Para-nitrophenyl phosphate

There is relatively good agreement concerning the phosphate requirement for calcium release and ADP synthesis. The reaction is activated maximally at a phosphate concentration of approximately 1 mM at 20°C at pH 7.0 when excess magnesium is present182. Apparent phosphate affinities have also been obtained from the phosphate concentrations which are required for the inhibition of the hydrolysis of weak substrates like ITP and para-nitrophenyl phosphate. Data for phosphate binding obtained from direct binding studies are not available. 

Catalytic activity was observed when a 625-membered undecapeptide library was complexed with Zr(IV). Beads with active catalysts were easily identified under a low-power microscope and the structures of the active peptides were analyzed by Edmann degradation. The activity of four Zr(IV)-peptide complexes was further proven and quantified in solution-phase experiments not only with the examined indigo derivative but also with the corresponding para-nitrophenyl phosphate. The group extended the screening for phosphodiesterase activity and enabled discovery of solid phase bound peptide-Eu3+ complexes as hydrolyzing agents [15]. 

F27. Fraenkel, H. H., and Peters, R. L., A modified calcium-cobalt method for the demonstration of alkaline phosphatase. The use of para-nitrophenyl phosphate as a substrate. Am. J. Clin. Paihol. 42, 324-327 (1964). 

Majority of phosphatase assays employed in HTS today rely on the ability of these enzymes to catalyze dephosphorylation of artificial chromogenic or flnorogenic snbstrates. Their popnlarity is dne to simplicity, high sensitivity, and robnsmess of these assays. The most popular chromogenic substrate is para-nitrophenyl phosphate (pNPP) (Fig. 3a). It is stable in aqueous solutions at different pH values and is readily hydrolyzed by majority of phosphatases [20-22]. Hydrolysis of pNPP results in generation of para-nitrophenol, which has pii =7.16 and once deprotonated becomes yellow-colored para-nitrophenolate ion with absorbance at 405 nm. It can be monitored continuously at pH values above 7 assays with lower pH values are performed in the end-point format, requiring alkalinization of solution prior to measuring absorbance. 

Fig. 3 Structures of chromogenic phosphatase substrates (a) Para-nitrophenyl phosphate (b) phenolphthalein monophosphate (c) thymolphthalein monophosphate...

The ELISA technique has been employed for detecting antibodies in serum bound to B[a]PDE-DNA adducts. The USERIA method involves measuring the immunological response of B[a]PDE-DNA in the presence of rabbit anti-serum, alkaline phosphatase enzyme, and radiolabeled para nitrophenyl phosphate (PNPP). The radioactivity of the hydrolyzed tritiated PNPP is measured by a scintillation counter. Both ELISA and USERIA methods have been employed to detect PAH-DNA adducts at 10"15 mol levels in the blood and tissues of humans occupationally exposed to PAH (Amin et al. 1982 Harris et al. 1985 ... 

TOBACCO [NicoTiANA TABACUM L.) Fractions M-Ia and M-Ib from acid phosphatase of tobacco cells did not hydrolyse phosphate monoesters except para-nitrophenyl phosphate, but hydrolysed phosphoric anhydrides (inorganic pyrophosphate, thiamine pyrophosphate, nucleoside di- and triphosphates). In addition, both fractions hydrolysed bis-para-nitrophenyl phosphate. Fraction M-II, a non-specific acid phosphatase, had broad substrate specificity it hydrolysed pyrophosphate, thiamine pyrophosphate, uridine diphosphate, adenosine triphosphate, uridine triphosphate, and cytidine triphosphate, at rates similar to para-nitrophenyl phosphate (Ninomiya et al., 1977). 

RICE ORYZA SATIVA L.) The activity of the add phosphatase purified from the aleurone particle of rice was assessed using various substrates para-nitrophenyl phosphate was readily hydrolysed, as were adenosine triphosphate and pyrophosphate. Among the myo-inositol phosphates, inositol trisphos-phate was hydrolysed at the highest rate (29% of para-nitrophenyl phosphate). The enzyme was also characterized by low or little activity towards (3-glycerophosphate, glucose 6-phosphate and nucleotide phosphates (Yamagata et al, 1980). 

The pH optimum for hydrolysis by the major acid phosphatase (EC 3.1.3.2) associated with the aleurone particle of rice grains Oryza sativa Japonica cv. Koshihikari) was 4.8 (Yamagata et al., 1980). The values were respectively 1.74 mM for para-nitrophenyl phosphate and 5.26 mM for adenosine triphosphate. They decreased slightly with an increase in the number of phosphate groups of various inositol phosphates, being 0.43 and 11.76 mM for myoinositol hexakisphosphate and myo-inositol monophosphate, respectively. In addition, the 17 values for myo-inositol bis- and tri-sphosphate were high (564-611 pmol phos-phate/min/mg protein). 

In contrast to the fractions M-la and M-Ib isolated from the extracellular acid phosphatase of tobacco XD-6 cells, which had maximum activity at pH 6.8, the fraction M-//exhibited a pH optimum at 5.8, indicating an acid phosphatase. When a temperature of 55°C (instead of 30°C) was used for the para-nitrophenyl phosphatase activity of the three enzyme fractions from phosphate-supplied culture, the activity of M-la was rather stable, whereas fractions M-lb and M-11 were inactivated by about 35% and 90%, respectively, in 30 min. The Lineweaver-Burk plot of the rate of para-nitrophenyl phosphate hydrolysis by enzyme fractions from a phosphate-supplied culture showed that the apparent Michaelis constant of fractions M-la and M-lb was 0.9 mM, whereas that of M-11 was 0.3 mM (Ninomiya et al., 1977). 

Regarding bacteria, the purified alkaline phosphatase (EC 3.1.3.1) associated with the outer membrane of Lysobacter enzymogenes UASM 495 (ATCC 29487) was most active at pH 8.5 and two values (0.056 and 0.34 mM) were estimated from kinetic studies using para-nitrophenyl phosphate (von Tigerstrom and Stelmas-chuk, 1986). 

The classical method of measuring phosphatase activity by the hydrolysis of para-nitrophenyl phosphate could be irrelevant for estimating the potential hydrolysis of natural organic phosphorus compounds such as myo-inositol hexakisphosphate, since the relative activity of phytase compared to para-nitrophenyl phosphatase activity may be several orders of magnitude lower (Beck et al., 1989 Joner et al., 2000 Tib-bett, 2002). 

Several independent techniques have been used to identify bacterial alkaline phosphatase activity in natural bacterial assemblages. Phosphatase activity perse has often been detected using para-nitrophenyl phosphate as a substrate enzyme activity is expressed as rate of formation of the hydrolytic product, para-nitrophenol, which is easily detected spectrophotometrically because it absorbs light strongly at = 395 10 nm at pH > 9 (see Huber and Kidby, 1984, for a review of variations on this theme). Other substrates that have been used to detect alkaline phosphatase activity... 

Nedoma et al. (2003) compared values of cell-specific activity (surface phosphomonoesterase activity per cell) obtained in their own study of two waterbodies in the Czech Republic (10-2260 fmol/cell/h, methylumbelliferyl phosphate) with those in the literature. These include (expressed as fmol/cell/h) Phaeocystis, 2.5 (methyl fluorescin phosphate van Boekel and Veld-huis, 1990) Nannochloris, 0.5-5.5 [para-nitrophenyl phosphate Lubifin et al.,... 

Staurastrum chaetoceras, 10 para-nitrophenyl phosphate Spijkerman and Coesel, 1998) Chlamydomonas reinhardtii, 3000-22,000 (methyl fluorescin phosphate Joseph et al., 1994). 

It is only safe to compare the results of studies on different organisms done at different assay concentrations if it is assumed that enzyme activity responds to environmental factors in the same way over the whole concentration range. However, it has been shown for a mammalian tissue (Fedde and Whyte, 1990) that this is not so, with the pH optimum for phosphomonoesterase (assayed with para-nitrophenyl phosphate) being two pH units lower using a low than a high substrate concentration. A similar effect was reported for the freshwater diatom Synedra acus (Hantke and Melzer,... 

Bis-para-nitrophenyl phosphate synthetic colorimetric monoester widely used to determine phosphodiesterase activity in soils and plant material. 

Farrell, H.M. Jr., Behe, M.J., and Enyeart, J.A., Binding of para-nitrophenyl phosphate and other aromatic compounds by beta-lactoglobulin, J. Dairy Sci., 70, 252, 1987. 

C. Wells were washed with PBS containing 0.1% Tween-20 (PBST) and subsequently incubated with anti-rabbit-alkaline phosphatase conjugate for 1 h at 37 C. Para-nitrophenyl phosphate substrate was added to wells after a final PBST wash, and the absorbance read at 405 nm after 30 min at room temperature. Data are presented as the mean reading of triplicate samples SD. 

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