Deoxyglucose Assay Using Enzymatic Cycling in Oil Wells

The basic enzymatic cycling reaction involves three steps in order to measure deoxyglucose. In the first step, a 0.1- to 0.2-^L aliquot is removed from the neutralized acid extraction and used in the specific reaction sequence, ending in reduction of a pyridine nucleotide. This reaction sequence is found in Fig. 1 and described in detail here. The second step is the enzymatic cycling or amplification step. NADPH is alternatively oxidized and reduced as seen in Fig. 2. In each oxidation/reduction cycle, 1 mol each of 6-phosphogluconate and glutamate is produced. A cycling rate of150,000 cycles is achieved at room temperature overnight using 150 ig/mL glutamate dehydrogenase and 15 ig/mL G6PDH. After the desired multiple of amplification, the enzymes are inactivated in alkali with heat, and in the third step or the indicator step, the 6-phosphogluconate is measured by the fluorescence of the NADPH generated in the conversion of 6-phosphoglu-conate dehydrogenase. Each of these three steps will be described in detail.

1. The first step in the cycling reaction generates NADPH from deoxyglucose. This assay involves three steps, as seen in Fig. 1. First, in the oil well apparatus, a 0.1-^L aliquot of a 2X 6-phosphate removal reagent is added to 0.1 ^L of extract. This addition converts all the 6-phosphate compounds [2-deoxyglucose-6-phos-phate (DG-6-P) and glucose 6-phosphate (G-6-P)] to 6-phopshogluconates via an excess of the enzyme glucose-6-phosphate dehydrogenase (L. mesenteroides). This reaction occurs at room temperature over 40 min.

2. Following completion of the first reaction, 0.05 ^L of 0.21 NHCl is added and the reaction mix heated to 80°C for 20 min to destroy the formed NADPH to avoid interference with subsequent steps. NaOH (0.21 N, 0.05^L) is then added to neutralize the solution.

3. In the third step, 0.1 ^L of a 4X removal reagent is added to the reaction and the reaction was allowed to occur over 20 min at room temperature. In this two-step reaction, hexokinase is added to convert the remaining free glucose and free DG to the

Fig.1. Description of the reactions used to measure 2-DG uptake.

6-phosphate compounds. Phosphoglucoseisomerase then selectively converts the formed G6P to fructose-6-P but does not convert DG6P. This phosphoglucoseiso-merase reaction is then driven to completion by adding phosphofructokinase to convert the fructose-6-P to fructose-1,6-bisphosphate. ATP, pyruvate kinase, and phosphoenolpyruvate are added to drive both the phosphofructokinase and hexokinase reactions to completion by replenishing ATP levels. Following the 20 min at room temperature, the reaction is heated to 80°C for 20 min to destroy the enzymes and prevent the back reactions.

4. In the next step, 0.1 ^L of a 5X DG6P reagent is added to convert the remaining DG6P from the previous step, to deoxy-6-phosphogluconate, generating an equimolar amount of NADPH. This reaction is performed at room temperature for 40 min. In the final step, 0.1 ^L of 0.3 N NaOH is added and the reaction is heated to 80°C for 20 min to destroy the enzymes and the excess NADP+.

5. The NADPH generated in the final enzymatic reaction is then enzymatically amplified in the second step and the principle of these reactions is illustrated in Fig. 2. NADPH is alternatively oxidized by glutamate dehydrogenase (GDH) and reduced by G6PDH. In each cycle, 1 mol of 6-phosphogluconate and glutamate is produced.

Fig. 2. Depiction of the cycling and indicator reaction used to measure 2-DG uptake in single embryos.

Cycling rates of 150,000 cycles are achieved using 150 ig/mL GDH and 15 ig/mL G6PDH at room temperature overnight. A 10-^L aliquot of the cycling reagent is added to the 0.6-^L reaction from the DG assay. This reagent consists of imidazole buffer with 150 ig/mL beef liver glutamate dehydrogenase (GDH) and 15 ig/mL Leuconostoc G6PDH added at room temperature overnight to give a cycling rate of 150,000 cycles. The reaction is stopped by adding 1 ^L of 1 N NaOH and heating to 80°C for 30 min (see Notes 2 and 3).

6. The third and final step in the cycling reaction is the indicator reaction. In this reaction, one of the cycling reaction products, 6-phosphogluconate (6-P-gluconate), is measured by a simple fluorometric assay using 6-phosphogluconate dehydrogenase to convert the substrate to ribulose 5-phosphate, CO2, and NADPH. A volume of 10 ^L of the cycling reaction is added to 1 mL of the indicator, 6-phosphogluconate reagent. The fluorescence of the generated NADPH is measured directly in a 1-mL volume in 10 X 75-mm fluorimeter tubes by use of a Farrand fluorimeter (see Notes 4 and 5).

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