Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Leishmania amazonensis: characterization of an ecto-phosphatase activity

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.     

Localisation of High Acid Phosphotyrosine Phosphatase Activity in Afferent Arterioles and Glomeruli of Human Kidney

Summary Endothelial cells contain a variety of specific protein tyrosine phosphatases and an acid phosphatase differing from other known phosphatases. The highest activity of this acid phosphatase with artificial or unspecific substrates is present in the afferent arterioles and glomeruli of human kidney, and the activity is inhibited by nephrotoxic fluoride concentrations, suggesting that it plays a role in circulatory regulation. Here the activity was characterised with physiological substrates. An incubation mixture containing phosphotyrosine or phosphoserine was stable at pH 5 when phosphate-precipitating lead was chelated with tartrate. The activities were studied in frozen sections. Only phosphotyrosine was hydrolysed by some cells. High activity of tartrate-resistant phosphotyrosine phosphatase was present in lymphocytes, endothelial cells of afferent arterioles, and glomerular mesangial cells of kidney, decidual cells, and alveolar macrophages. In lymphocytes the activity was fluoride-resistant and vanadate-sensitive, in other cells fluoride- and vanadate-sensitive. In decidual cells and alveolar macrophages, the activity is due to specific osteoclastic/macrophagic tartrate-resistant acid phosphatase, in lymphocytes to specific protein tyrosine phosphatases, and in endothelial and mesangial cells to a protein tyrosine phosphatase-like acid phosphatase. The results suggest that in endothelial cells of the afferent arterioles, mesangial cells, and lymphocytes the cellular activities are regulated by high constitutive phosphotyrosine phosphatase activity and this may be related to the exceptional cyclosporin A sensitivity of these cells.     

Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells.

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.     

Acid phosphatase and phosphoamino acid phosphatases in murine erythroleukaemic cells

The activities of acid and alkaline phosphatases and phosphotyrosine, phosphoserine and phosphothreonine phosphatases were measured in Friend murine erythroleukaemic (MEL) cells. The effects of treating the cells with dimethyl sulphoxide (DMSO), an inducer of differentiation, were examined. In untreated cells alkaline phosphatase activity was undetectable, though there were significant amounts of acid phosphatase (76 +/- 15 mU/mg protein) and phosphotyrosine phosphatase (16 +/- 0.9mU/mg protein); phosphoserine and phosphothreonine phosphatase activities (9 +/- 0.4 and 7 +/- 0.6mU/mg protein, respectively) were lower than for phosphotyrosine phosphatase. Addition of 1 or 2% DMSO to the culture medium resulted in the expected cell death within 2 weeks. With 0.5% DMSO, cells remained viable for at least 8 weeks, but while some appeared to have smaller nuclei and retained their rounded appearance, others became fibroblastic within several days and adhered to the culture vessel. The treated cells which had kept their morphology showed no difference in acid phosphatase activities as compared with untreated controls; phosphotyrosine phosphatase was lower (9 +/- 0.8mU/mg protein) and phosphoserine and phosphothreonine phophatases higher (11 +/- 0.5 and 10 +/- 0.4mU/mg protein, respectively) than in the controls. The Km values for p-nitrophenyl phosphate were similar in untreated and treated cells (0.069 and 0.068mM, respectively); for phosphotyrosine the Km value was lower in the treated cells (0.97mM) than in the controls (1.9mM).     

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.     

Purification and properties of an acid phosphatase from Entamoeba histolytica HM-1:IMSS

Entamoeba histolytica contains and secretes acid phosphatase, which has been proposed as a virulence factor in some pathogenic microorganisms. In this work, we purified and characterised a membrane-bound acid phosphatase (MAP) from E. histolytica HM-1:IMSS and studied the effect of different chemical compounds on the secreted acid phosphatase and MAP activities. MAP purification was accomplished by detergent solubilisation, and affinity and ion exchange chromatographies. The enzyme showed a pI of 5.5-6.2, an optimum pH of 5.5, and a Km value of 1.14 mM with p-nitrophenyl phosphate.     

Phosphoamino acid phosphatases in human normal and leukaemic lymphocytes

The activities of phosphoamino acid phosphatases were measured in lymphocytes from normal adults. For cells from white individuals the mean value (+/- SD) for phosphotyrosine phosphatase activity was 342 +/- 120 mU/mg protein; mean values for phosphothreonine and phosphoserine phosphatase activities were 35.6 +/- 17.1 and 21.6 +/- 10.2 mU/mg protein, respectively. The corresponding activities were similar to these for both the Indian and Bantu population groups. The activity of phosphotyrosine phosphatase was significantly lower in lymphocytes from white children with acute lymphocytic leukaemia than in cells from normal children or adults. For phosphothreonine and phosphoserine phosphatases, there was no significant difference between activities obtained in lymphocytes from normal and leukaemic children.     

The 18 kDa cytosolic acid phosphatase from bovine liver has phosphotyrosine phosphatase activity on the autophosphorylated epidermal growth factor receptor

In this paper we demonstrate that the cytosofic low-M_r acid phosphatase purified from bovine liver has phosphotyrosine protein phosphatase acitivity on ³²P-autophosphorylated epidermal growth factor (EGF) receptor. This activity was significantly inhibited by orthovanadate and p-hydroxymercuribenzoate; the latter result indicates that free sulfhydryl groups are required for phosphotyrosine phosphatase activity. The enzyme was active in a broad pH range, with maximum activity between pH 5.5 and 7.5. The apparent K_m for ³²P-EGF receptor dephosphorylation was 4 nM. The enzyme appeared to be specific for phosphotyrosine in that it dephosphorylated the autophosphorylated EGF receptor and L-phosphotyrosine, but not ³²P-Ser-casein, L-phosphoserine or L-phosphothreonine. These data suggest that the cytosolic low-M_r acid phosphatase might play a regulatory role in EGF receptor-dependent transmembrane signalling.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Characterization of a tyrosine phosphatase activity in the oogenesis of Periplaneta americana

In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.     

Giardia lamblia: Characterization of ecto-phosphatase activities

Ecto-phosphatase activities of Giardia lamblia were characterized in intact cells, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 8.4 ± 0.8 nmol p-NP/h/10⁷ cells. The ecto-phosphatase activities were inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium fluoride and sodium molybdate and by inorganic phosphate, the final product of the reaction. Experiments using a classical inhibitor of phosphotyrosine phosphatase, sodium orthovanadate, also showed that the ecto-phosphatase activity was inhibited in a dose-dependent manner. Different phosphorylated amino acids were used as substrates for the G. lamblia ecto-phosphatase activities the highest rate of phosphate release was achieved using phosphotyrosine. Not only p-NPP hydrolysis but also phosphotyrosine hydrolysis was inhibited by sodium orthovanadate. Phosphotyrosine but not phospho-serine or phospho-threonine inhibited the p-nitrophenylphosphatase activity. We also observed a positive correlation between the ecto-phosphatase activity and the capacity to encystation of G. lamblia trophozoites.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Ecto-phosphatase activity on the cell surface of Crithidia deanei

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.     

Secretory hydrolases of Entamoeba histolytica

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.     

Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica , which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica .     

The human red cell acid phosphatase is a phosphotyrosine protein phosphatase which dephosphorylates the membrane protein band 3

Human red cell cytosol acid phosphatase activity is supported by a main enzyme which can be extracted by DEAE and phosphocellulose chromatography. It uses pNPP as a substrate and is a protein phosphatase specific to phosphotyrosine. It dephosphorylates the tyrosine-phosphorylated cytosolic fragment of membrane protein 3. When taken together, these results suggest that the physiological role of red cell acid phosphatase is the FB3 phosphotyrosine dephosphorylation. Whatever it may be phosphotyrosine protein phosphatase activity is the first role of red cell acid phosphatase to be demonstrated.     

Characterization of a prostate-specific tyrosine phosphatase by mutagenesis and expression in human prostate cancer cells

The cellular form of human prostatic acid phosphatase (PAcP) is a neutral protein-tyrosine phosphatase (PTP) and may play a key role in regulating the growth and androgen responsiveness of prostate cancer cells. The functional role of the enzyme is at least due in part to its dephosphorylation of c-ErbB-2, an in vivo substrate of the enzyme. In this study, we investigated the molecular mechanism of phosphotyrosine dephosphorylation by cellular PAcP. We mutated several amino acid residues including one cysteine residue that was proposed to be involved in the PTP activity of the enzyme by serving as the phosphate acceptor. The cDNA constructs of mutant enzymes were transiently transfected into C-81 LNCaP and PC-3 human prostate cancer cells that lack the endogenous PAcP expression. The phosphotyrosine level of ErbB-2 in these transfected cells was subsequently analyzed. Our results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant PAcP is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2. In contrast, cells expressing C183A, C281A, or wild-type PAcP had a decreased phosphotyrosine level of ErbB-2, compared with the control cells. Similar results were obtained from in vitro dephosphorylation of immunoprecipitated ErbB-2 by these mutant enzymes. Furthermore, transient expression of C183A, C281A, or the wild-type enzyme, but not H12A or D258A, decreased the growth rate of C-81 LNCaP cells. The data collectively indicate that His-12 and Asp-258, but not Cys-183 or Cys-281, are required for the PTP activity of PAcP.     

Secretion of Acid Phosphatase by x4xenic Entamoeba histolytica NIH-200 and Properties of the Extracellular Enzyme

Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupnc chloride, ethylene diamine-tetra acetic acid, ammonium molybdale and cysteine: however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.