with respect to a role of mesangial cells in inflammatory processes.
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PMID:Effect of polyamines on mesangial cell ecto-5'-nucleotidase and ecto-ATPase activity. 1218 6

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.
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PMID:Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat. 1254 43

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.
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PMID:Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation. 1267 11

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P2X and P2Y and PA1. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5'-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre-pubertal, mid-pubertal and 'young adult' (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.
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PMID:Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation. 1284 38

CD73 (ecto-5'-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-alpha produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-alpha treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1beta, TNF-alpha, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-alpha. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-alpha is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.
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PMID:IFN-alpha induced adenosine production on the endothelium: a mechanism mediated by CD73 (ecto-5'-nucleotidase) up-regulation. 1473 46

Studies on the purinergic system normally deal with adenine-based purines, namely, adenine nucleotides and adenosine. However, a guanine-based purinergic system may also have important neuromodulatory roles. Guanine-based purines exert trophic effects on neural cells, protect brain slices in a model of hypoxia and stimulate glutamate uptake. In vivo, both guanosine 5'-monophosphate (GMP) and guanosine (GUO) protected against seizures. In this study, we investigated if the anticonvulsant effect of GMP is mediated by guanosine and if guanosine or GMP treatments were able to increase adenosine levels. Intraperitoneal (i.p.) treatments with 7.5 mg/kg GMP or guanosine prevented 50% of seizures by quinolinic acid (QA) and increased guanosine cerebrospinal fluid (CSF) levels around twofold and threefold, respectively; GMP and adenosine levels remained unchanged. Intracerebroventricular treatment with 960 nmol GMP prevented 80% of seizures and the 5'-nucleotidase inhibitor alpha-beta-methyleneadenosine 5'-diphosphate (AOPCP), when injected 3 min before, reduced this anticonvulsant effect to 30% protection as well as significantly decreased the conversion of GMP into guanosine measured in the CSF. This study shows that the previously reported effect of GMP as an anticonvulsant seems to be related to its ability to generate guanosine through the action of ecto-5'-nucleotidase.
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PMID:Anticonvulsant effect of GMP depends on its conversion to guanosine. 1504 76


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