Hydrolysis Of Nitrophenylphosphate Research Articles

Multinuclear diffusion NMR spectroscopy and DFT modeling: a powerful combination for unraveling the mechanism of phosphoester bond hydrolysis catalyzed by metal-substituted polyoxometalates.

A detailed reaction mechanism is proposed for the hydrolysis of the phosphoester bonds in the DNA model substrate bis(4-nitrophenyl) phosphate (BNPP) in the presence of the Zr(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]⋅7 H2O (ZrK 2:2) at pD 6.4. Low-temperature (31)P DOSY spectra at pD 6.4 gave the first experimental evidence for the presence of ZrK 1:1 in fast equilibrium with ZrK 2:2 in purely aqueous solution. Moreover, theoretical calculations identified the ZrK 1:1 form as the potentially active species in solution. The reaction intermediates involved in the hydrolysis were identified by means of (1)H/(31)P NMR studies, including EXSY and DOSY NMR spectroscopy, which were supported by DFT calculations. This experimental/theoretical approach enabled the determination of the structures of four intermediate species in which the starting compound BNPP, nitrophenyl phosphate (NPP), or the end product phosphate (P) is coordinated to ZrK 1:1. In the proposed reaction mechanism, BNPP initially coordinates to ZrK 1:1 in a monodentate fashion, which results in hydrolysis of the first phosphoester bond in BNPP and formation of NPP. EXSY NMR studies showed that the bidentate complex between NPP and ZrK 1:1 is in equilibrium with monobound and free NPP. Subsequently, hydrolysis of NPP results in P, which is in equilibrium with its monobound form.

Cofactor interactions in the activation of tissue non-specific alkaline phosphatase: Synergistic effects of Zn<sup>2+</sup> and Mg<i>2+</i> ions

The interactions of Mg2+ and Zn2+ ions in the activation of non-specific tissue alkaline phosphatase were investigated using crude extracts of rat kidney. Activation of alkaline phosphatase by the metal ions was accompanied by changes in the kinetic parameters of nitrophenylphosphate hydrolysis. The results suggest some synergistic interactions between Mg2+ and Zn2+ ions in promoting the hydrolysis of p-nitrophenylphosphate by alkaline phosphatase. The results show that assays of alkaline phosphatase activity in homogenised tissue samples will give better responses if both Mg2+ and Zn2+ ions are included in the reactions

Nitric oxide assisted hydrolysis of nitrophenylphosphate

In this study the use of sodium nitroprusside as nitric oxide donor in solutions was utilized. It has been established that maximum release of nitric oxide is achieved under irradiation by UV light at 254 nm. The synergistic effects of cobalt trimethylene diamine and nitroprusside towards the hydrolysis of nitrophenylphosphate under the above conditions for different cobalt to nitrophenylphosphate ratio were investigated. This study demonstrates that besides the effect of hydroxyl radicals, the direct interaction of nitric oxide with the phosphorous center also play a role in decontamination reactions of poorly biodegradable phosphate esters in natural waters, due to phototransformation.

Identification of two thylakoid-associated phosphatases with protein phosphatase activity in chloroplasts of the soybean ( Glycine max)

Two thylakoid-associated phosphatases from soybean ( Glycine max) have been purified and characterized. Both enzymes were prepared by ultrasonication of thylakoids, (NH 4) 2SO 4 fractionation, gel filtration, cation, and anion exchange chromatography. Phosphatase I and II copurified during the first steps of purification and were separated by ion exchange chromatography on CM cellulose. The phosphatases have a molecular mass of 70 kDa. They exhibit a pH optimum at 5.5 and 6, respectively. Mg 2+ ions are not required for activity. Several phosphorylated substrates are hydrolyzed, including phenyl and 4-nitrophenyl phosphate, 3-phosphoglycerate, and fructose 1,6-bisphosphate. The thylakoid-associated phosphatases also possess phosphoprotein phosphatase activity towards 32P-labelled casein and chloroplast LHC II as substrates. Fluoride inhibited the hydrolysis of nitrophenylphosphate and [ 32P]casein but not the protein phosphatase activity towards 32P-LHC II. The protein serine/threonine phosphatase inhibitor okadaic acid did not affect these phosphatases at all.

Surface and Intracellular Pools of Na,K-ATPase Catalytic and Immuno-activities in Rat Exorbital Lacrimal Gland

The subcellular distribution of Na,K-ATPase in rat lacrimal gland acinar cells was surveyed by subcellar fractionation followed by determination of two Na,K-ATPase catalytic activities, K+-dependent p -nitrophenylphosphate hydrolysis and ouabain-sensitive ATP hydrolysis, by Western blotting of isolated membrane fractions, and by analysis of lacrimal tissue with immunocytochemical methods at the light and EM levels. Both catalytic activities distributed in parallel after centrifugation on sorbitol gradients. They were associated with membrane samples that appeared to be derived from: (1) acinar cell basal-lateral membranes; (2) the Golgi complex: and (3) endocytic compartments. β1-subunit immunoreactivity closely paralleled catalytic activity. The α1-subunit immunoreactivity distribution suggested the presence of α1-subunits in αβ complexes and excess α1 subunits which were not assembled with β subunits and not catalytically active. In the putative basal-lateral membrane and endocytic samples, α1-reactivity was associated primarily with a 100-kDa band, while in the Golgi samples it was associated primarily with 40 and 60-kDa bands. β1-reactivity was also heterogeneous, with reactivity in basal-lateral membrane and putative endocytic samples associated with a broad band of 50-54 kDa, and reactivity in Golgi samples associated with discrete bands of 50, 52, and 54 kDa. Staining with anti-holoenzyme and anti-α1-subunit antibodies yielded strong indirect immunofluorescence signals both in plasma membrane and in intracellular regions of acinar cells, β1-like immunoreactivity was concentrated in cytoplasmic regions of acinar cells. Immunogold electron microscopy revealed positive staining with anti-holoenzyme in Golgi membranes of acinar cells and in basal-lateral membranes of duct cells. These data support the hypothesis that lacrimal acinar cells contain substantial cytoplasmic pools of Na,K-ATPase and that there is a location-dependent heterogeneity which is not detected by immunocytochemical methods.

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the ( [formula omitted] and the Na +, K + pump

Inhibition by vanadate of the K +-dependent p- nitrophenylphosphatase activity catalyzed by the ( Na + + K +)- ATPase partially purified from pig kidney showed competitive behavior with the substrate. K + and Mg 2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K +-dependent p- nitrophenyl phosphate hydrolysis (Na +, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p- nitrophenyl phosphate and Mg 2+, or when the protective ligands were tested alone, the activation of p- nitrophenyl phosphate hydrolysis by K + followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K + on the p- nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K + concentration, the p- nitrophenylphosphatase activity declined at high K + concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na + (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K + -K + exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na + plus ATP, K + promoted vanadate inhibition of p- nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na + plus ATP, the biphasic response of p- nitrophenyl phosphate hydrolysis is due to K + acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb + influx as a function of the external Rb + concentration failed to develop when there was no Na + in the extracellular media. In addition, in the absence of extracellular Na +, external Rb + did not influence the magnitude of inhibition. The present findings indicate that external K + favors vanadate inhibition by displacing Na + from unspecified extracellular membrane sites.

Molecular properties and active form of nonspecific acid phosphatase from Schizosaccharomyces pombe.

Equilibrium sedimentation experiments of the native acid phosphatase indicate a dimer-tetramer dissociating nonequilibrating system with a dimer Mr = 180,000 g/mol. The hydrolysis of nitrophenylphosphate was used to determine the sedimentation coefficient of the active species. The s20,w value for the species which degrades nitrophenylphosphate is 13.52 +/- 0.46 S in 1% sucrose and 13.72 +/- 0.11 S in 1.3 M sodium chloride, corresponding to the Svedberg value of the tetramer species. Several lines of evidence are presented which, together with previous data, indicate that the Schizosaccharomyces pombe nonspecific acid phosphatase is composed of 4 identical or nearly identical polypeptide chains: a, equilibrium sedimentation analysis of the enzyme in denaturing agents indicates the presence of homogeneous material having Mr = 90,800 g/mol; b, digestion with carboxypeptidase A releases 0.82 mol of tyrosine/monomer molecular weight. Concomitant phosphatase inactivation occurred during the splitting off of the tyrosyl terminal residue. Furthermore, a unique NH2-terminal residue (histidine) was determined.

Dissociation between Ca 2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca 2+-ATPase activities with high-affinity sites for Ca 2+ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca 2+, the substrate specificity of Ca 2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca 2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca 2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and p- nitrophenylphosphate at Ca 2+ concentrations below 25 μM. At 0.2 mM Ca 2+ the rates of ATP, ADP, AMP and p- nitrophenylphosphate hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca 2+, but at 0.2 mM Ca 2+, Ca 2+-induced hydrolysis of ADP and AMP was greater than either ATP or p- nitrophenylphosphate . Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca 2+-stimulated ATP hydrolysis at 1 μM Ca 2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca 2+, Ca 2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca 2+-stimulated ATP hydrolysis, either at low or at high Ca 2+ concentrations. Chlorpromazine fully inhibited Ca 2+-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca 2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca 2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca 2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca 2+ concentrations, but this Ca 2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.

Some kinetic aspects of the mechanism of hydrolysis of phosphoric acid esters by nonspecific acid phosphatase from Schizosaccharomyces pombe

1. 1. The kinetics of the hydrolysis of nitrophenylphosphate by nonspecific acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2.) from Schizosaccharomices pombe was studied. 2. 2. The kinetic parameters, K m and V, were determined as well as the inhibition constants, K i, for the inhibitors, phosphate and fluoride, as a function of pH. 3. 3. The results, interpreted according to the theories of Dixon and Waley indicated the presence of three ionizable groups on the enzyme itself and one on the enzyme-substrate complex. 4. 4. A model of the hydrolysis of phosphoric acid monoesters by the S. pombe acid phosphatase is proposed based on the ionization state of the reactants and on the results of the inhibition by the competitive inhibitors.

Chromatographic Fractionation and Partial Characterization of Acid Phosphatase in Guinea-Pig Epidermis

Two chromatographically and enzymatically distinct forms of acid phosphatase have been separated by gel filtration from an extract of guinea-pig epidermis. These enzymes, termed APase 1 and APase 2 , had pH optima for p -nitrophenyl phosphate hydrolysis around 5.0. APase 1 was inhibited by fluoride, L(+) tartrate, and lead nitrate, but was insensitive to formaldehyde and glutaraldehyde. In contrast, Apase 2 was insensitive to fluoride, L(+) tartrate, and lead nitrate, but was sensitive to formaldehyde and glutaraldehyde. K m values for APase 1 and APase 2 with p -nitrophenyl phosphate as substrate were 0.09mM and 0.125 mM, respectively.

L'activité pyrophosphatasique de la phosphatase alcaline du cerveau

Resume 1. 1. The pyrophosphatase activity of alkaline phosphatase from bovine brain depends on Mg 2+ bound to the enzyme, rather than on the formation of the complex ion, MgP 2 O 7 2− . Indeed, the Mg 2+ -enzyme (the enzyme preincubated with Mg 2+ ) hydrolyses the Mg 2+ -free pyrophosphate and its activity reaches a maximum value without Mg 2+ added to the assay medium. The Mg 2+ -free enzyme hydrolyses the MgP 2 O 7 2− complex, but a weaker activity is observed. Its activity towards P 2 O 7 4− only reaches 1–5% of the value obtained with the Mg 2+ -enzyme. 2. 2. When present at relatively high concentration, MgP 2 O 7 2− is not hydrolyzed, but is able to activate the rate of p -nitrophenyl phosphate hydrolysis at pH 8.5, indicating that by its binding, MgP 2 O 7 2− has the same activating effect as Mg 2+ . 3. 3. Among divalent metal ions which can bind to the enzyme, Mg 2+ is the most effective in inducing the pyrophosphatase activity. Mn 2+ , Ni 2+ , Co 2+ and particularly Zn 2+ have weaker effects. 4. 4. It is concluded that the true substrate of the pyrophosphatase activity of alkaline phosphatases is P 2 O 7 4− , which behaves like a phosphomonoester.

Glucose-6-phosphatase activity in a soluble fraction from cotyledon tissue of Brassica nigra

A soluble fraction from cotyledon tissue of black mustard ( Brassica nigra) was found to catalyze the hydrolysis of glucose 6-phosphate. In an attempt to determine whether this reaction was catalyzed by a distinct glucose-6-phosphate ( d-glucose 6-phosphate phosphohydrolase, EC 3.1.3.9) or by an acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2), various characteristics of glucose 6-phosphate and p- nitrophenyl phosphate hydrolysis were compared. Both phosphatase activities exhibited a similar distribution pattern in subcellular fractions and in fractions obtained by precipitation with (NH 4) 2SO 4. The activities also were compared with respect to the effects of reaction mixture pH in the absence and presence of 1 mM KF-4 mM EDTA and the effect of partial inactivation by various enzyme pretreatments. Glucose 6-phosphate and p- nitrophenyl phosphate were mutually competitive inhibitors for the hydrolysis of the other compound. The K m and K f values were, respectively, 4.6 mM and 4.6 mM for glucose 6-phosphate and 0.72 mM and 0.79 mM for p- nitrophenyl phosphate. A substrate-specificity study indicated that p- nitrophenyl phosphate and phenyl phosphate were hydrolyzed more rapidly than glucose 6-phosphate. The soluble enzyme preparation did not exhibit PP 1-glucose phosphotransferase activity. Glucose did not inhibit the hydrolysis of glucose 6-phosphate. It is concluded that the observed hydrolysis of glucose 6-phosphate was catalyzed by an acid phosphatase. The possible existence in plants of a distinct glucose-6-phosphatase is discussed.

Activation and inhibition processes of alkaline phosphatase from bovine brain by metal ions (Mg 2+ and Zn 2)

1. 1 Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC3.1.3.1) from bovine brain is strongly activated by Mg 2+ (5–4-fold). In this respect the enzymes behaves like that of kidney or bone marrow but is different from the intestinal and placental enzymes which are much less activated. 2. 2 The rate of the activation process is pH-dependent. At pH 10.0, the pH optimum of p nitrophenyl phosphate hydrolysis, the activation process is rapid. At lower pH values, it is slow and can be described by an exponential relationship vs time. The results suggest that the activation by Mg 2+ proceeds through a binding of the metal with free enzyme which might be followed by a conformational change, rather than by an action on the substrate. 3. 3 Other metal ions can also bind to the brain enzyme. Mn 2+, Co 2+, Ni 2+ with an activating effect, Zn 2+ with an inhibitory effect. They all bind to the enzyme at the same site and also induce the supposed conformational change. However, Zn 2+ is able to produce interactions, perhaps with P i which is one of the products of hydrolysis, which might explain its inhibitory effect.

P-nitrophenyl phosphate hydrolysis and calcium ion transport in fragmented sarcoplasmic reticulum.

The calcium ion pump of fragmented sarcoplasmic reticulum can be coupled to hydrolysis of p-nitrophenyl phosphate, in the absence of added adenosine triphosphate. Comparison of the activities obtained with the two substrates suggests an analogous mechanism of transport. Independent of the substrate, a 2 : 1 ratio between calcium ion transport and substrate hydrolysis is displayed by the system, and an identical amount of work is required for ion transport against a given gradient. A phosphate ester appears necessary for substrate utilization in the pump mechanism, whereas the structure of the substrate determines the rates of activity and the affinity of the system for calcium ion.