Pyrophosphatase assay

The enzyme is routinely assayed by a modification of the Fiske and SubbaRow technique for measuring Pi production from PP (5). Cooper-man has developed a continuous automated pH-stat pyrophosphatase assay which is very convenient for repetitive analyses (0). Kinetic studies over a wide range of conditions require a more sensitive assay such as the production of 32Pi from 32PP, as used by Moe and Butler (9). 

The different ionic forms of PPt which are present in significant amounts at pH 6-9 in the presence of Mg2, as activating cation, and KC1, to adjust the ionic strength, are shown in Fig. 1. Moe and Butler have attempted to assess the catalytic significance of these forms and of free Mg2 which is also present (9). The values of the equilibrium constants for all these interactions have been evaluated under conditions similar to those of the pyrophosphatase assay (SI), and a computer program has been devised to calculate the concentration of each of these ionic species as a function of pH and of total added PPi and MgCl2 (9). Rates of PPi hydrolysis were determined over a wide range of Mg2 and PP, concentrations at pH 7.4, 8.1, and 9.0. The observed rates of PP, hydrolysis and computed concentrations of the ionic species... 

A flow assay was reported for determination of inorganic pyrophosphate a pyrophosphatase was coimmobilized with luciferase on Sepharose beads with continuous flow of saturating concentrations of substrates. The instrument allowed automation with a throughput of approximately one sample every 4 min. 

Vance DH, Czamik AW (1994) Real-time assay of inorganic pyrophosphatase using a high-affinity chelation-enhanced fluorescence chemosensor. J Am Chem Soc 116 9397-9398... 

Lieberherr, M., Vreven, J., Vaes, G. The acid and alkaline phosphatases, inorganic pyrophosphatases and phosphoprotein phosphatase of bone. I. Characterization and assay. Bio-chim. Biophys. Acta 293, 160 (1973)... 

Fia. 5. Reaction of E. coli pyrophosphatase with TNBS at pH 8.5 in 0.5 M NaHCOs. The reaction was followed in a spectrophotometer, and the concentrations of e-TNP-lysine were determined from the optical density at 345 m/i. The solid line shows these data as percentage of total protein lysines by 6 hr the optical density had stabilized at the level indicated by the arrow on the right-hand ordinate. Aliquots were removed at the times shown (circles) and assayed for remaining enzymic activity. 

Escherichia coli pyrophosphatase can be assayed in vitro by means of a simple two-step procedure. The enzyme is first incubated with PP in the presence of a divalent cation, and the amount of liberated Pi is then determined colorimetrically by the method of Fiske and Subba-Row 12, 52). 

The various compounds were tested in the routine assay under all of the conditions noted in Table IV liberation of Pi after incubation with large excesses of purified enzyme was not detected. Compounds of groups (2) and (3), containing phosphodiester or internal phosphoanhydride linkages, were additionally tested with human semen phosphomonoesterase after incubation with E. coli pyrophosphatase. No Pi was liberated after this dual incubation, indicating absence of phosphodiesterase or coenzyme-degrading activity in the pyrophosphatase (12). 

Recently, Czamik et al. have reported the use of the acyclic protonated amine host 9 as a chemosensor of pyrophosphate. Typical fluorescence sensing methods rely on the ability of a complexed anion to quench the fluorophore. The fluorescence intensity of host 9, however, is actually increased upon complexation of anions and its 2200-fold selectivity of pyrophosphate over phosphate allows for real-time assay of pyrophosphate hydrolysis by inorganic pyrophosphatase.18... 

Hydrolytic driving force. The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic reactions such as the synthesis of DNA. This hydrolytic reaction is catalyzed in Escherichia coli hy a pyrophosphatase that has a mass of 120 kd and consists of six identical subunits. For this enzyme, a unit of activity is defined as the amount of enzyme that hydrolyzes 10 pmol of pyrophosphate in 15 minutes at 37°C under standard assay conditions. The purified enzyme has a of 2800 units per milligram of enzyme. 

Problems in these assays that cannot be attributed to interfering NADases or pyrophosphatases are usually attributable to a single component (i.e. inactive toxin or ARF) and can be identified using data from the controls. When A ild-type CT or LT display reduced (or zero) basal activity, one of several conditions may be the cause. 

Vance and Czamik have also developed a new chemosensor for pyrophosphate based on an anthracene chromophore (Figure 91) [118]. This compound exhibits low fluorescence. However, upon binding pyrophosphate, the fluorescence increases considerably. It has been shown by Vance and Czamik that inorganic pyrophosphatase can be assayed in real time using this chemosensor since the inorganic phosphate produced does not affect the fluorescence of this molecule. 

U/ml inorganic pyrophosphatase from Baker s yeast (Sigma-Aldrich, St. Louis, MO, USA). Resuspend 500 U lyophilized enzyme and buffer salts in 250 pi water. Store at -80 °C in 5 pi aliquots. Store thawed aliquot on ice and discard at the end of assay day. 

Prepare 0.8 U/ml diluted pyrophosphatase solution by diluting 4 pi of 2,000 U/ml pyrophosphatase solution in 10 ml Assay Buffer in 15 ml conical tube. Mix by pipetting. 

Fig. 2 Sample ATP titration curve. Change in ODess values are plotted against initial ATP concentration in 6 pi assay volume, and the data fitted to Michaelis-Menten equation. /C, values are 13.3 1.1,6.7 1.4,4.4 1.8, and 2.4 0.4 pM for 0.78 (filled upright triangid), 0.39 (filled inverted triangld, 0.195 (filled circid), and 0.098 nM (filled squard) ENPP1, in the presence of 0.4 U/ml pyrophosphatase...

Prepare Assay buffer and diluted pyrophosphatase solution similar to Subheading 3.1, steps 1 and 2. 

Pyrophosphorylase activity of the DNA has been determined by an indirect and a direct approach. In the indirect one, the PPj exchange with deoxynucleo-side triphosphate is measured. In the direct approach, cleavage of DNA to the respective pyrophosphatase is assayed. 

PET sensor for dihydrogen pyrophosphate dianion. Both of these cases require careful pH control for success. Also, sensor B may be used as an assay for the pyrophosphatase enzyme since the hydrolysis products will be released from the receptor pair with concomitant loss of fluorescence. 

---