Substrates phosphatase activity measurement

Alkaline phosphatase activity measurements The activity of the enzyme alkahne phosphatase is determined by using the substrate p-aminophenylphosphate (RAPP). The product of the enzymatic reaction, PAP, is oxidized on the working electrode at 220 mV. This oxidation current is monitored. 

The attachment of the ferrocene groups to small molecules has been used in two main classes of biosensor in the first, the conjugate is an enzyme substrate such that a shift in its redox potential occurs following enzymatic modification. An example is ferrocenylethyl phosphate (Figure 9) that facilitates the electrochemical assay of alkaline phosphatase, a common enzyme label for immunoassays. The redox potential of the alcohol product is lower than the phosphate ester and hence by poising the potential, the product can be selectively measured and so the alkaline phosphatase activity measured. The sensitivity of this method was further enhanced by the use of stripping voltammetry to detect the product. ... 

The third enzyme in the pathway, KD0-8-phosphate phosphatase, has been purified to homogeneity (26). Because of its abosolute specificity, it should be a focal point for chemotherapeutic studies. jThe apparent for KD0-8-phosp te was+ etermined to be 5.8 x 10 M in the presence of 1.0 mM Co or Mg. This specific KD0-8-phosphate phosphatase was separated from enzymes, present in crude extracts, having phosphatase activity on other phosphorylated compounds by column chromatography on DGAE-Sephadex (26). Three distinct peaks of activity were detected. Fractions from each peak were pooled and the rates for the hydrolysis of five compounds were measured. Peak A possessed phosphatase activity for D-glucose-6-phosphate, D-arabinose-5-phosphate, D-ribose-5-phosphate and j-nitrophenylphosphate Peak B dephosphorylated D-arabinose-5-phosphate, D-ribose-5-phosphate and D-glucose-6-phos-phate. Peak C, which was well separated from the other two peaks, could only utilize KD0-8-phosphate as a substrate. KD0-8-phos-phate was not hydrolyzed by the phosphatases present in peaks A and B. 

Phosphotyrosine phosphatase activity was measured at 30°C in a total volume of 100 /x.L containing 24 mM imidazole (pH 7.2), 1 mM EDTA, 1 mM dithiothreitol, 100 jug of bovine serum albumin, 50 fiM dansyl phosphopeptide, and extracts containing enzyme activity. After 15 minutes, the reaction was terminated by the addition of 20 /x.L of 30% (w/v) trichloroacetic acid. The mixture was centrifuged before injection for HPLC analysis. The reaction was linear with respect to both time and enzyme concentration up to at least 30% substrate conversion. 

A considerable number of procedures have been utilized to assay the acid phosphatase activity of serum, blood cells, and tissues. These have involved different substrates or concentrations of substrates, different temperatures, buffers, or variations in other conditions. If the same acid phosphatase were being measured, then the results were naturally not comparable. But the possibility also exists that closely related but different acid phosphatases were present within the same tissue or in different tissues, and the rate of action of these acid phosphatases depended on the particular substrates, buffers that were employed, or other conditions of the reaction. It seems most appropriate then to preface our review and consideration of the literature by describing briefly the conditions characterizing the most frequently used procedures for the determination of acid phosphatase activity, particularly in the serum. Other methods, or modifications of those to be presented here, will be described in later sections of this review. 

With enzyme determinations, standards for calibration purposes and for checking of instrumental performance make use of separately prepared solutions of one of the reactants or the products of the enzyme-catalyzed reaction. For instance, a solution of phenol may be standardized and thereafter used itself as the standard in determinations of acid and alkaline phosphatase, in methods employing phenyl phosphate as substrate and depending on the measurement of the amount of phenol liberated. This standard phenol solution, however, cannot be taken through all the steps in the determination of phosphatase, and a separate control solution must be used to check the performance of the overall technique if this control were omitted, it would be possible, for instance, for a buffer to be incorrectly prepared and for erroneous levels of phosphatase activity to be found. There is no substitute, therefore, in the control of enzyme determinations for the inclusion of a sample (of serum, urine, etc.) previously investigated for its level of enzyme activity. For long-term monitoring of an enzyme method, the repeated analysis of aliquots of a standardized enzyme preparation is most useful, provided... 

The treated cells were placed into the electrochemical chambers and the substrate PAPP was added to a 1 mg/ml final concentration at a total volume of 100 nL. Alkaline phosphatase activity was measured by monitored the PAP oxidation current. The electrochemical chips are disposable and were replaced every experiment. 

Fig. 12.4 HT-29 colon cancer cells response to BA, AN-7, and AN-9. Amperometiic response curves for monitoring of alkaline phosphatase activity using the electrochemical array chip. The HT-29 colon cancer cells were exposed to the differentiation agents Butyric acid (2.5 mM), AN-7 and AN-9 (50 pM). The HT-29 cells with the substrate PAPP were placed into the 100 nL volume electrochemical chambers on the chip. Current was measured using the amperometric technique at 220 mV...

The intrinsic substrate specificity of protein phosphatases, when measured in vitro, is quite low. Much of the specificity of substrate dephosphorylation is achieved by targeting or scaffolding subunits that serve to localize the phosphatase in proximity to particular substrates, and also to reduce its activity towards other potential substrates. 

Furthermore, a useful way of assessing the magnitude of promiscuous activities is the rate acceleration iKsa/Kncm) or catalytic proficiency ( cat/- M/ uncat)- These parameters are indicative because they take into account the inherent reactivity of the substrate. In many cases, promiscuous activities occur, or are measured, with highly reactive substrates. Such activities are in a way expected. However, there are many cases in which promiscuous activities take place with substrates that are less activated than the native one. Examples include, the amidase activity of esterases such as lipases (Table 1, entry 7), the phosphodiesterase activities of P. diminuta PTE and alkaline phosphatase (Table 1, entries 10 and 8), and the PTE activities or various lactonases (Table 1, entries 11-13 and the notable fact that some of these lactonases do not hydrolyze the more activated aryl esters). In such cases, the chemical challenge posed by a less activated substrate is reflected in the more favorable comparisons of rate accelerations, or catalytic proficiencies, for the native versus the promiscuous substrates. 

The measurement of alkaline phosphatase activity (APA) of target phytoplankton is a recently developed bioassay that has been used to determine the algicidal effects of polyphenols from Eurasian watermilfoil (Myriophyllum spicatum) [80]. Phytoplankton produce extracellular enzymes, such as alkaline phosphatase, to provide additional sources of nutrients. Fluorescence spectrometry is used to measure APA, with methylumbeliferyl-phosphate used as substrate and mixed with the algal or cyanobacterial suspension and the suspected inhibitor. 

Because phosphatase activity is often a good indicator of phosphorus limitation, measurements of activity in field materials have been used for diverse purposes ranging from routine monitoring to attempts to understand the phosphorus dynamics of complex communities, such as periphyton in the Everglades (Newman et al., 2003). Possible substrates for assays using spectrophotometry or fluorimetry have been mentioned above. A detailed account of the procedure for using ELFP and subsequent quantification of fluorescence associated with individual cells was given by Nedoma et al. (2003). 

Determination of Alkaline Phosphatase Activity. Vomeronasal and olfactory epithelia were removed and transferred to 0.9% NaCl solution where they were maintained at 4°C. All soluble forms of enzymes were washed out from the surface of the receptor tissue (see Chukhrai et al., 1992 for details). Using procedures that are described in detail in Chukhrai et al. (1992), alkaline phosphatase activity in olfactory and vomeronasal epithelia was determined at pH=8.3 in 0.9% bicarbonate buffer. Disodium p-ni-trophenol phosphate (Sigma) was used as a substrate its initial concentration was 8.1 x 10" M in the buffer. The increase of p-nitrophenol (the product of p-nitrophenylphosphate hydrolysis) was measured with a double-beam spectrophotometer at 400 nm every 30 sec. The velocity of the reaction was determined by estimating the angle of the slope (optical units [OU]/min). The obtained values were divided into the extinction coefficient (C ) under corresponding pH values to obtain the reaction velocity values in pM/min. Effective parameters of the Michaelis-Menten equation were compared using standard procedures (Chukhrai et al., 1992). 

Phosphoglycolate phosphatase activity in 18-7F was 15% of the activity measured in wild type (data not shown). This amount of activity could be due to non-specific phosphatase activity which was estimated in the cell extracts using nitrophenylphosphate as the substrate. Non-specific phosphatase activity was the same in wild type and in the mutant. Mixing experiments indicated that the reduced enzyme activity in the mutant was not due to the presence of an inhibitor (data not shown). 

There are no characteristic abnormalities of serum or of formed blood elements in ML. Non-specific hypercholesterolemia has been seen (Hagberg 1963), but sulfatides are present in normal amounts (Svennerholm and Svennerholm 1962). The ratio of cerebrosides sulfatides, which is abnormal in the central nervous system, is normal in serum (Hagberg 1963). Some relevant serum enzymes have been studied by Austin et al. (1965 a, b). According to these authors acid phosphatase activity was normal when measured with p-nitrophenylphosphate as substrate. The activity of arylsulfatase B was three to four times that of type A, and thus similar to findings in normal serum. 

Most of the substrates that have been used in measuring alkaline phosphatase activity have also been used to measure acid phosphatase, e.g. p-nitrophenylphosphate, phenylphosphate and a-naphthyl phosphate. In most cases of acid phosphatase estimation, it is the level of the prostatic phosphatase which needs to be known, and so specific inhibitors are included in the reaction mixtures. The prostatic isoenzyme is strongly inhibited by tartrate and so in many methods it is tartrate-labile acid phosphatase which is measured. On the pther hand, the red cell enzyme which contributes significantly to the total serum activity is inhibited by formaldehyde and cupric ions. Many laboratories therefore measure formaldehyde-stable acid phosphatase as an indication of prostatic acid phosphatase. 

A multitude of methods exist for alkaline phosphatase measurement using a variety of substrates and conditions. This accounts for the variety of different units in which alkaline phosphatase activity can be expressed. 

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