P-nitrophenyl phosphatase

Bansal SK, Desaiah D. 1982. Effects of chlordecone and its structural analogs on p-nitrophenyl phosphatase. Toxicol Lett 12(2-3) 83-90. 

To characterize the preparation, determine marker enzymes, e.g., p-nitrophenyl phosphatase (PNPase), ouabain-sensitive Na,K-dependent ATPase, or dihydropyridine receptor complex (L-type voltage dependent calcium channel). 

Frosolono and Pawlowski (1977) studied biochemical changes in various lung fractions prepared from rats exposed to phosgene at concentrations near to or above the LCtso. A number of enzymes showed decreased activity in all fractions these included p-nitrophenyl phosphatase, cytochrome c oxidase, ATPase and lactate dehydrogenase (LDH). The serum LDH rose. It was suggested that either inhibition of enzyme activity or loss of enzyme from cells would account for these changes. The data available did not allow... 

Recently, Robinson (1969) has made a kinetic study of p-nitrophenyl-phosphatase in a partially purified ATPase preparation from rat brain from which he proposes that for ATPase there is a concerted process with interdependent coupled cation activators and not two sequential mechanisms each with its own cation activator. Thus, instead of a Na site being translocated to become an external K site in the enzyme molecule, Na+ andK sites would coexist. 

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

In spite of the above mentioned Co(EII) compounds, kinetically labile metal complexes may provide fast product/substrate exchange and some of these systems show real catalytic activity. In native dinuclear phosphatases Mg(II), Mn(II), Fe(II/III), or Zn(II) ions are present in the active centers. Although the aqua complexes of the weakest Lewis acids, Mg(H) and Mn(II), show measurable acceleration of e.g. the transesterification of 2-hydroxypropyl p-nitrophenyl phosphate HPNP, [Mn(II)] = 0.004 M, kobs/ uncat = 73 at pH 7 and 310 K, [38] or the hydrolysis of S -uridyluridine (UpU) [39], only a few structural [40] but no functional phosphatase-mimicking dinuclear complexes have been reported with these metal ions. 

The two most common enzyme labels for secondary antibodies are alkaline phosphatase (AP) used in conjunction with the substrate p-nitrophenyl phosphate, which results in a yellow reaction product, and horseradish peroxidase (HRP) used in conjunction with the substrate ABTS and H2O2, which results in blue-green reaction product see Chapter 23). 

Detection reagent (substrate) for enzyme-labeled antibody 2,2-azino-di(3-ethyl-benzthiazohne sulfonate-6) (ABTS) 0.3 g/L in 0.15 M sodium citrate with 0.1% H2O2 for the detection of horseradish peroxidase-labeled secondary antibody or p-nitrophenyl phosphate (pNPP) 1 g/L in 1M diethanolamine in water for the detection of an alkaline phosphatase-labeled antibody (Kirkegaard and Perry Laboratories). 

In subsequent years, much evidence has been adduced to support this mechanism. Alkaline phosphatase and, by analogy, other serine enzymes, are directly phosphorylated on serine serine phosphate is not an artifact (Kennedy and Koshland, 1957). In the presence of nitrophenyl acetate, chymotrypsin is acetylated on serine, and the resulting acetylchymotrypsin has been isolated (Balls and Aldrich, 1955 Balls and Wood, 1956). Similarly, the action of p-nitrophenyl pivalate gave rise to pivaloyl chymotrypsin, which could be crystallized (Balls et al., 1957). Neurath and workers showed that acetylchymotrypsin is hydrolyzed at pH 5.5, but that it is reversibly denatured by 8 M urea the denatured derivative is inert to hydrolysis and even to hydroxylamine, whereas the renatured protein, obtained by... 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

Substrate products can be classified as either soluble or precipitating. Soluble peroxidase substrates include o-phenylenediamine, which is converted into a yellow product 2,2 -azino-(3-ethyl)-benzothiazoline-sulfonic acid, which is converted into a green product and tetramethylbenzidine, which is converted into a blue product. Precipitating substrates for peroxidase include 4-chloronaphthol, which yields a blue precipitate and aminoethylcarbizole, which forms a red precipitate. Alkaline phosphatase is most frequently used with p-nitrophenyl phosphate to give a yellow-orange soluble product, or with 5-bromo-4-chloro-3-indo-lyl-phosphate p-toluidine salt to yield an insoluble blue product. 

Hog spleen acid DNase, as obtained by the above procedure, is completely free of contaminating phosphatase, exonuclease, and adenosine deaminase activities. The enzyme has a weak intrinsic hydrolytic activity on bis(p-nitrophenyl) phosphate and the p-nitrophenyl derivatives of deoxyribonucleoside 3 -phosphates (see Section III,D,3). 

In studies with alkaline phosphatase it has been found that the enzymic activity measured by the release of p-nitrophenol from p-nitrophenyl phosphate increases with the concentration of tris buffer much faster than it increases with the ionic strength of other salts such as NaCl and Mg2SO< (4, 50). This behavior of tris was shown by Dayan and Wilson (122, 123) to result from a transphosphorylation reaction, where 0.5 M tris reacts with phosphoryl enzyme to form tris phosphate at the same rate as does 55 M water to form orthophosphate. 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

As with Km, the effect of pH on Fmax cannot be described by a simple ionization curve. With calf intestinal phosphatase, the log ym8X curve for a monoester substrate is sigmoid (143, 162) or, in the case of synovial phosphatase, extremely shallow (76). Both curves approach a maximum value at alkaline pH. Barman and Gutfreund, however, found that milk phosphatase had an optimum at pH 10 with only 60% activity at pH 11 (83). This is by no means typical since placental phosphatase has been shown to be fully active with the same substrate, p-nitrophenyl phosphate at pH 11.5 (85). With PP as substrate there is evidence that an optimum in Vmax is reached at considerably lower pH values (8.5-9.2) (116, 117, 164). A pH-activity curve for calf intestinal phosphatase is given in Fig. 3. Features to note are the plateau in activity around pH 7, corresponding to a minimum in the phosphorylation rate constant, and a change in rate determining step at about pH 6 (165). 

For the hydrolysis of p-nitrophenyl phosphate by placental phosphatase at pH 10.5 the corresponding figure is 10,380 cal/mole (101a). Taking into account changes in ionization of the enzyme, a value of 9800 cal/mole for 4-methylumbelliferyl phosphate and calf intestinal phosphatase was derived (143). The comparable values for nonenzymic hydrolysis of monoanions of aryl phosphates are 27,000-31,000 cal/mole... 

A detailed study of the effects of dioxane and ethanol on calf intestinal phosphatase showed that Fmax for p-nitrophenyl phosphate decreased... 

Most of these observations have since been verified. Phosphorylation by substrate has been shown to occur under acid conditions by using a stopped-flow technique (118, 165) as illustrated in Fig. 4. Under alkaline conditions the phosphoryl enzyme cannot normally be observed or isolated because the rate of dephosphorylation exceeds the maximum rate of phosphorylation (170). One interesting aspect is that the pH-rate profiles for phosphorylation and dephosphorylation are quite different, as is the case for E. coli alkaline phosphatase (171). Barman and Gut-freund studied the formation and breakdown of milk phosphoryl phosphatase using a rapid-quenching technique and concluded that dephosphorylation could not be rate limiting for the hydrolysis of p-nitrophenyl phosphate at pH 7 (S3). 

Most investigators utilize p-nitrophenyl or a-naphthyl phosphate as substrate. The determination of serum prostatic acid phosphatase was developed by Fishman and Lemer (34) based on the d-(+)-tartrate inhibition of prostatic enzyme discussed below. Babson et al. (35, 36) demonstrated that a-naphthyl phosphate was much more easily split by prostatic than red cell phosphatase. Table V (35) shows the results obtained when prostatic or red cell phosphatase was added to human serum which had been incubated at pH 8.6 for 1 hr at 37° to destroy all endogeneous phosphatase activity. The table shows the superiority of a-naphthyl phosphate as substrate. 

Fig. 1. Prostatic acid phosphatase activity as a function of pH ( ) phenyl phosphate (O) p-nitrophenyl phosphate and (A) /8-glycerophosphate. Buffers Ac, acetate Cit, citrate and tris. From Nigam et al. (88).

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

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