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Pyrophosphate formation from acetyl phosphate and orthophosphate: Evidence for heterogeneous catalysis
Abstract
The formation of [32P]pyrophosphate from acetyl phosphate and [32P]orthophosphate was studied under conditions in which phosphate-metal salts or acetyl phosphate-metal salts precipitate. In the absence of precipitates in purely aqueous media, the initial rate constant of transphosphorylation (kobs) was extremely small and the formation of pyrophosphate was detected only in the presence of calcium. In various combinations, conditions such as high pH, high concentrations of reactants, and the presence of dimethyl sulfoxide caused three types of precipitates to form. In completely aqueous solution with an excess of orthophosphate, the crystals formed at high pH contained 3 mol of calcium for 2 mol of phosphate and they were poorly effective at promoting phosphorolysis. In the presence of dimethyl sulfoxide, the ratio of calcium to phosphate in the sediment was 1:1 and phosphorolysis proceeded at a high rate. In either solvent, an excess of acetyl phosphate caused precipitation of a complex containing 1 mol of acetyl phosphate to 1 mol of calcium. In aqueous media the rate constant of phosphorolysis increased with increasing precipitation of the acetyl phosphate-calcium complex. With destabilization of the anions by dimethyl sulfoxide the increase in kobs for a given amount of acetyl phosphate-calcium precipitated was 200-fold higher. Magnesium did not form precipitates and was ineffective in promoting transphosphorylation in completely aqueous media, either in the presence of excess phosphate or in the presence of excess acetyl phosphate. However, when precipitation of phosphate-magnesium or acetyl phosphate-magnesium was promoted by addition of dimethyl sulfoxide, phosphorolysis was observed with rate constants as high as those found in the presence of calcium. These results indicate that phosphorolysis of acetyl phosphate occurs at higher rates on the surface of solid structures, through highly specific interactions involving acetyl phosphate, orthophosphate, and divalent cations.
... Previous studies have reported the formation of PPi using condensing agents such as cyanate (Miller and Parris 1964;Beck and Orgel 1965), dicyandiamide (Steinman et al. 1965), dithionite (Gabel 1968), urea (Osterberg andOrgel 1972;Handschuh et al. 1973), and thioesters (Weber 1981(Weber , 1982. Other authors have synthesized PPi from molecules with an energy-rich phosphoryl group such as ATP and ADP (Lowenstein 1958(Lowenstein , 1960Krane and Glimcher 1962;Burley 1965), cyanovinyl phosphate (Fen-is 1968), acetyl phosphate (Etaix and Buvet 1975;Vieyra et al. 1985;Meyer-Fernandes and Vieyra 1988), and phospho(enol)pyruvate (Hermes-Lima and Vieyra 1989). All these reactions of PPi synthesis depend on the prior formation of condensing or phosphorylating agents. ...
... Precipitation of ~2Pi was quantified as described by Meyer-Fernandes and Vieyra (1988). The detection limit is about l~ of solid Pi. ...
... Figure 2 shows that the condensation reaction at pH 9.0 in the presence of 5 mM MgCI2 in-Creases with the increase in Me2SO concentration, being parallel to the formation of precipitated Pi. The formation of Pi precipitates in Me2SO-containing media was previously described by Meyer-Fernandes and Vieyra (1988). ...
The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of32PPi from32Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.
... Determinations of pH in Me2SO were done as described by Vieyra et al. (1985). Precipitation of 32Pi and Mg was quantified according Meyer-Fernandes and Vieyra (1988) and the Mg:P molar ratio of the precipitates was calculated as described for the Ca:P ratio (Hermes-Lima and Vieyra 1989), except that precipitation of Mg was measured using atomic absorption spectroscopy. Briefly, samples containing 0.8-1 mi of the reaction media were stirred by vortexing, and a 0.l-ml aliquot was immediately removed and counted for 3zp in a liquid scintillation counter. ...
... In a previous report, the highest value for enthalpy of activation for PPi formation from acetyl phosphate was observed in homogeneous conditions, in marked contrast with the low values found in the presence of precipitates (Table 1). Therefore, the precipitate structures with Mg z+ ions on the surface can promote a decrease in the activation barrier for PPi formation, perhaps by approximation of the reacting phosphate groups and by overcoming the electrostatic repulsion between the reacting anions ( Fig. 7) (Krane and Glimcher 1962;Jencks 1969;Meyer-Fernandes and Vieyra 1988). In the phosphorolytic cleavage by Pi of ADP, ATP, and linear tripolyphosphate to form PPi, the enthalpy of activation decreased from 190 kJ.mol-J to values ranging from 30 to 60 kJ.mol-1, providing that formation of a solid catalyst occurred (Vieyra 1982). ...
... The data of Figs. 1, 5, 6, and 7 and of Table 1 suggest that the Pi.Mg structure not only provides a reactant (phosphate) in a model very reminiscent of the phosphoenzyme intermediate formed during the catalytic cycle of ion-motive P-ATPases (Pickart and Jencks 1984;Pedersen and Carafoli 1987), but that it also catalyzes the transphosphorylation reaction. However, one problem in comparing the kinetic properties of magnesium phosphate precipitates with those of enzymes is that the insoluble matrix is consumed in the course of the reaction and that the product (PPi) remains bound to the surface of the precipitated Pi.Mg complex as previously measured (Meyer-Fernandes and Vieyra 1988). (The product pyruvate is released from the surface; Hermes-Lima 1989.) ...
The enzyme-like kinetic properties of precipitated magnesium phosphate as a catalyst for formation of pyrophosphate (PPi) from phospho(enol)pyruvate (PEP) are described. This synthesis occurs at a low temperature (37 degrees C) and represents a model that may help us understand the relevance to chemical evolution of minerals as ancient catalysts whose functions could have been taken over by contemporary enzymes. An insoluble Pi.Mg matrix was formed in a medium with 80% of the water replaced by dimethyl sulfoxide as a way of simulating conditions in a drying pond. Phospho(enol)pyruvate adsorbs onto the Pi.Mg surface according to a Langmuir isotherm, and the PEP concentration dependence of PPi formation follows a Michaelian-like function. A yield of 33% for transformation of the initially adsorbed PEP into PPi was attained after 4 days of incubation with equimolecular concentrations of Pi, MgCl2, and PEP. The magnesium concentration dependence for Pi and Mg precipitation, for adsorption of PEP onto solid Pi.Mg, and for PPi formation showed complex cooperative behavior. These results taken as a whole lead to the conclusion that the Pi.Mg surface not only provides a reactant for PPi formation but also catalyzes the reaction.
... The PPase activity was calculated by subtracting the nonspecific PPi hydrolysis measured in the absence of the parasite cells. In the experiments in which high concentrations of metals were tested, the possibility of precipitate formation was checked as previously described [56]. Under the conditions employed in this study, in the reaction medium containing 100 mM sucrose, 20 mM KCl, 50 mM Tris-HCl (pH 7.2) and 1 mM PPi, no phosphate precipitates were observed in the presence of these cations. ...
... The ecto-ATPase activity was calculated from the total activity, which was measured in the presence of MgCl 2 , minus the basal activity, which was measured in the absence of MgCl 2 . This activity was determined by measuring the hydrolysis of [c 32 P] ATP (10 4 Bq/nmol ATP) [56]. The experiments were initiated by the addition of living cells and were terminated by the addition of 1.0 ml of a cold mixture containing 25% charcoal in 1.0 M HCl. ...
In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H+-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H+-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl2. In the presence of MgCl2, this activity was inhibited by millimolar concentrations of CaCl2. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.
... Ca 2+ -binding levels were measured by filtration. SRV (0.2-0.4 mg/ml) were first phosphorylated for 15 min as above except that non-radioactive P i was used and 1 mM 3 H-glucose was added to determine the wet volume of the filters (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Then the SRV suspended in phosphorylation buffer were supplied with 45 Ca 2+ and filtered at the desired times. ...
... To test the specificity of Ca 2+ binding, we first checked that retention of insoluble CaP i complexes on filters during the filtration experiments was not the source of apparent Ca 2+ binding, particularly in 30-40% Me 2 SO [29]. With 0.1 mM P i , which promoted the formation of 4.5 nmol E-P/mg, i.e., nearly maximal phosphorylation (Figs. ...
Sarcoplasmic reticulum (SR) Ca2+-ATPase was phosphorylated by Pi at pH 8.0 in the presence of dimethyl sulfoxide (Me2SO). Under these conditions, it was possible to measure transient 45Ca2+ binding to the phosphoenzyme. Binding reached 1.2 Ca2+ per phosphoenzyme (E-PCax) within 10 min in 30% Me2SO, 20 mM MgCl2 and 0.1 mM Pi and the phosphoenzyme only decreased by 23% during this period. This Ca2+ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca2+-ATPase. At 40% Me2SO, simultaneous addition of Ca2+ and ADP increased Ca2+ binding up to almost four Ca2+ per phosphoenzyme (ADPE-PCay), revealing a species bearing simultaneously four Ca2+ sites. Both E-PCax and ADPE-PCay were further identified as distinct species by (2',3'-O-2(2,4,6-trinitrophenyl)adenosine 5'-triphosphate) fluorescence, which revealed long-range modifications in the Ca2+-transport sites induced by ADP binding to E-P. In addition, E-PCax was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me2SO was diluted. These findings indicate that more than two functional Ca2+-sites exist on the functional Ca2+-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca2+-ATPase allowing simultaneous binding of Ca2+ at lumenal and cytosolic sites. The stoichiometries for Ca2+ binding found here could either be interpreted as binding of four Ca2+ on a Ca2+-ATPase monomer considered as the functional unit or as binding of two Ca2+ per monomer of a functional dimer.
... This solution was measured spectrophotometrically at 650 nm, and a Pi curve was used as a standard. In experiments that tested high concentrations of MnCl 2 and CaCl 2 , the possibility of precipitate formation was checked as described previously (Meyer-Fernandes and Vieyra, 1988). In reaction media containing 50 mM HEPES (pH 7.4), 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 1 mM PPi, no phosphate precipitates were observed in the presence of these cations under the conditions used. ...
Several ecto-enzymatic activities have been described in the plasma membrane of the protozoan Leishmania amazonensis, which is the major etiological agent of diffuse cutaneous leishmaniasis in South America. These enzymes, including ecto-phosphatases, contribute to the survival of the parasite by participating in phosphate metabolism. This work identifies and characterizes the extracellular hydrolysis of inorganic pyrophosphate related to the ecto-pyrophosphatase activity of the promastigote form of L. amazonensis. This ecto-pyrophosphatase activity is insensitive to MnCl2 but is strongly stimulated by MgCl2. This stimulation was not observed during the hydrolysis of p-nitrophenyl phosphate (p-NPP) or β-glycerophosphate, two substrates for different ecto-phosphatases present in the L. amazonensis plasma membrane. Furthermore, extracellular PPi hydrolysis is more efficient at alkaline pHs, while p-NPP hydrolysis occurs mainly at acidic pHs. These results led us to conclude that extracellular PPi is hydrolyzed not by non-specific ecto-phosphatases but rather by a genuine ecto-pyrophosphatase. In the presence of 5 mM MgCl2, the ecto-pyrophosphatase activity from L. amazonensis is sensitive to micromolar concentrations of NaF and millimolar concentrations of CaCl2. Moreover, this activity is significantly higher during the first days of L. amazonensis culture, which suggests a possible role for this enzyme in parasite growth.
... The modulation of the ecto-phosphatase enzymes by divalent metals was measured in the presence of 5 mM MgCl 2 , CaCl 2 , and ZnCl 2 or 1 mM EDTA and EGTA as chelating agents. The formation of precipitates was evaluated as described by Meyer- Fernandes and Vieyra (1988). Under these conditions, no phosphate precipitates were observed in the reaction mixture when these cations were tested. ...
Strigomonas culicis is a monoxenous trypanosomatid that co-evolves with a symbiotic bacterium in a mutualistic relationship that is characterized by intense metabolic exchanges between both partners. Strigomonas culicis infects and colonizes the Aedes aegypti mosquito midgut, reaches its hemocoel and then invades the salivary glands. An artificial aposymbiotic strain is unable to colonize insects, reinforcing the idea that the bacterium influences the protozoan surface composition and cell interaction. Here, we report the characterization of the hydrolytic activity of ecto-phosphatases evaluated in symbiont-bearing and aposymbiotic strains of S. culicis by incubating the protozoa with p-nitrophenyl phosphate (pNPP) at different pH levels, in the presence of phosphatase inhibitors, and with several divalent metals. The symbiont-bearing and aposymbiotic cells differ in their ecto-phosphatase enzymes, based on their activities and specificities. Furthermore, the ability of the protozoan to bind to the mosquito midgut and salivary glands was impaired by ecto-phosphatase inhibition. Taken together, our data suggest that the symbiont influences the host protozoan ecto-phosphatase activity and indicate a possible role of this enzyme during mosquito tissue colonization by S. culicis.
... The concentration of released p-nitrophenolate ion in the reaction was determined using a standard curve of p-nitrophenolate ion for comparison. In the experiments where high concentrations of FeCl 2 , MgCl 2 , CaCl 2 , SrCl 2 , ZnCl 2 , CdCl 2 , CoCl 2 and CuCl 2 were used, the possible formation of precipitates was investigated as described previously [31]. ...
Metarhizium anisopliae is an entomopathogenic fungus with the ability to infect a broad range of arthropods, and have evolved distinct strategies for their attachment to hosts. Here, we describe the characterisation of ecto-phosphatase activity on the conidia surface of M. anisopliae and its relevance in the host interaction process. Ecto-phosphatase activity was linear for 60 min and during this time, was linear with the increase of cell density. The optimum pH was in the acidic range and some divalent metals, such as Cu(2+), Cd(2+) and Zn(2+), inhibited ecto-phosphatase activity. The activity was also reduced by phosphatase inhibitors. Importantly, the inhibition of phosphatase activity in conidia reduced the adhesion to Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) integument and, consequently and indirectly, M. anisopliae infection. The results herein presented show, for the first time, the importance of ecto-phosphatase activity in M. anisopliae conidia and provide the first evidence of its direct involvement in adhesion and host infection.
... The hydrolysis of other nucleotides was also calculated by subtracting the nonspecific nucleotide hydrolysis measured in the absence of parasites. In the experiments where high concentrations of Ca 2+ , Mn 2+ and Sr 2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra 1988). Under the conditions employed, in a reaction medium containing 50 mM HEPES pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. ...
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41±0.13 nmol Pi h–1 107 cells–1). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 4.05±0.17 nmol Pi h–1 107 cells–1. Mg2+-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93±0.26 mM MgCl2. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not CaCl2 or SrCl2. The apparent K
m for Mg-ATP2– was 0.26±0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg2+-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg2+-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4′-diisothiocyanostylbene 2′-2′-disulfonic acid as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.
... The concentration of Pi released in the reaction was determined using a standard curve of Pi for comparison. In the experiments where divalent cations such as, Mn 2+ , Ca 2+ , Sr 2+ , Cd 2+ , Cu 2+ , Fe 2+ and Fe 3+ were tested, the possible formation of precipitates was investigated as described previously (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed here, in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM Dglucose , 50 mM HEPES–Tris buffer, pH 7.2 and 5 mM 3 0 AMP, no phosphate precipitates were observed in the presence of these cations. ...
Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.
... The values obtained for the 5 nucleotidase activity at higher concentrations of 5 AMP using both methods (Fiske and Subbarow, 1925;Lanzeta et al., 1979) were the same. In the experiments where high concentrations of Mn 2+ , Ca 2+ and Sr 2+ were tested, the possible formation of precipitates was investigated as described previously (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed here, in the reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose, 50 mM Hepes-Tris buffer, pH 7.2 and 5 mM 5 AMP, no phosphate precipitates were observed in the presence of these cations. ...
Tritrichomonas foetus is the causative agent of sexually transmitted trichomoniasis in cattle. In females, the infection can be associated with infertility, vaginitis, endometritis, abortion or pyometra, leading to significant economic losses in cattle raising. T. foetus is devoid of the ability to synthesize purine nucleotides de novo, depending instead on salvaging purines from the host environment. Ecto-5'-nucleotidase catalyzes the final step of extracellular nucleotide degradation, the hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and Pi. In this work we show that living, intact cells of T. foetus were able to hydrolyze 5'AMP at a rate of 12.57 ± 1.23 nmol Pi × h(-1) × 10(-7) cells at pH 7.2 and the 5'AMP hydrolysis is due to a plasma membrane-bound ecto-enzyme activity. The apparent K(m) for 5'AMP was 0.49 ± 0.06 mM. In addition to 5'AMP, the enzyme hydrolyzed all substrate monophosphates tested except 3'AMP. No divalent metals or metal chelators were able to modulate enzyme activity. Phosphatase inhibitors did not have an effect on ecto-5'-nucleotidase activity while ammonium molybdate did inhibit the activity in a dose dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. These results indicate the existence of an ecto-5'-nucleotidase that may play a role in the salvage of purines.
... We also measured the hydrolysis of other nucleoside monophosphates, using 5 0-CMP, 5 0-IMP, 5 0-GMP, 5 0-UMP or 3 0-AMP as substrates under the same conditions described above. In experiments in which high concentrations of Mn 21 , Ca 21 and Sr 21 were tested, the possible formation of precipitates was checked as described previously (Meyer-Fernandes & Vieyra, 1988). In the reaction media containing 50 mM MES-Hepes-Tris (pH 4.5), 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 5 mM AMP, no phosphate precipitates were observed in the presence of these cations under the conditions used. ...
Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.
... We also measured the hydrolysis of other monophosphates, using 5 0 -CMP, 5 0 -IMP, 5 0 -GMP, 5 0 -UMP or 3 0 -AMP as substrates, under the same conditions described above. In the experiments where high concentrations of MgCl 2 , CaCl 2 , MnCl 2 and SrCl 2 were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed here, in the reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50 mM Hepes-Tris buffer, pH 7.2 and 5 mM 5 0 -AMP, no phosphate precipitates were observed in the presence of these cations. ...
In this work, we biochemically characterized the ecto-5'-nucleotidase activity present on the surface of the living trophozoites of Giardia duodenalis. Two sequences of the 5'-nucleotidase family protein were identified in the Giardia genome. Anti-mouse CD73 showed a high reaction with the cell surface of parasites. At pH 7.2, intact cells were able to hydrolyze 5'-AMP at a rate of 10.66 ± 0.92 nmol Pi/h/10(7) cells. AMP is the best substrate for this enzyme, and the optimum pH lies in the acidic range. No divalent cations had an effect on the ecto-5'-nucleotidase activity, and the same was seen for NaF, an acid phosphatase inhibitor. Ammonium molybdate, a potent inhibitor of nucleotidases, inhibited the enzyme activity in a dose-dependent manner. The presence of adenosine in the culture medium negatively modulated the enzyme. The results indicate the existence of an ecto-5'-nucleotidase that could play a role in the salvage of purines.
... spectrophotometry and radioactivity) were the same. In the experiments where high concentrations of Mn 21 , Ca 21 and Sr 21 were tested, the possible formation of precipitates was checked as described previously (Meyer-Fernandes & Vieyra, 1988). In the reaction media containing 50 mM HEPES (pH 7.2), 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations under the conditions used. ...
In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.
... The values obtained for p-nitrophenylphosphatase activity measured using both methods are exactly the same. In the experiments where high concentrations of MgCl 2 , CaCl 2 , MnCl 2 , ZnCl 2 , CoCl 2 , FeCl 2 , CuCl 2 and NiCl 2 were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed here, in the reaction medium containing 100 mM sucrose, 20 mM KCl, 50 mM TrisHCl (pH 7.2) and 5 mM p-NPP, no phosphate precipitates were observed in the presence of these cations. ...
In this work, we showed that living cells of Trypanosoma rangeli express different ecto-phosphatase activities in response to different inorganic phosphate (Pi) concentrations in the culture medium. The ecto-phosphatase activity from T. rangeli grown at low-Pi concentration was inhibited by the increase of the pH, while the ecto-phosphatase of the cells grown at high Pi concentration was not modulated by the change of the pH of the medium. Okadaic acid inhibited only the ecto-phosphatase activity from cells grown at low-Pi concentration but not the ecto-phosphatase activity from cells grown at high-Pi concentration. Accordingly, phosphatase activity from T. rangeli grown at low Pi concentration was able to hydrolyze P-serine and P-threonine at high rate but not P-tyrosine. The phosphatase activity from T. rangeli grown at high-Pi concentration was able to hydrolyze P-serine, P-threonine and P-tyrosine with the same rate. The addition of anterior midgut homogenate of Rhodnius prolixus on the epimastigotes suspension inhibited the enzyme activity of T. rangeli grown at low-Pi concentration. On the other hand, anterior midgut homogenate had no effect in the ecto-phosphatase of T. rangeli maintained at high-Pi concentration. Altogether, the results described here indicate that ecto-phosphatase activities hydrolyzing phosphorylated compounds present in the extracellular medium of T. rangeli are regulated by the external Pi concentration.
... Methods. Precipitation of [ 32 P]Pi was quantified according to Meyer-Fernandes and Vieyra (1988). Precipitation of calcium was measured using [ 45 Ca]CaCl 2 as tracer, using the same procedure employed for quantifying sedimented [ 32 P]Pi. ...
Phospho(enol)pyruvate (PEP) undergoes transphosphorylation to form pyrophosphate (PPi) and adenosine 5'-diphosphate (5'-ADP) with high yields in the presence of an adsorbent surface of calcium phosphate (Pi.Ca), which is considered to be an ancient mineral with catalytic properties. PPi formation is a result of the phosphorolytic cleavage of the enol phosphate group of PEP by precipitated Pi. The synthesis of PPi is dependent on the amount of the solid matrix; it increases with the amount of adsorbed PEP and upon addition of dimethyl sulfoxide (Me2SO), a molecule with high dipolar moment. Although it is saturated with PEP at neutral pH, the phosphorylating Pi.Ca surface becomes effective only in alkaline conditions. In a parallel reaction, PEP phosphorylates 5'-AMP to 5'-ADP with a yield that is sevenfold higher in the presence of the Pi.Ca surface than in its absence, indicating that the solid matrix promotes interaction between adsorbed molecules with a high potential for phosphoryl transfer. In contrast to phosphorolysis, this latter reaction is stimulated by Me2SO only in homogeneous solution. It is concluded that phosphate minerals may have coadjuvated in reactions involving different phosphorylated compounds and that molecules with high dipolar moment may have acted in mildly alkaline, primitive aqueous environments to modulate phosphoryl transfer reactions catalyzed by phosphate minerals.
... The values obtained for ATPase activities measured using both methods (colorimetric and radioactive) are exactly the same. In the experiments in which high concentrations of Ca 2ϩ , Mn 2ϩ , and Fe 3ϩ were tested, possible precipitates formed were checked as previously described (26). Under the conditions employed, in the reaction medium containing 50 mM Hepes, pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. ...
In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol P(i)/h/10(5) cells). Interestingly, in the presence of 5 mM MgCl(2) an ecto-ATPase activity of 300 nmol P(i)/h/10(5) cells was observed. The addition of MgCl(2) to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl(2). Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2) but not by SrCl(2), ZnCl(2), or FeCl(3). In fact, FeCl(3) inhibited both Mg(2+)-dependent and Mg(2+)-independent ecto-ATPase activities. The Mg(2+)-independent ATPase activity was unaffected by pH in the range between 6.4 and 8. 4, in which the cells were viable. However, the Mg(2+)-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5'-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg(2+) (basal activity), the apparent K(m) for ATP(4-) was 0.053 +/- 0.008 mM, whereas at saturating MgCl(2) concentrations, the corresponding apparent K(m) for Mg-ATP(2-) for Mg(2+)-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM +/- 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P(2) purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4'-4'-diisothiocyanostylbenzene-2'-2'-disulfonic acid. These two reagents inhibited the Mg(2+)-independent and the Mg(2+)-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg(2+)-dependent ecto-ATPase but not the Mg(2+)-independent ATPase activity.
... The values obtained for the ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Ca 2+ , Mn 2+ and Sr 2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. ...
In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.
... The values obtained for the ATPase activities measured using both methods (spectrophotometric and radioactive) were the same. In the experiments where high concentrations of Ca 2ϩ , Mn 2ϩ , Zn 2ϩ , and Sr 2ϩ were tested, possible precipitates formed were checked, as previously described [31]. Under the conditions employed no phosphate precipitates were observed in the presence of these cations. ...
In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.
... The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments in which high concentrations of Mn 2+ , Ca 2+ and Sr 2+ were tested, possible precipitates were checked as previously described (Meyer-Fernandes and Vieyra 1988). Under the conditions employed in the reaction medium containing 50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. ...
In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42±0.31 nmol Pi/h×108 cells). ATP hydrolysis was stimulated by MgCl2, and the Mg-dependent ecto-ATPase activity was 27.15±2.91 nmol Pi/h×108 cells. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The apparent K
m for Mg-ATP2− was 0.61 mM, and free Mg2+ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4′.diisothiocyanostylbene 2′-2′-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg2+-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.
... The values obtained for ATPase activities measured using both methods (i.e., spectrophotometry and radioactivity) were exactly the same. In the experiments where high concentrations of Mn 2+ , Ca 2+ and Sr 2+ were tested, possible formation of precipitates was checked as described previously [34]. In the reaction media containing 50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations under the conditions employed. ...
Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. In the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36+/-3.36nmol Pi/hx10(8) cells. In the presence of 5 mM MgCl(2), this activity was enhanced around 70 times, and an apparent K(m) for Mg-ATP corresponding to 0.61mM was determined. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases, V-ATPases, Na(+)-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). In the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ecto-ATPase that may have a role in fungal physiology.
... The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Mn 2+ , Ca 2+ , Sr 2+ , and Zn 2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50 mM Tris-HCl buVer, pH 7.2, 100 mM sucrose, 20 mM KCl, and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. ...
In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.
... The values obtained for the ATPase activities measured using both methods (spectrophotometric and radioactive) were the same. In experiments where high concentrations of Ca 2+ , Mn 2+ , Sr 2+ , and Zn 2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988), and no phosphate precipitates were observed. ...
In this study, we describe the ability of intact fat body of an insect, Rhodnius prolixus, to hydrolyze extracellular ATP. In these fat bodies, the ATP hydrolysis was low in the absence of any divalent metal, and was stimulated by MgCl(2). Both activities (in the absence or presence of MgCl(2)) were linear with time for at least 30 min. In order to confirm the observed nucleotidase activities as ecto-nucleotidases, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid). This reagent inhibited both nucleotidase activities and its inhibitory effect was suppressed by ATP. Both ecto-nucleotidase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin, ouabain, vanadate, molybdate, sodium fluoride, levamizole, tartrate, p-NPP, sodium phosphate, and suramin. Concanavalin A, activator of some ecto-ATPases, was able to stimulate the Mg(2+)-independent nucleotidase activity, but not the Mg(2+)-dependent one. The Mg(2+)-independent nucleotidase activity was enhanced with increases in the pH in the range between 6.4-8.0, but the Mg(2+)-dependent nucleotidase activity was not affected. Besides MgCl(2) , the ecto-ATPase activity was also stimulated by CaCl(2),() MnCl(2), and SrCl(2), but not by ZnCl(2). ATP, ADP, and AMP were the best substrates for the Mg(2+)-dependent ecto-nucleotidase activity, and CTP, GTP, and UTP produced very low reaction rates. However, the Mg(2+)-independent nucleotidase activity recognized all these nucleotides producing similar reaction rates, but GTP was a less efficient substrate. The possible role of the two ecto-nucleotidase activities present on the cell surface of fat body of Rhodnius prolixus, which are distinguished by their substrate specificity and their response to Mg(2+), is discussed.
... In the experiments where concentrations of MgCl 2 , CaCl 2 , MnCl 2 , ZnCl 2 , SrCl 2 , CuCl 2 , and NiCl 2 were high, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed in the reaction medium containing 100 mM sucrose, 20 mM KCl, 50 mM Tris-HCl, pH 7.2, and 5 mM p-NPP, no phosphate precipitates were observed in the presence of these cations. ...
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.
... The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Mn 2+ , Zn 2+ , Ca 2+ , and Sr 2+ were tested, possible precipitates formed were checked as previously described (Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50 mM Tris–HCl, pH 7.2, 20 mM KCl, 100 mM sucrose and 5.0 mM ATP, no phosphate precipitates were observed in the presence of these cations. ...
In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.
... The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Mn 2+ and Ca 2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50.0 mM Hepes, pH 7.2, 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, and 5 mM ATP, no phosphate precipitates where observed in the presence of these cations. ...
In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78+/-0.08 nmol Pi x h(-1)x10(-6) cells). The ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53+/-0.07 mM. ATP was the best substrate for this enzyme. The apparent K(m) for ATP was 0.21+/-0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg(2+)-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A(1) (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5 AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl(2). Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.
The ecto-phosphatases belong to a group of enzymes closely associated with the cell surface that has its catalytic site facing the extracellular environment, where different phosphorylated substrates can be hydrolyzed. In the present work, we biochemically characterized the ecto-phosphatase activity of the freshwater microalgae Euglena gracilis, a model microorganism, ubiquitously distributed and resistant to several environmental stressors. The ecto-phosphatase activity is acidic, stimulated by copper and presents the following apparent kinetic parameters: Km = 2.52 ± 0.12 mM p-NPP and Vmax = 3.62 ± 0.06 nmol p-NP × h-1 × 106 cells. We observed that zinc, orthovanadate, molybdate, fluoride, and inorganic phosphate inhibit the ecto-phosphatase activity with different magnitudes. Fluoroaluminate complexes are also inhibitors of this ecto-phosphatase activity. They can be formed in the enzyme reaction conditions and are likely to occur in a natural environment where E. gracilis can be found. The ecto-phosphatase activity is constant through the culture growth phases and is negatively modulated after continuous subculturing in the dark when a shift from phototrophic to the heterotrophic metabolism is likely. The analysis of those biochemical parameters may contribute to understanding the role of E. gracilis ecto-phosphatase activity in natural environments.
Building up on an available synthetic methodology, phosphate monoesters ROPO3H2 have been synthesized in good yields. The synthetic procedure employed features acetic anhydride mediated activation of phosphoric acid in the presence of alcohols, leading to the formation of phosphate monoesters. The products have been isolated as their cyclohexyl amine salts, [CyNH3]2[(MeO)PO3]‧3H2O (1) and [CyNH3][(RO)PO3H] (Cy = cyclohexyl; R = Et (2), iPr (3), or tBu (4)). Neutralization of 1-4 by readily available inexpensive ion exchange resin Amberlite produces monoalkyl phosphates (RO)P(O)(OH)2 (R = Me (5); Et (6), iPr (7), or tBu (8)). Thermally labile 1-4 and 7 have been structurally characterized by single crystal X-ray diffraction studies. Due to intrinsic thermal instability owing to β-H elimination, these compounds can be used as ligands for the preparation of single-source precursors for ceramic phosphates by reacting them with suitable metals ions. It is also possible to isolate co-crystals of the anionic and neutral forms of these phosphates as it has been demonstrated in the isolation and structural characterization of [(iPrO)PO3H2]·{[CyNH3] [(iPrO)PO3H]} (9). To demonstrate the utility of monoalkylphosphates in the low-temperature synthesis of metal phosphate bioceramics, isopropyl phosphate 7 has been employed to prepare calcium phosphate [{Ca((iPrO)PO3)(OH2)}·H2O]n (10), which undergoes neat thermal decomposition in two stages to lose water and propene to yield β- Ca2P2O7 at low temperatures (280 oC)
It has been postulated that adsorption and surface catalysis, as well as repeated drying and wetting cycles, were essential in the synthesis, interconversion and co-evolution of phosphorylated molecules, including ‘energy-rich’ compounds. We have investigated the formation of pyrophosphate from phospho(enol)pyruvate and orthophosphate. The reaction occurred within hours at 37°C, required the adsorption of phospho(enol)pyruvate onto sedimented phosphate, exhibited Michaelian-like behavior and showed positive cooperativity with respect to divalent cation concentration. Thus in ‘mild’, near-equilibrium conditions, the de-solvated surfaces of phosphate crystals can catalyze the formation of pyrophosphate with a kinetic behavior similar to that found in contemporary enzymes. The experimental system we describe may represent a model for the prebiotic catalysis of transphosphorylations.
Iron(II) minerals have been found to catalyse the formation of pyrophosphate from activated phosphate compounds and inorganic phosphate at near neutral pH in water. Iron(II) phosphate catalyses the formation of pyrophosphate, from either acetyl phosphate or phosphoenolpyruvate as the activated phosphoryl donor, in yields of up to 25% and 14% respectively. Under similar conditions, these minerals also retard the hydrolysis of pyrophosphate. The catalysis of pyrophosphate formation is tolerant of sulfide ions: pyrophosphate being produced from acetyl phosphate in 12% yield in the presence of equimolar amounts of sulfide. These reactions mimic the extant biosynthesis of ATP from acetyl phosphate or phosphoenolpyruvate; they counter the concern that hydrolysis of polyphosphates would out-compete their accumulation under prebiotic conditions; and hence suggest a possible prebiotic route to polyphosphates under conditions that may have occurred on the early earth.
In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3'AMP 10 times more than 5'AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3'nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3'nucleotidase activity was not observed on ecto-5'nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3'nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage-parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3'nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite-macrophage interaction.
In this paper it is shown that the adsorption of 5'-adenosine monophosphate (5'-AMP) onto precipitated calcium phosphate exhibits a sigmoidal profile as revealed by isotherms at 45 degrees C. This result indicates a cooperative behavior in the adsorption of 5'-AMP. The relationship between adsorption capacity and surface area of the sedimented matrix may be interpreted as an indication that there is a monolayer of the absorbed nucleotide on the solid surface. The pH dependence of adsorption suggests that the negatively charged phosphoryl group of 5'-AMP interacts with a positively charged site (possibly Ca2+) on the matrix surface. The adsorption of the nucleotide is markedly decreased at pH values above 8.0. The Dixon-like plot of the effect of pH suggests an inhibitory role of hydroxyl ions in the adsorption of 5'-AMP. At pH 7.5, other anions such as pyrophosphate, tripolyphosphate and carbamyl phosphate also inhibit the adsorption of the nucleotide, probably by interacting with its adsorption site. We suggest that these phosphorylated molecules could have played a role in chemical evolution by modulating the amount of nucleotides adsorbed onto mineral surfaces. The significance of these phenomena in chemical evolution is discussed.
The plasma membrane of cells contains enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using living cells. In this work we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (5.39 +/- 0.71 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 30.75 +/- 2.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.21 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.98 mM and free Mg(2+) did not increase the ecto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells were viable, the acid phosphatase activity decreased, while the Mg(2+)-dependent ATPase activity increased. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. A comparison between the Mg(2+)-dependent ATPase activity of virulent and avirulent promastigotes showed that avirulent promastigotes were less efficient than the virulent promastigotes in hydrolyzing ATP.
The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO-) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO- and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5'-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.
In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h−1 mg−1 mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h−1 mg−1) in the presence of 5 mM MgCl2, with values of V
max and apparent K
m for Mg-ATP2−corresponding to 541.9 ± 48.6 nmol Pi h−1 mg−1 cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg2+-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1 (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg2+-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P2 purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.
A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.
The equilibrium between inorganic pyrophosphate and inorganic orthophosphate was determined at pH values varying between 6.0 and 8.0, in the presence of different concentrations of MgCl2, mixtures of MgCl2 and CaCl2, and different organic solvents. The reactions were catalyzed by yeast inorganic pyrophosphatase. It was found that at 35 degrees C, depending on the conditions used, the observed equilibrium constant of pyrophosphate hydrolysis vary from a value higher than 4 X 10(3) M (delta Goobs more negative than -5.1 kcal/mol) to a value as low as 3 M (delta Goobs -0.7 kcal/mol). The experimental data were used to compute the equilibrium constants of the reactions involving different ionic species. The data presented are interpreted according to the concept that the Keq of hydrolysis of a high energy compound depends on the difference in solvation energy of reactants and products.
This chapter describes the application of ¹⁸O methods to oxidative phosphorylation. The procedure described involves isolation of the Pi as KH2PO4, and conversion of the phosphate oxygen to CO2 for mass spectrometry by heating with guanidine hydrochloride. The procedure is also applicable for the measurement of ¹⁸O content of HOH. The stated ¹⁸O content of the water supplied may be taken as a convenient approximation for calculations. Standards may be prepared by equilibrating water with CO2 by shaking for several days; addition of certain salts hastens the equilibration. Use of bicarbonate as a source of CO2 is convenient as it makes arrangement for addition of known amounts of gaseous CO2 unnecessary. Other useful procedures for determination of ¹⁸O in Pi include unaided equilibrium with water or equilibration with water hastened by a hot Pt wire, electric discharge, or sulfite. Direct conversion of phosphate oxygen to gases suitable for mass spectrometry include heating Ba3 (PO4)2 with C to yield CO, heating of KH2PO4 with Hg (CN)2 to yield CO2, heating of Ag3PO4 to give O2, and treatment of BiPO4 with BrF3 to giveO2. The chapter examines the preparation of CO2 standards, preparation of Pi-l8O, and measurement of ¹⁸O in Pi or in HOH.
The following evidence supports the hypothesis that the hydrolysis of simple acyl phosphates near neutrality proceeds through monomeric metaphosphate ion, the dianion (AcP-) by direct elimination of carboxylate ion and the monoanion (AcP-) by proton transfer and elimination of carboxylic acid, while the hydrolysis of the acetyl phenyl phosphate monoanion (AcPhP-) proceeds through a nucleophilic reaction of water with the acyl group: (1) Values of ΔS‡ for hydrolysis are 3.8 e.u. for AcP-, -3.6 e.u. for AcP- and -28.8 e.u. for AcPhP-. (2) There is no significant solvent deuterium isotope effect on the hydrolysis of AcP- and AcP-, while the hydrolysis of AcPhP- is decreased 2.5-fold in deuterium oxide. (3) The hydrolysis of simple acyl phosphates is much faster than that of other phosphate esters and AcPhP-. (4) The rate of hydrolysis of substituted benzoyl phosphate dianions is increased by electron-withdrawing substituents (p = 1.2), while hydrolysis of the corresponding monoanions is much less sensitive to structure. (5) Solvolysis of AcP- and AcP- is known to proceed with P-O bond cleavage, while the solvolysis of AcPhP- in 90% methanol proceeds with C-O cleavage. (6) The rates of hydrolysis of AcP- and AcP- are slightly increased by concentrated salt solutions, while that of AcPhP- is decreased. (7) The addition of acetonitrile to the reaction medium has little or no effect on the rates of hydrolysis of AcP- and AcP-, while it considerably decreases that of AcPhP-. (8) Values of ΔV‡ for hydrolysis are -1.0 cm.3/mole for AcP-, -0.6 cm.3/mole for AcP- and -19 cm.3/mole for AcPhP-. (9) Solvolysis of AcP- in 7 M NaClO4 gives pyrophosphate.
Energy calculations are made for a number of anion-dipole aromatic nucleophilic substitutions utilizing the concept of an intermediate complex. Recorded data, wherever available, for bond energies, electron affinities and solvation energies are used together with an allowance for electronic reorganization, to calculate energies of initial and final states and an intermediate complex. In a consistent way these are related to transition states and activation energies. The results, which are shown to be in accord with experiment, cover several important systems including methanolysis of aryl halides, aryl halide exchange, the formation of stable intermediates, and the change in relationship of reactivity of fluoro-and other halogeno-compounds with change in reagent. Solvent effects are also discussed. A clearer understanding of the factors which influence nucleophilic strength emerges and this is discussed also. The principles involved should permit wider applications than are considered in the present paper.
The formation of pyrophosphate (PPi) from the anions of acetyl phosphate (AcP) and orthophosphate (Pi) in concentrated aqueous sodium perchlorate is approximately first order in each reactant. The pH dependence of the reaction in the presence of 0.25 M Pi monoanion and 6.4 M sodium perchlorate at 54°C shows different yields of PPi from AcP monoanion and dianion. This result is inconsistent with trapping of a free metaphosphate intermediate by Pi monoanion and water. The results are consistent with a concerted mechanism for PPi formation and yields of PPi from the different ionic species of 11.7% for AcP2- and Pi2-, 7.8% for AcP2- and Pi-, and 4.2% for AcP- and Pi-. Measurement of rate constants and partitioning with AcP2-, 0.25 M Pi2-, and 6.4 M sodium perchlorate, in the presence and absence of pyridine, show that the phosphorylated pyridine monoanion yields 40% PPi, whereas AcP2- yields 10% PPi under the same conditions. The formation of PPi from AcP and Pi dianions is at least fourth order in sodium ion in concentrated sodium perchlorate solutions. The corresponding reaction of the monoanions is second order in sodium ion. The order of effectiveness of salts for facilitation of the formation of PPi from the dianions of AcP and Pi is NaClO4 > NaCl, KCl, CsCl, and NaCl > CsCl. However, increasing the concentration of sodium perchlorate from 0.1 to 6.6 M changes the rate constant for hydrolysis of AcP mono- and dianion by <30% at 39 and 547deg;C. Values of ΔH≠ = 29 kcal mol-1 and ΔS≠ = +12 eu for PPi formation from the dianions of AcP and Pi and ΔH≠ = 19 kcal mol-1 and ΔS≠ = -20 eu for PPi formation from the monoanions were obtained from the yields of PPi and the rates of hydrolysis at 39 and 54°C. The high concentrations of sodium ion decrease electrostatic repulsion between the anions of AcP and Pi and may catalyze the formation of PPi by bridging the anions of AcP and Pi. These results provide no evidence for the existence of a metaphosphate intermediate in reactions in aqueous solution.
Incubation of aqueous Ca2+, carbamylphosphate and ADP results in ATP formation with yields up to 20%. The dependence of the yield on the reaction parameters suggests that this transphosphorylation reaction proceeds heterogeneously on the surface of the calcium-carbamylphosphate precipitate
The formation of pyrophosphate as a result of nucleophilic attack by orthophosphate at the acylphosphate bond of acetyl phosphate was detectable in completely aqueous media, and was enhanced by dimethyl sulfoxide. The reaction had an absolute requirement for divalent cations, the rate constant of phosphorolysis being dependent on the species and concentration of cations as well as on temperature and pH. The amount of pyrophosphate formed depended on both the acetyl phosphate and orthophosphate concentrations. In purely aqueous media, phosphorolysis was barely detectable in the presence of Mg2+, and its rate increased 40-fold when Mg2+ was replaced by Ca2+ or Sr2+. In the presence of Mg2+ the rate of phosphorolysis increased 400-fold when 50 to 80% of the water was replaced by dimethyl sulfoxide. In the latter case, the rate also increased as the pH was raised from 4.0 to 9.0. The entropy of activation was large and negative in the presence of Mg2+ or Ca2+, indicating that the nucleophile is involved in the rate-limiting step of the reaction. Since this thermodynamic parameter became large and positive in the presence of Ca2+ when dimethyl sulfoxide was omitted, it is inferred that the transition state of the same reaction may be changed by the solvent composition and the solvation of reactants.
In 70% dimethyl sulfoxide there is a rapid transfer of the terminal phosphate of ATP to either water or inorganic phosphate. The hydrolysis of ATP requires the simultaneous presence of Mg2+, a dicarboxylic acid and arsenate. The transfer reaction requires only Mg2+ but is stimulated by a dicarboxylic acid. The product of the transfer reaction appears to be exclusively inorganic pyrophosphate. These reactions may serve as model systems for phosphate transfer and ATPase activity in oxidative and photosynthetic phosphorylation.
- Lipmann
- Fiske