Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10 percent serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1 percent serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3 - 10 days was approximately 1.08 ± 0.04 æg/female and 0.1 ± 0.05 æg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Proteínas das glândulas salivares do Anopheles dirus B (Diptera: Culicidae), vetor da malária humana foram determinadas e analisadas. A quantidade de proteínas das glândulas salivares em mosquitos com três a 10 dias de idade foi de aproximadamente 1,08 ± 0,04 æg/ fêmea e de 0,1 ± 0,05 æg/macho. As glândulas salivares de ambos os sexos mostraram organização morfológica semelhante à de outros mosquitos anofelinos. Em fêmeas, apirase acumula-se nas regiões distais, enquanto alfa-glucosidase foi encontrada na região proximal dos lóbulos laterais. Esta distribuição diferencial das enzimas analisadas reflete a especialização de diferentes regiões para alimentação de açucares e sangue. Análise SDS-PAGE revelou que pelo menos sete proteínas foram encontradas nas glândulas salivares de fêmeas, das quais cada região morfológica continha diferentes proteínas principais. Perfis eletroforéticos de proteínas semelhantes foram detectados comparando-se mosquitos não alimentados e alimentados por sangue, sugerindo que não existe proteína específica induzida pelo mesmo. Análise por gel poliacrilamida bi-dimensional mostrou a mais abundante proteína de glândulas salivares com aproximadamente 35 kilodaltons de massa molecular e ponto isoelétrico de aproximadamente 4,0. Estes resultados dão informações básicas que levariam a estudos adicionais sobre o papel das proteínas salivares do An. dirus B na transmissão da doença e hematofagia.

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney

ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metais, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similaiities in Mr, bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specifjc ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

ATP diphosphohydrolase activity in synaptosomes from cerebral cortex of rats subjected to chemically induced phenylketonuria

ATP diphosphohydrolase (apyrase)(EC3.6.1.5) activity was measured in synaptosomes from cerebral cortex of Wistar rats of both sexes subjected to experimental phenylketonuria, i.e., chemical hyperphenylaninemia induced by subcutaneous administration of 5.2 µmol phenylalanine/g body weight (twice a day) plus 0.9 µmol p-chlorophenylalanine/g body weight (once a day). ATP diphosphohydrolase specific activity (nmol Pi min-1 mg protein-1) of synaptosomes was significantly decreased compared to controls for both ATp (from 147.6 to 129.9) and ADP (from 70.2 to 63.1) hydrolysis one hour after single administration of the drugs to 35-day old rats. Chronic treatment was performed from the 6th to the 28th postpartum day. The enzyme specific activity of synaptosomes was measured one week after the last administration of the drugs and was significantly reduced compared to controls for both ATP (from 164.1 to 150.2) and ADP (from 76.3 to 62.1) hydrolysis. The in vitro effects of the drugs on the synaptosome enzyme specific activity were also investigated. Phenylalnine alone or associated with p-chlorophenylalanine significantly reduced enzyme specific activity for both ATP (from 150.2 to 136.0) and ADP (from 70.5 to 59.3) nucleotides as substrates. Since ATP diphosphohrolase seems to play an important role in neurotransmission, these findings may be related to the neurological dysfunction characteristic of human phenylketonuria

ATP diphosphohydrolases: an overview

This review aimed at discussing the protocols used to characterize and to distinguish ATP diphosphohydrolases from other enzymes which can promote the degradation of ATP and ADP, since there is a confusion about the identily of this enzyme and ATPases.

Effects of undernutrition during suckling on ATP and ADP hydrolysis by synaptosomes from the cerebral cortex of adult rats

Early undernutrition can cause permanent functional changes in the nervous system. Alterations in enzymes involved in neurotransmiter metabolism have been reported to result from early undernutrition. In a previous study, we demonstrated that undernutrition during suckling decreaseATP and ADP hydrolysis by synaptosomes from cerebral cortex by abouth 20% of the value found in 20-day-old well-nourished rats (j.B.T. Rocha, C.F. Melo, J.J.F.Sarkis and R.D. Dias, British Journal of Nutrition, 63:273-283, 1990). In the present study, we investigated whether this deficit persists in synaptosomes from cerebral cortex of nutritionally rehabilitated adult rats. rats were undernourished from birth to 25 days of life by feeding their dams a 7% casein (w/w) diet, while well-nourished offspring were fed by mothers maintained on a 28% casein diet. In contrast to the results previously obtained in young rats, the synaptosomes obtained from the cerebral cortex of early undernourished adult rats hydrolyzed ATP and ADP more efficiently than did those obtained from well-nourished rats. Specific activity (nmol min-1 mg protein-1, mean ñ SD) was 114.9ñ9.5 for undernourished rats (N=8) for ATP, and 50.4ñ6.1 (N=8) vs 38.8ñ4.5 (N=8) for ADP. These results suggest that the deficits found in young rats disappear in rehabilitation adult rats

Synaptosomal apyrase in the hypiothalamus of adult rats

1. The synaptosomal fraction isolated from hypothalamus of adult rats on sucrose density gradient hydrolyzes the labile phosphatase from ATP and ADP, thereby satisfying the general definition of apyrase activity. 2. The parallel behavior of ATPase and ADPase activities under different reaction conditions suggests the presence of a "true" apyrase enzyme. The optimum conditions for the are the same for both nucleotides: pH 8.0, 0.6 mM nucleotide and 1.5 mM cation. At temperatures between 10 and 40-C, both activities increase with no change in the ATP/ADP hydrolysis ratio. Thermal inactivation or inhibition of the enzyme activity by iodoacetamide, p-hydroxynercuribenzoate or 2- mercaptoethanol affected the hydrolysis of both substrates in a similar manner. 3- Adenylate Kinase and phyrophosphatase activities were not detected in the preparation. 4. The enzyme is located on the outer surface of the synaptosomal membrane: intact and lysed synaptosomes have similar activity and the supernatant obtained by centrifugation of intact synaptosomal preparations does not hydrolyze ATP or ADP