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Query: UNIPROT:P01189 (beta-endorphin)
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An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
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PMID:Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes. 1186 92

In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 +/- 0.47 nmol Pi/h x 10(8) cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent Km for p-NPP was 5.35 +/- 0.89 mM. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl2 and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.
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PMID:Ecto-phosphatase activity on the cell surface of Crithidia deanei. 1213 92

1. Different concentrations of non-phytate phosphorus (NPP, 2.5, 3.0, 3.5, 4.0 and 4.5 g/kg diet) were given to broilers (8 to 42 d of age) to establish regressions between dietary NPP concentration and body weight gain and tibia ash content. Second and third experiments were conducted to study the feasibility of utilisation of different phosphatic fertilisers [ammonium phosphate (AP), ammonium polyphosphate (APP), single super phosphate (SSP), NPK (17:17:17, NPK) and NP (28:28:0, NPK)] in commercial broilers (8 to 42 d) and White Leghorn layers (252 to 364 d). 2. Phosphatic fertilisers were incorporated both in broiler (10 g calcium and 4.5 g NPP/kg) and layer (35 g calcium and 3.5 g NPP/kg) diets by replacing dicalcium phosphate (DCP) in toto. 3. The logarithmic curves obtained for predicting the body weight gain and tibia ash content at different levels of NPP used in experiment 1 were Y = 156.27 + 2,468.8 logX (r2= 0.958) and Y = 530.82 + 144.26 log X (r2 = 0.916), respectively. 4. Body weight gain and food intake in broilers given APP- or NP-supplemented diets were comparable to these in the DCP-fed group. Feeding of NPK, AP or SSP resulted in significant depression in weight gain and food intake and high excreta moisture content. Food/gain, Ca and P contents in tibia ash and serum were not influenced by the use of phosphatic fertilisers as P sources in broiler diets. 5. Tibia ash content in broilers fed on diets containing fertilisers was either similar to or significantly higher than that in the DCP-fed group. Broilers on AP or SSP retained more P and had higher tibia ash content than those on DCP. AP, SSP or NPK caused degenerative and necrotic changes in liver, kidney and intestine of broilers. 6. Relative bio-availability of P from APP or NP was better for body weight gain than AP, SSP or NPK, while the reverse was true for bone calcification. 7. APP and NP gave hen-d egg production similar to that of DCP-fed layers. Food intake was significantly reduced in layers fed on diets containing fertilisers. However, food/egg mass, egg weight and serum Ca and inorganic P contents were not influenced by inclusion of fertilisers in layer diets. 8. Except for AP, inclusion of fertilisers in layer diets reduced shell weight and shell thickness compared with the DCP-fed group. However, no apparent eggshell defects were found which could be attributable to diet. 9. Results of these experiments suggest that APP and NP can be used as the sole source of P both in broiler and layer diets, replacing DCP in toto. However, when utilising these P sources in layers, due attention should be given to shell quality. Fertilisers containing high F (AP and SSP) or K (NPK) reduced performance in broilers and layers and caused microscopic changes in liver, kidney and intestine in broilers.
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PMID:Relative bio-availability and utilisation of phosphatic fertilisers as sources of phosphorus in broilers and layers. 1273 31

We have characterized a phosphatase activity present on the external surface of intact Malpighian tubules in Rhodnius prolixus. This phosphatase hydrolyses the substrate p-nitrophenyl phosphate at a rate of 3.38 +/- 0.07 nmol Pi x mg(-1) x min(-1). Phosphatase activity decreased with the increase of the pH from 6.4 to 7.6 pH, a range in which tubules cellular integrity was maintained for at least 1 h. Classical inhibitors of acid phosphatase, such as ammonium molybdate, fluoride, vanadate, mpV-PIC, and bpV-PHEN, caused different patters of inhibition. The ecto-phosphatase present an apparent Km of 1.67 +/- 0.34 mM and Vmax of 5.71 +/- 0.37 nmol Pi x mg(-1) x min(-1) for p-NPP. Zinc chloride inhibited 78.2% of ecto-phosphatase activity, with Ki of 0.35 mM. Such inhibition was reversed by incubation with cysteine and GSH, but not DTT, serine, and GSSG, showing that cysteine residues are important for enzymatic activity. Phosphatase activity increased 141% three days after blood meal, and returned to basal levels 2 days later. These results suggest that ecto-phosphatase activity could be involved in a diuretic mechanism, essential in the initial days after a blood meal for the control of Rhodnius homeostasis.
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PMID:Characterization of an ecto-phosphatase activity in malpighian tubules of hematophagous bug Rhodnius prolixus. 1535 54

Trypanosoma rangeli is a parasite of a numerous wild and domestic animals, presenting wide geographical distribution and high immunological cross-reactivity with Trypanosoma cruzi, which may lead to misdiagnosis. T. rangeli has a complex life cycle, involving distinct morphological and functional forms in the vector. Here, we characterized the cell surface polypeptides and the phosphatase activities in short and long epimastigotes forms of T. rangeli, using intact living parasites. The surface protein profile revealed by the incubation of parasites with biotin showed a preferential expression of the 97, 70, 50, 45, 25-22, and 15 kDa biotinylated polypeptides in the long forms, in contrast to the 55 and 28 kDa biotinylated polypeptides synthesized by the short epimastigotes. Additionally, flow cytometry analysis showed that the short forms had relatively lower biotin surface binding than long ones. The involvement of phosphatases with the trypanosomatid differentiation has been proposed. In this sense, T. rangeli living parasites were able to hydrolyze the artificial substrate p-nitrophenylphosphate at a rate of 25.57+/-2.03 and 10.09+/-0.93 nmol p-NPP x h(-1) x 10(7) cells for the short and long epimastigotes, respectively. These phosphatase activities were linear with time for at least 60 min and the optimum pH lies in the acid range. Classical inhibitors of acid phosphatases, such as ammonium molybdate, sodium fluoride, and zinc chloride, showed a significant decrease in these phosphatase activities, with different patterns of inhibition. Additionally, these phosphatase activities presented different kinetic parameters (Km and Vmax) and distinct sensitivities to divalent cations. Both epimastigotes were unable to release phosphatase to the extracellular environment. Cytochemical analysis demonstrated the localization of these enzymes on the parasite surfaces (cell body and flagellum) and in intracellular vacuoles, resembling acidocalcisomes.
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PMID:Trypanosoma rangeli: Differential expression of cell surface polypeptides and ecto-phosphatase activity in short and long epimastigote forms. 1644

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.
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PMID:Leishmania amazonensis: characterization of an ecto-phosphatase activity. 1681 76

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.
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PMID:Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina. 1712 1

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41+/-2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl2, MnCl2 and ZnCl2 were able to increase the (p-NPP) hydrolysis while CdCl2 and CuCl2 inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.
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PMID:Mycelial forms of Pseudallescheria boydii present ectophosphatase activities. 1742 13

Biochemical properties of a termostable alkaline phosphatase obtained from the mycelium extract of A. caespitosus were described. The enzyme was purified 42-fold with 32% recovery by DEAE-cellulose and concanavalin A-Sepharose chromatography. The molar mass estimated by Sephacryl S-200 or by 7% SDS-PAGE was 138 kDa and 71 kDa, respectively, indicating a homodimer. Temperature and pH optima were 80 degrees C and pH 9.0. This enzyme was highly glycosylated (approximately 74% saccharide content). The activity was enhanced by Mg2+ (19-139%), NH4+ (64%), Na+ (51%) and Mn2+ (38%). 4-Nitrophenyl phosphate (4-NPP) was preferentially hydrolyzed, but glucose 1-phosphate (93%), UTP (67%) and O-phosphoamino acids also acted as substrates. V(lim) and K(m) were 3.78 nkat per mg protein and 270 micromol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 micromol/L in the presence of Mg2+, using 4-NPP as substrate. The purified alkaline phosphatase removed the 5'-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology.
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PMID:Purification and biochemical characterization of a mycelial alkaline phosphatase without DNAase activity produced by Aspergillus caespitosus. 1770 60

In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).
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PMID:A Mg(2+)-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate. 1859 5


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