Pyrophosphate assay

Several chemical-assay methods (15,23,50) for tetraethyl pyrophosphate were recently developed and applied by seven collaborating laboratories to samples of representative commercial products and to a sample of purified tetraethyl pyrophosphate which served as a common standard. Concordant results, which correlated well with bioassay results,... 

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. 

The highest PGM recovery was achieved using collector PM443, which is an amine + ester-modified xanthate. Among the chromium slime depressants evaluated, modified mixtures of organic acids, RQ depressants and a low-molecular-weight polyacrylic acid + pyrophosphate mixture were there. The effect of different chromium depressants on chromium assays of the PGM concentrate are illustrated in Figure 18.7. 

S. O. Obare and C. J. Murphy, A Two-Color Fluorescent Lithium Ion Sensor, Inorg. Chem. 2001,40, 6080 L. Fabbrizzi, N. Marcotte, F. Stomeo, and A. Taglietti, Pyrophosphate Detection in Water by Fluorescence Competition Assays, Angew. Chem. Int. Ed 2002,41, 3811. 

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... 

In order to find lead inhibitors against PGTs [15], Piet Herdewijn et al. [22] synthesized a series of substrate analogues of lipid I and lipid II with variations in lipid, pyrophosphate, and peptide moieties. Although many efforts were spent in designing and synthesizing lipid I- and lipid II-based inhibitors [23-26], the PGT inhibition assay performed with Escherichia coli PBP1B in vitro and antibacterial activity... 

One of the simplest methods of estimation of PolyPs in extracts is based on the assay of Pi, which is released from the PolyPs by hydrolysis with 1 M HC1 at 90 °C for 10 min. The Pi released under these conditions is defined as labile phosphorus . If the compounds containing organic labile phosphorus (i.e. nucleotide phosphates, sugar phosphates, etc.) were removed from the extracts by adsorption on Norit charcoal, the increase in Pj content after hydrolysis can be attributed to PolyP and pyrophosphate (PPi). Estimation of the PPj content (Mansurova, 1989) before hydrolysis may be needed in some cases for more precise calculations of the PolyP content. Pi may be determined by one of the well-known chemical methods (Fiske and Subarrow, 1925 Weil-Malerbe and Green, 1951). 

Glickman, J.F. and A. Schmid. 2007. Famesyl pyrophosphate synthase real-time kinetics and inhibition by nitrogen-containing bisphosphonates in a scintillation assay. Assay Drug Dev. Technol. 5, 205-214. 

MgATP is the substrate, and AMP and pyrophosphate (PPi) are the products. Since this activity is usually assayed at a pH of 7.5 using a Tris-HCI buffer system, the reaction tube will contain ATP, Mg, and Tris-HCI as illustrated in Figure 4.1. 

The application of the HPLC assay method to studies on reaction mechanisms has been limited, and the reader is referred to the work of Sloan (1984). Sloan and his colleagues studied the formation of IMP or GMP (and pyrophosphate) from the substrates phosphoribosylpyrophosphate (PRPP) and either hypoxanthine or guanine. These reactions, catalyzed by hypoxan-thine/guanine phosphoribosyltransferase (GHPRTase), were studied by HPLC after a method was developed to separate all the reactants and products simultaneously. 

In the enzyme catalysis of the first committed step in the de novo synthesis of purines, an amino group from L-glutamine is transferred to 5-phosphoribosyl-l-pyrophosphate to form glutamate and 5-phosphoribosyl-1-amine. The assay includes glycinamide ribonucleotide synthetase, which converts 5-phosphoribosyl-l-amine to glycinamide ribonucleotide, which is the reaction product quantitated. 

Dickinson, D.B., Hyman, D., and Gonzales, J.W., 1977, Isolation of uridine 5(-pyrophosphate glucuronic acid pyrophosphorylase and its assay using 32P-pyrophosphate. Plant Physiol. 59 1082-1084. 

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. 

Fig. 6.5. Time-course of ATP synthesis in a suspension of PPase, ATPase liposomes. 25 /nl liposomes (about 0.07 mg protein per ml) were suspended in a medium containing 1 ml 0.2 M glycylglycine (pH 7.8), 0.2 ml luciferin/luciferase assay, 20 fjl 100 mM sodium phosphate, 10 pi 10 mM ADP and 50 pi 10 mM sodium pyrophosphate. The final concentration of MgClj was about 10 mM. At the arrow, 25 pi liposomes were added. The resulting luminescence was measured in an LKB luminometer 1250. The light output was calibrated by addition of a known amount of ATP. (From Ref. 98.)...

The hydrolysis of inorganic pyrophosphate has been carried out in cacodylate buffer, pH 5.5 [179], trichloroacetic acid was then added to terminate the reaction, denatured protein was sedimented by centrifugation, and suitable aliquots of the supernatant solution were assayed for inorganic phosphate by the method of Fiske and SubbaRow [163]. 

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