Phosphate exchange assay

There are two commonly used assay procedures for investigating amino acid activating enzymes. These are (a) the hydroxamate assay 97, 99, 100, 103, 104) where an extract is incubated in the presence of high concentrations (1-2 M) of hydroxylamine, and the ATP-and amino acid-dependent formation of hydroxamate is measured the latter is best determined by the method of Schweet et al. 105) , and (5) the pyrophosphate exchange assay 97, 99, 100), in which an amino acid-dependent exchange of labeled pyrophosphate with the two terminal phosphates of ATP is measured. 

Label a set of tubes according to the standards and samples to be used. Add 250 pi of each standard and sample to the appropriate tubes. If the samples are in a buffer that may significantly change the pH of the reaction buffer, the samples should be buffer-exchanged or dialyzed into 0.1M sodium phosphate, pH 8.0, before running the assay. 

Glycerophosphoethanolamine. The water-soluble fraction is assayed for total phosphate (and as a control for inorganic phosphate). The latter is a check on whether the reaction proceeded as expected. It can be purified by ion exchange chromatography and then examined as follows. 

Day 2 Precipitation of Pi, Cation Exchange with Dowex 50, and Initial Glucose-1-Phosphate Assays... 

Figure 5.9 Enzymatic activities of fractions following HPLC chromatography. A partially purified preparation was fractionated by ion-exchange HPLC (AX-300) with a mobile phase of 0.1 M potassium phosphate. Proteins were eluted with a gradient of sodium acetate. Column eluent was monitored at 280 nm. Fractions were collected, and each fraction was assayed for three different activities.

Measurement of the activity of this enzyme by the HPLC assay method requires separation of the hypoxanthine and the IMP, which can be easily accomplished by several methods, including reversed-phase or ion-exchange HPLC. Jahngen and Rossomando (1984) used a reversed-phase Ci8 column with a mobile phase of 10 mM potassium phosphate (pH 5.6), and 10% methanol. Detection was at 254 nm, and the separation obtained is shown in Figure 9.98. 

The HPLC enzyme assay method provides an alternative procedure with which to approach this problem. Clearly, to be able to assay these activities by the HPLC method, it is necessary to separate ATP, ADP, and AMP. This separation can be easily accomplished by ion-exchange HPLC eluted isocratically with a mobile phase containing a phosphate buffer and sufficient concentration of salt to elute the ATP. Under these conditions, the order of elution of the compounds would be AMP first, ADP next, and ATP last. 

In this assay both the formation of product and the loss of substrate were followed. The compounds were separated on an ion-exchange HPLC column equilibrated with 0.5M phosphate buffer of pH 4.0. The eluent was monitored at 254 nm. 

The Pj liberated by acid or enzymatic hydrolysis of the inositol phosphates may also be assayed. An anion-exchange HPLC column, which separates the inositol phosphates, is coupled to a second column that contains immobilized alkaline phosphatase. The Pj that is released by the enzyme is measured color-imetrically. The system can detect 1 nmol of inositol phosphates in a single sample to indicate levels of IP3 of 13 to 40 nmol/g of tissue. The sensitivity of the phosphate assay is increased to the picomolar level with malachite green as a reagent (Dean and Beaven, 1989 Palmer and Wakelam, 1989). 

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