Hexokinase glucose-6-phosphate dehydrogenase

Neese JW, Duncan P, Bayse D. Development and Evaluation of a Hexokinase/Glucose-6-Phosphate Dehydrogenase Procedure for Use as a National Glucose Reference Method. HEW PubHcation No. (CDC) 77-8330, Atlanta Centers for Disease Control, 1976. 

Hexokinase/glucose-6-phosphate dehydrogenase ATP, ADP, glucose 6-phosphate, 6-phosphogluconate, NADP, NADPH K14... 

Recently, a universal enzyme-coupled fluorescence assay for glycosyl transferases was developed. This method is extremely cost-effective and is based on the quantification of nucleotides produced in the glycosyl transfer reaction. The guanosine diphosphate (GDP), uridine diphosphate (UDP), and cytidine monophosphate (CMP) are phos-phorylated with nucleotide kinase in the presence of excess of ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase,glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for the conversion of resazurin to resorufin, which is then quantified by fluorescence measurement. 

Two or more linked enzyme reactions can lead to a change in the concentration of NADH or NADPH that is equivalent to the concentration of the original analyte. The reference glucose measurement using hexokinase [9001-51-8] and glucose-6-phosphate dehydrogenase [9001-40-5] is an example ... 

Fig. 4. Schematic of a multisequence biosensor in which the target glucose is first converted to glucose-6-phosphate, G6P, in the test solution by hexokinase. G6P then reacts selectively with glucose-6-phosphate dehydrogenase immobilized on the quartz crystal surface. Electrons released in the reaction then chemically reduce the Pmssian blue film (see Fig. 3), forcing an uptake of potassium ions. The resulting mass increase is manifested as a...

Figure 7-10. Coupled enzyme assay for hexokinase activity. The production of glucose 6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP". When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose 6-phosphate by hexokinase.

Another common procedure which is used for glucose assay is the hexokinase procedure in which the glucose is phosphory-lated by means of ATP and then dehydrogenated with glucose-6-phosphate dehydrogenase measuring the abosrption of NADPH... 

A kinetic procedure employing the reverse reaction is coupled to the enzymes hexokinase and glucose-6-phosphate dehydrogenase, as used by Nielson and Ludvigson after the method of Oliver (J ). This procedure was later modified and optimized by Rosalki (38). 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

Glucose-6-phosphate dehydrogenase is used as an indicator enzyme in the hexokinase assay of glucose... 

Hexokinase 280 kU/l/glucose-6-phosphate dehydrogenase 140 kU/1 hexokinase and glucose-6-phosphate dehydrogenase from yeast, suspended in 3.2 M ammonium sulfate solution. Dilute stock solutions with 3.2 M ammonium sulfate solution according to specified activity to 280 kU/1 and 140 kU/1, respectively. 

The preparation of these solutions is described in the text in section 4.6.7.2. Pipette these successively into 1-cm cuvettes. The absorbance of the mixtures is read twice (at 340 nm) before (ODi) and after (OD2) the addition of glucose-phosphate isomerase. G6P-DH glucose-6-phosphate dehydrogenase, HK hexokinase, NADP nicotinamide adenine dinucleotide, PGI glucose phosphate isomerase, TEA triethanolamine... 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

Fig. 9. Sequence of transformations catalysed by the supramolecular ATP-generating system [38, AcP, Mg2, ADP] (38 = [24]-N6C>2) and the enzymes hexokinase (HK), glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phospho-gluconate dehydrogenase (6-P-GDH) [5.32],...

Rat liver mitochondria were isolated as described (4). The initial rate of ATP synthesis associated with the oxidation of succinate was followed by monitoring fluorometrically the ATP-linked NADPH production in the presence of hexokinase and glucose-6-phosphate dehydrogenase (10). Control experiments showed no interference from unexpected reduction of NADP+ or from electron backflow. Possible ATP formation via mitochondrial adenylate... 

Note HxK, G6PDH, GlcDH, and GaK are hexokinase, D-glucose-6-phosphate dehydrogenase, glucose dehydrogenase, and gluconate kinase. 

Exley et al. [61] found aluminum in practically all reagents used in a study of the inhibition of hexokinase activity by this element. The way to overcome the problem was cleaning the solutions using an aminophosphonate chelation resin. The procedure reduced the contamination of ATP and NADP to approximately 5% and 10% of their initial values, respectively, but the resin was ineffective in removing aluminum from magnesium acetate or the enzyme glucose 6-phosphate dehydrogenase. Probably the conditions were not favorable for the resin to pick up the aluminum ions from these solutions. It is important to remember that, if there is an affinity between aluminum and the species in solution, a competition between this species and the resin will take place. 

Bondar, R. J. L., and Mead, D. C. (1974). Evaluation of Glucose-6-Phosphate Dehydrogenase from Leu-conostoc mesenteroides in the Hexokinase Method for Determining Glucose in Seram. Clin Chem 20 586. 

Stevens and Stevens (1979) measured the hydration dependence of glucose-6-phosphate dehydrogenase, hexokinase, fumarate hydratase (fumarase), and glucose-6-phosphate isomerase (phosphoglucose isom-erase) over the range 0.1-0.6 h. Serum albumin was used as a carrier protein to buffer the water content. The hydration isotherms of the enzymes and the serum albumin were assumed to be similar. For the first three enzymes activity was detected (0.05% of full solution activity) near 0.2 h. Activity was measurable for the isomerase at 0.15 h. In all cases, even at 0.3 h, the activity in the powder was less than 5% of the solution rate. Diffusion of substrates in the powder may be rate limiting. The amount of albumin in the powder affected the rate. 

To 2.0 ml of a glucose solution, 1.0 ml of solution containing excess ATP, NADP", MgCh, hexokinase, and glucose-6-phosphate dehydrogenase was added. The absorbance of the final solution (in a 1 cm cuvette) increased to 0.91 at 340 nm. Calculate the concentration of glucose in the original solution. 

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