A radioassay for phosphofructokinase-1 activity in cell

J. Biochem. Biophys. Methods 50 Ž2002. 129–140 www.elsevier.comrlocaterjbbm

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme Mauro Sola-Penna a,) , Ana Cristina dos Santos b, Gutemberg G. Alves a,b, Tatiana El-Bacha a,b, Joana Faber-Barata a,b, Monica F. Pereira a , Fredson C. Serejo a , Andrea T. Da Poian b, Martha Sorenson b a

Laboratorio ´ de Enzimologia e Controle do Metabolismo (LabECoM), Departamento de Farmacos, ´ Faculdade de Farmacia, UniÕersidade Federal do Rio de Janeiro, Ilha do Fundao, ´ ˜ Rio de Janeiro, RJ 21944-910, Brazil b Departamento de Bioquımica Medica, Instituto de Ciencias Biomedicas, UniÕersidade Federal do Rio de ´ ´ ˆ ´ Janeiro, Rio de Janeiro, RJ 21941-590, Brazil Received 11 May 2001; accepted 14 June 2001

Abstract Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32 P in the gamma position Žw g-32 PxATP., and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32 P Žw1-32 Pxfructose-1,6-bisphosphate.. Activated charcoal is used to adsorb unreacted w g-32 PxATP, and the radioactive product in the supernatant, w1-32 Pxfructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Phosphofructokinase-1; w g-32 PxATP; Glycolytic enzyme; Fructose-6-phosphate; Radioassay

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Corresponding author. Tel.: q55-21-260-9192x219; fax: q55-21-562-6445. E-mail address: [email protected] ŽM. Sola-Penna..

0165-022Xr02r$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 1 8 0 - 4

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M. Sola-Penna et al.r J. Biochem. Biophys. Methods 50 (2002) 129–140

1. Introduction Phosphofructokinase-1 ŽATP:D-fructose-6-phosphate 1 phosphotransferase, PFK-1, EC 2.7.1.11. is a key regulatory glycolytic enzyme. It is characterized by allosteric kinetics, a complex oligomeric structure, and multiple modes of regulation w1–4x. The interactions of phosphofructokinase-1 and other glycolytic enzymes with subcellular components, in particular cytoskeletal structures, have been studied extensively w4–19x. It is now understood that the glycolytic pathway is not composed of freely soluble enzymes, but occurs instead in both soluble and cytoskeletal fractions. For example, phosphofructokinase and aldolase, when associated with actin, become more active and are not susceptible to allosteric regulation w5–7x. Therefore, the phenomenon of association of glycolytic enzymes with cellular components is tightly linked to metabolic regulation w8–10x. Basically, three assays have been developed for measuring PFK-1 activity w20x: Ža. the continuous spectrophotometric or fluorimetric measurement of NADH disappearance, in which fructose-1,6-bisphosphate formation is coupled to aldolase, a-glycerophosphate dehydrogenase, and triosephosphate isomerase reactions, yielding 2 mol of NADH oxidized for each mole of fructose-1,6-bisphosphatase formed; Žb. the continuous spectrophotometric or fluorimetric measurement of ADP production using pyruvate kinase and lactate dehydrogenase; and Žc. a continuous measurement of Hq production by use of a pH stat. Among these assay methods, the least sensitive is the measurement of Hq production w20x. The methods more commonly employed because of their convenience and rapidity are those that measure NADH disappearance coupled to fructose-1,6-bisphosphate or ADP production w20x. However, the continuous methods are limited by the susceptibility of auxiliary enzymes to the assay conditions. Measurements performed in crude homogenates, under extreme conditions of pH or osmolarity, or in the presence of ligands require extensive controls in order to avoid artifacts due to changes in the activity of auxiliary enzymes. In addition, non-covalent binding of the auxiliary enzymes to the phosphofructokinase itself has the potential for altering phosphofructokinase activity. Here, we describe a method for measuring phosphofructokinase-1 activity that is adapted from one originally used for ATPase activity measurements w22x. This new version employs w g-32 PxATP and fructose-6-phosphate as substrates for PFK-1, generating fructose-1,6-bisphosphate labeled with 32 P in position 1. After adsorption of unreacted ATP on activated charcoal, the radioactivity remaining in the supernatant can be attributed to the fructose 1,6-bisphosphate formed.

2. Material and methods 2.1. Materials ATP, Tris, fructose-6-phosphate, fructose-1,6-bisphosphate, fructose-2,6-bisphosphate, aldolase, triosephosphate isomerase, a-glycerophosphate dehydrogenase, and


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NADH were purchased from Sigma ŽSt. Louis, MO, USA.. Rabbit muscle PFK-1 was from three different sources: Sigma; ICN Pharmaceuticals ŽCosta Mesa, CA, USA.; and prepared in our laboratory according to Kemp w21x. 32 Pi was purchased from Instituto de Pesquisas Energeticas e Nucleares ŽSao ´ ˜ Paulo, Brazil.. w g-32 PxATP was prepared according to Ref. w23x. Other reagents were of the highest purity available. 2.2. Radioassay for phosphofructokinase-1 actiÕity Phosphofructokinase-1 activity was measured by adapting the method developed by Grubmeyer and Penefsky w22x for ATPase activity. Except where specified, assays were performed in a medium containing: 50 mM Tris–HCl ŽpH 7.4., 5 mM MgCl 2 , 5 mM ŽNH 4 . 2 SO4 , 1 mM fructose 6-P, 0.1 mM w g-32 PxATP Ž4 mCirmmol. and either purified PFK-1 Ž2 mg protein. or cell-free extracts Ž100 mg protein. in a final volume of 0.4 ml, at 37 8C. Reaction was quenched after 30 s when purified enzyme was used and after 2 min when cell-free extracts were used. Quenching was achieved by addition of 1 ml of activated charcoal suspended in 0.1 N HCl Ž25 g activated charcoal per 100 ml 0.1 N HCl.. The suspension was centrifuged at 1500 = g in a refrigerated clinical centrifuge for 15 min, and 0.4 ml of the supernatant was counted in a liquid scintillation counter. Blanks for each tube were performed in parallel in the absence of fructose-6-phosphate. 2.3. Spectrophotometric assay for PFK-1 actiÕity The assay medium contained: 50 mM Tris–HCl ŽpH 7.4., 5 mM MgCl 2 , 5 mM ŽNH 4 . 2 SO4 , 1 mM fructose 6-P, 0.1 mM ATP, 5 mM NADH, 1.5 U aldolase, 2.5 U triosephosphate isomerase, 4 U a-glycerophosphate dehydrogenase and 2 mg of purified PFK-1 in a final volume of 1 ml. Reaction was started by addition of PFK-1, and NADH oxidation was followed by measuring the decrease in absorbance at 340 nm in a Hitachi Model U-2001 spectrophotometer ŽHitachi Instruments, San Jose, CA, USA.. 2.4. Inorganic phosphate Inorganic phosphate was determined colorimetrically w24x. 2.5. Thin-layer chromatography Ascending thin-layer chromatography ŽTLC. was performed on pre-coated silica gel 60 F254 plates Ž20 = 20 cm, Merck KgaA, Germany., using as solvent system isopropanolracetonerlactic–phosphoric acid Ž4:1:4, vrvrv.. Lactic–phosphoric acid was prepared as a solution containing 10 mM lactic acid and 5 mM phosphoric acid. Fructose 6-phosphate Ž10 mg., fructose 1,6-bisphosphate Ž10 mg. and ATP Žwith w g-32 PxATP as a tracer; 10 mg. were used as standards. Duplicate aliquots containing 50 mM Tris–HCl ŽpH 7.4., 5 mM MgCl 2 , 5 mM ŽNH 4 . 2 SO4 , 1 mM fructose 6-P, 0.1 mM w g-32 PxATP Ž4 mCirmmol. and 2 mg of purified PFK-1 in a final volume of 0.4 ml

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were allowed to react for 30 s at 37 8C; one tube was quenched with 1 ml of 0.1 N HCl, and the other with 1 ml of activated charcoal in 0.1 N HCl. Both were centrifuged at 1500 = g on a refrigerated clinical centrifuge for 15 min, and 0.4 ml of the supernatant was frozen in liquid nitrogen and lyophilized overnight. The lyophilized residue was dissolved in 10 ml water and spotted on a TLC plate. After development for 3.5 h at room temperature, the TLC plate was dried and revealed for sugars by spraying a solution of 20 mg orcinol in 100 ml H 2 SO4 Ž20%, vrv. and incubating at 100 8C for 10 min. The radioactive spots were detected by autoradiography on Kodak XK-1 film exposed for 24 h at room temperature. 2.6. Cell-free muscle homogenate A rabbit killed by cervical dislocation was bled by cutting the blood vessels of the neck, and the muscles of the hind legs and back were quickly removed, cleaned to remove fat and connective tissue and chilled in ice. The muscle mass was passed through a meat grinder, weighed, and homogenized for 30 s in a blender in the presence of 2 volumes of 30 mM NaF, 4 mM EDTA and 1 mM dithiothreitol, at pH 7.5. The suspension was homogenized a second time in a Polytron ŽBrinkmann Instruments, Westbury, NY, USA. for 30 s, aliquoted and immediately used or stored at y70 8C for further experiments. 2.7. Red blood cells Human blood Ž5 ml. was freshly collected from volunteers in tubes containing a few drops of EDTA and centrifuged at 1500 = g, and the supernatant was discarded. The remaining cells were washed three times with 2 ml of 0.9% Žwrv. NaCl solution. The pellet was frozen in liquid nitrogen, thawed with 1 ml of a cold solution containing 0.25 M sucrose, 1 mM dithiothreitol and 20 mM NaF ŽpH 7.5., and used immediately. 2.8. Vero cells Vero cells were grown in monolayers at 37 8C in a CO 2 incubator, using Dulbecco’s modified Eagle’s medium ŽDMEM. supplemented with 10% Žvrv. fetal bovine serum ŽCultilab, Campinas, SP, Brazil., 100 mgrml ampicillin, 5 mgrml gentamycin, and buffered with 45 mM sodium bicarbonate ŽpH 7.4.. Two days before use, cells were harvested from stock culture flasks after trypsinization, diluted 1:4 with DMEM, transferred to 35-cm2 culture flasks, and grown to confluence at 37 8C. Confluent monolayers of Vero cells were incubated at 37 8C with fresh DMEM supplemented with 10% fetal bovine serum for 6 h. The cells were scraped and harvested by centrifugation at 4000 = g, for 5 min. For each flask, extracts were prepared in 0.3 ml of lysis buffer Ž50 mM Tris–HCl, 250 mM NaCl, pH 7.5, containing 0.1% Triton X-100, 5 mM EDTA, 10 mM EGTA, 50 mM NaF, 1 mM NaVO4 , 50 mgrml PMSF, 2 mgrml leupeptin, 1 mgrml aprotinin..


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3. Results and discussion 3.1. Radioassay for PFK-1 actiÕity The method proposed here is an adaptation of a classical method w22x for measurement of ATP hydrolysis catalyzed by ATPases. As originally developed, this method employs w g-32 PxATP, which releases ADP and 32 Pi when hydrolyzed, and the 32 Pi is separated from the non-hydrolyzed w g-32 PxATP by adsorbing the latter on activated charcoal in 0.1 N HCl. After centrifugation, the supernatant Žcontaining 32 Pi . is counted in a liquid scintillation counter. Here, we adapted this method for measurement of PFK-1 activity by incubating fructose 6-phosphate and w g-32 PxATP in the presence of PFK-1. The products released by the enzyme are ADP and w1-32 Pxfructose-1,6-bisphosphate, which is separated from the non-hydrolyzed w g-32 PxATP by adsorbing the latter on activated charcoal in 0.1 N HCl. After centrifugation, the radioactivity remaining in the supernatant is from w1-32 Pxfructose-1,6-bisphosphate. Controls are run in parallel in the absence of fructose-6-phosphate, and subtracted from values obtained with complete assay medium. 3.2. PFK-1 actiÕity of purified enzyme analyzed by the radiometric and spectrophotometric assays The reaction rate of purified PFK-1 was analyzed by the traditional continuous spectrophotometric method and the new radiometric method. The spectrophotometric assay provides a continuous trace, but values obtained at specific times were registered and plotted to allow comparison with the radiometric method. As shown in Fig. 1, both

Fig. 1. Time course of PFK-1 activity measured by radiometric and spectrophotometric methods. Activities were determined as specified under Section 2. Žv .: PFK-1 activity measured by radioassay; ŽI. PFK-1 activity measured by the spectrophotometric method; and Ž`. Pi determination colorimetrically as described in Section 2. Values are means of at least five independent experiments and bars represent the standard errors.

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methods give very similar results and are equivalent in sensitivity and precision. Since the radioassay will also detect ATPase activities, we also measured the Pi production during the PFK-1 reaction. This measurement was performed in control experiments in the absence of fructose-6-phosphate as well as in the complete assay medium, using a colorimetric method for Pi w24x. There was no Pi production measurable either case. Fig. 1 shows these data over a 5-min period, during which activity declined to nearly zero. 3.3. Characterization of the radioactiÕe product of the radiometric method In order to confirm that the radioactivity remaining in the supernatant after activated charcoal treatment of the reaction mixture was from w1-32 Pxfructose-1,6-bisphosphate, thin-layer chromatography was performed on reaction mixture treated or not with activated charcoal. The colorimetric reaction for sugars ŽFig. 2A, lane 4. reveals three spots in the reaction mixture not treated with activated charcoal, which exhibit the same mobility as standards for fructose 6-phosphate Žlane 1., fructose 1,6-bisphosphate Žlane 2. and ATP Žlane 3.. Autoradiography of the TLC plate ŽFig. 2B. shows that only the

Fig. 2. Thin-layer chromatography and autoradiography of the products of reaction of PFK-1 assayed by the radiometric method. Panel A: thin-layer chromatogram exposed to orcinol–H 2 SO4 Žsee Section 2.. Lanes 1, 2 and 3: fructose-6-phosphate, fructose-1,6-bisphosphate and ATP standards, respectively; lane 4: whole reaction mixture; and lane 5: reaction mixture Žsupernatant. after activated charcoal treatment. Panel B: autoradiography of the thin-layer chromatogram in panel A.


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spots corresponding to fructose 1,6-bisphosphate and ATP were radioactive. After treatment with activated charcoal ŽFig. 2A and B, lane 5., the ATP spot disappeared. This experiment shows that the radioactivity remaining in the supernatant after treatment with activated charcoal is due to formation of w1-32 Pxfructose-1,6-bisphosphate. 3.4. Catalytic properties of purified PFK-1 eÕaluated by the radiometric assay Catalytic properties of purified muscle PFK-1 were analyzed by the new radioassay. Fig. 3 summarizes these data, which show the affinity for each substrate ŽATP and fructose-6-phosphate. and the activation by fructose-2,6-bisphosphate. Fig. 3A shows both stimulatory and inhibitory ATP-dependent components. Activation and inhibition constants for ATP calculated from fitted curves were 0.05 and 1.8 mM, respectively, and are close to parameters published elsewhere w25,26x. Activation by fructose-6-phosphate was analyzed at two different ATP concentrations: 0.1 mM, when the catalytic site for ATP is nearly saturated, and at 1 mM ATP, when the inhibitory site is partially saturated and the affinity for fructose-6-phosphate is diminished ŽFig. 3B.. The activation constant for fructose-6-phosphate increased from 0.015 to 0.05 mM when ATP was raised from 0.1 to 1 mM. These data are in accordance with those previously reported for this enzyme w25,26x Fig. 3C shows the activation of PFK-1 by fructose-2,6-bisphosphate: the activation constant is 20 nM, and the effect is saturated with less than 100 nM of activator as classically reported w25,26x. 3.5. Comparison between the radiometric and spectrophotometric methods in the presence of a modulating compound This method is particularly useful when tested conditions can directly affect the function of auxiliary enzymes, thereby disguising the actual effects on phosphofructoki-

Fig. 3. Kinetic properties of purified muscle PFK-1 assayed by the radiometric method. Panel A: ATP concentration dependence assayed as indicated under Section 2, except that the ATP concentrations are those shown on the abscissa. Panel B: fructose-6-phosphate dependence assayed in the presence of 0.1 mM ATP Žv . and 1 mM ATP Ž`.. Panel C: fructose-2,6-bisphosphate dependence assayed in the presence of 3 mM ATP and 1 mM fructose-6-phosphate. Values are means of at least five independent experiments and bars represent the standard errors.

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nase-1. This is clearly seen when we analyze the effects of trehalose on this enzyme. Trehalose is a non-reducing disaccharide of glucose, widely distributed among living systems w16x. We have previously reported that this osmolyte modulates the activity of several enzymes, an effect that is normally associated with stabilization of the enzyme w27,28x. Fig. 4A shows the effect of trehalose on PFK-1 activity measured by radioassay. It can be seen that this osmolyte stimulates the PFK-1 activity in a dose-dependent manner. On the other hand, trehalose appears to inhibit PFK-1 when the activity is measured by the traditional spectrophotometric method ŽFig. 4B.. Fig. 4C shows the effect of trehalose directly on the combined auxiliary enzymes when PFK-1 is omitted. Reaction catalyzed by the auxiliary enzymes was guaranteed by addition of 0.1 mM fructose-1,6-bisphosphate, simulating the PFK-1 reaction. The decrease in NADH catalyzed by the coupled enzymes Žaldolase, a-glycerophosphate dehydrogenase, and triose-phosphate isomerase. is inhibited by trehalose with a concentration profile that is very similar to that shown in Fig. 4B. Thus, the inhibition observed in Fig. 4B is an artifact due to the inhibition of auxiliary enzymes promoted by trehalose ŽFig. 4C.. 3.6. Phosphofructokinase-1 actiÕity measured by radioassay in muscle homogenates, lysed erythrocytes and Vero cell extracts In these experiments, the new radioassay was used to measure PFK-1 activity in different cell-free systems. Fig. 5 shows the time course and fructose-6-phosphate dependence of PFK-1 activity in a homogenate of rabbit muscle, in lysed fresh red blood cells, and in an extract from lysed Vero cells. Phosphofructokinase-1 activity was calculated from the difference between reaction mixtures with and without fructose-6phosphate, after stopping the reaction with activated charcoal as described. The time course of the PFK-1 activity in each cell-free system shows an increase in w1-32 Pxfructose-1,6-bisphosphate produced over a 5-min period Žfilled circles in Fig. 5A,C,E, for rabbit muscle, red blood cells and Vero cells, respectively.. Tubes run in parallel without fructose-6-phosphate show an increase in Pi Žempty circles in Fig. 5A,C,E., probably

Fig. 4. Effects of trehalose on PFK-1 activity measured by radioassay ŽA. and by the spectrophotometric method ŽB.. In C, effects of trehalose on the auxiliary enzyme system in the absence of PFK and 0.1 mM fructose-2,6-bisphosphate was added to all assays. Values are means of three independent experiments and bars represent the standard errors.


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Fig. 5. Time course and fructose-6-phosphate dependence of PFK-1 activity measured by radioassay in different cell-free systems. A and B: rabbit muscle homogenate. C and D: freshly lysed red blood cells. E and F: Vero cells lysate. Experiments are representative of a series. In panels A, C and E: Žv . fructose-1,6-bisphosphate produced Ždifference between assays in the presence and the absence of 1 mM fructose-6-phosphate. and Ž`. 32 Pi produced by hydrolysis of w g-32 PxATP Žin the absence of fructose-6-phosphate, as described in Ref. w22x..

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due to ATPase activity from these systems. The amount of Pi produced corresponds to approximately 20% of the amount of fructose-1,6-bisphosphate produced in the same period. Activation of PFK-1 activity by the substrate fructose-6-phosphate is clearly observed using the new radiometric method ŽFig. 5B,D,F.. These data indicate that the new radioassay may be useful for analyzing PFK-1 regulation in different tissues in the presence of diverse intracellular components. 3.7. Cost estimate An additional advantage of the radioassay compared to the traditional spectrophotometric method is suggested by cost estimates, based on U.S. catalog prices for the enzymes Žaldolase, a-glycerophosphate dehydrogenase, and triosephosphate isomerase. and NADH used in a 1-ml reaction mixture Žsince other reagents are the same in both methods.. The coupled enzyme mixture costs approximately US$6.50 per tube. On the other hand, based on the cost of the w g-32 PxATP, activated charcoal and a scintillation liquid Žtoluene, Triton X-100 and 1,4-bisw5-phenyl-2-oxazolylx-benzene., the radioassay costs approximately US$0.50 per 0.4 ml assay, less than 10% of the cost of the spectrophotometric method.

4. Simplified description of the method and its applications Phosphofructokinase-1 catalyzes one of the regulatory steps of glycolysis w4x. Its activity is affected by many substances that modulate glycolysis in different cells w4–10x. This enzyme associates with several proteins that may modulate and regulate its activity, including microtubules, actin, aldolase, pyruvate kinase and others w4–10x. The traditional methods employed to measure phosphofructokinase-1 activity use auxiliary enzymes that include aldolase or pyruvate kinase. Both enzymes can bind directly to phosphofructokinase-1 and alter its activity w5–7x. Here, we developed a direct method for phosphofructokinase-1 activity which eliminates the need for auxiliary enzymes. Kinetic properties of the enzyme are clearly observed ŽFigs. 1 and 2., and are similar to those found in using a coupled assay w25,26x. The radiometric method avoids artifacts due to alteration of auxiliary enzyme function, as is shown in the presence of trehalose ŽFig. 4.. In addition, it is cheaper than the coupled assay and can be used even in the presence of other cellular components.

Acknowledgements We thank Dr. Adalberto Vieyra for the use of his spectrophotometer. This work was supported by grants from Fundaçao ˜ Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro ŽFAPERJ., Fundaçao ˜ Universitaria ´ Jose´ Bonifacio ´ ŽFUJB., ŽCNPq. PRONEX, and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico ´ ´ to M.S.P. and M.S.; and Conselho Nacional de Desenvolvimento Cientıfico e ´ ŽCNPq.; Fundaçao Tecnologico ´ ˜ Carlos Chagas Filho de Amparo a` Pesquisa do Estado


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do Rio de Janeiro ŽFAPERJ., and Programa de Apoio ao Desenvolvimento Cientifico e ŽPADCT. to A.T.D.P. F.C.S. was the recipient of an undergraduate Tecnologico ´ fellowship from Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico ´ ´ ŽCNPq.. A.C.S., G.G.A., and T.E.B. were the recipients of graduate fellowships from Coordenaçao ˜ de Aperfeiçoamento de Pessoal de Nıvel ´ Superior ŽCAPES..

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