João José Freitas Sarkis

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats.

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.

Characterization of a synaptosomal ATP diphosphohydrolase from the electric organ of Torpedo marmorata

A true ecto-apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme was found in the synaptosomal fraction from the electric organ of the electric ray Torpedo marmorata. The activity could not be attributed to the combined action of different enzymes. The pH requirement and calcium dependence were the same for hydrolysis of both substrates ADP and ATP. The enzyme had an apparent Km value of 117 microM for ATP and of 123 microM for ADP. The involvement of nonspecific phosphatases in the hydrolysis of both substrates was excluded. The enzyme hydrolyses almost equally well different nucleoside di- and triphosphates. ATP and ADP hydrolysis was not inhibited by seven ATPase inhibitors, i.e., sodium azide, dinitrophenol, ruthenium red, oligomycin, ouabain, sodium orthovanadate and lanthanum.

ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) in human blood platelets

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca(2+) dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2 μg/d ouabain, 1.0 μM levamisole, 10 μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100 μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.

Inhibition of cytochrome c oxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro

L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.

Inhibition of succinate dehydrogenase and β-hydroxybutyrate dehydrogenase activities by methylmalonate in brain and liver of developing rats

SummaryThe effects of methylmalonate (MMA) on succinate dehydrogenase (SDH) and β-hydroxybutyrate dehydrogenase (HBDH) activities in brain and liver of 15-day-old rats were studied. The apparentKm of SDH for succinate was 0.45 mmol/L in brain and 0.34 mmol/L in liver. MMA inhibited the enzyme activity in both tissues withKi values of 4.5 mmol/L and 2.3 mmol/L in brain and liver, respectively, and the inhibition was of the reversible competitive type. The calculatedKm for HBDH with β-hydroxybutyrate as substrate was 1.26 mmol/L in brain and 0.36 mmol/L in liver. MMA inhibited the enzyme with aKi value of 0.015 mmol/L in brain and 0.275 mmol/L in liver. These results are probably relevant to our understanding of cerebral metabolism in methylmalonic acidaemic children, especially during ketoacidotic and hypoglycaemic crises, and may be related to the pathogenesis of cerebral dysfunction of methylmalonic acidaemia.

Characterization of an ATP diphosphohydrolase activity (APYRASE, EC 3.6.1.5) in rat blood platelets

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca2+-ATP and Ca2+-ADP as substrates. Ca2+-ATP and Ca2+-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca2+-ATPase and Ca2+-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.

Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: Kinetic characterization and biochemical properties

In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. Km and Vmax values for p-Nph-5′-TMP hydrolysis were found to be 106 ± 18 µM and 3.44 ± 0.18 nmol p-nitrophenol/min/mg (mean ± SD, n = 5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5′-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25 mM suramin inhibited p-Nph-5′-TMP, ATP and ADP hydrolysis, while 0.5 mM AMP decreased only p-Nph-5′-TMP hydrolysis. Besides, 5.0, 10 and 20 mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5 mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.

Solubilization and Characterization of an ATP Diphosphohydrolase (EC 3.6.1.5) from Rat Brain Synaptic Plasma Membranes

The effect of different detergents on the ATPase and ADPase activities from synaptic plasma membrane were investigated. Triton X-100, deoxycholate, CHAPS, Nonidet, N-octylglucoside and C12E8, which is commonly used to solubilize plasma membrane proteins, easily inactivated the ATPase and ADPase activities, while digitonin was not harmful to the enzyme. Treatment of the synaptic plasma membrane from rat brain with 0.5% digitonin solubilizes 80% of the proteins and 50% and 60% of ATPase and ADPase, respectively, with the following characteristics: stimulation by Ca2+ in the millimolar range, insensitivity to ATPase inhibitors (ouabain, olygomicyn, orthovanadate), inhibition with sodium azide and NEM and broad substrate specificity for the hydrolysis of nucleoside di- and triphosphate. To further characterize the enzyme solubilized, polyclonal antibodies specific for ATP diphosphohydrolase from potato tuber were tested. Western blot showed that two electrophoretic bands with a molecular mass close to 60-70 kDa had cross-immunoreactivity with antibodies against potato apyrase. The results presented here demonstrate for the first time the solubilization of ATPase and ADPase activities with characteristics of a true ATP diphosphohydrolase from synaptic plasma membrane from rat brain and with cross-immunoreactivity with antibodies against potato apyrase.

Activation of adenosine receptors in the posterior cingulate cortex impairs memory retrieval in the rat

Adenosine A1 and A2A receptor agonists and antagonists have been reported to alter learning and memory. The aim of our study was to investigate the involvement of adenosinergic system in memory retrieval into posterior cingulate cortex (PCC) of Wistar rats. To clarify this question, we tested specifics agonist and antagonists of adenosine A1 and A2A receptors in rats submitted to a one-trial inhibitory avoidance task. The stimulation of adenosine A1 and A2A receptors by CPA and CGS21680, respectively, impaired memory retrieval for inhibitory avoidance task, into PCC. These findings provide behavioral evidence for the role of adenosinergic system in the memory retrieval into PCC.

Altered ATP Hydrolysis Induced by Pentylenetetrazol Kindling in Rat Brain Synaptosomes

The ectonucleotidase pathway is an important metabolic source of extracellular adenosine. Adenosine has potent anticonvulsant effects on various models of epilepsy. One of these models is pentylenetetrazol (PTZ) kindling, in which repeated administration of subconvulsive doses of this drug induces progressive intensification of seizure activity. In this study, we examine the effect of a single convulsive injection (60 mg/kg, i.p.) or 10 successive (35 mg/kg, i.p.) injections of PTZ on synaptosomal ectonucleotidases. Our results have shown that no changes in ectonucleotidase activities were seen at 0, 1, and 24 h or at 5 days after a single convulsive PTZ injection. However, after PTZ-kindling, rats which were more resistant to seizure development presented an increase in ATP hydrolysis in synaptosomes from hippocampus and cerebral cortex (44% and 28%, respectively). These results suggest that changes in nucleotide hydrolysis may represent an important mechanism in the modulation of chronic epileptic activity in this model.