Erythrocyte glucose-6-phosphate dehydrogenase activity assay and affinity for its substrate under "physiological" conditions

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) activity and the affinity for its substrate glucose-6-phosphate were investigated under conditions similar to the physiological environment in terms of ionic strength (I: 0.188), cation concentration, pH 7.34, and temperature (37 o C). A 12.4, 10.4 and 21.4% decrease was observed in G6PD B, G6PD A + and G6PD A -activities, respectively. A Km increase of 95.1, 94.4 and 95.4% was observed in G6PD B, G6PD A + and G6PD A -, respectively, leading to a marked decrease in affinity.In conclusion, the observation of the reduced activity and affinity for its natural substrate reflects the actual pentose pathway rate. It also suggests a much lower NADPH generation, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor. Key words• Glucose-6-phosphate dehydrogenase • Red blood cells • G6PD activity and affinity Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is a pentose shunt enzyme involved in the maintenance of adequate concentrations of reduced nicotinamide adenine dinucleotide phosphate (NADPH). This nucleotide, together with glutathione reductase, keeps glutathione in its reduced form (GSH), protecting the red cell against oxidative stress. There are two normal G6PD variants, G6PD B and G6PD A + ,and other deficient polymorphic mutants such as G6PD A -, besides dozens of rare ones (1), some of which have been described by our group (2,3).The standard methods used to assay G6PD activity and affinity (Michaelis-Menten constant -Km) for its substrate are currently performed using reaction conditions of 145 mOs and ionic strength I: 0.06, pH 8.0, at 37 o C for activity assay and 25 o C for Km determination. In the present study the enzyme activity as well as its affinity were determined under nearly physiological conditions regarding osmolarity (290 mOs) and ionic strength (I: 0:188), 3 mM MgCl 2 , pH 7.34, at 37 o C. The activity assay was performed with NADP and glucose-6-phosphate (G6P) in excess. Under physiological conditions (37 o C and 290 mOs) there was a 10-12% decrease in the activity assay, as well as an affinity decrease of 50% when compared to the standard methods (25 o C and 145 mOs). Blood was collected in the preservative

The role of glucose-6-phosphate dehydrogenase in adipose tissue inflammation in obesity

“…21 Multiple lines of evidence suggest that G6PD-derived NADPH has a contradictory impact on ROS level in different cell types (Table 1). 12,[30][31][32][33][34][35][36][37][38][39][40] Previous reports highlight that cells with intrinsic susceptibility to ROS predominantly use G6PD-derived NADPH for antioxidant defense in response to oxidative stress. In the case of red blood cells (RBCs), there is constant production of ROS by spontaneous reaction and oxygen oxidation of ferrous iron (Fe 2C ) to ferric iron (Fe 3C ).…”

“…One of the distinct characteristics of RBCs is their large dependence on G6PD-derived NADPH to activate the antioxidant glutathione system as compared with other cells. 31 Since RBCs are not equipped with other NADPH-producing systems, when G6PD becomes dysfunctional, these RBCs become highly susceptible to oxidative stress. 31 Therefore, the most common complications of G6PD deficiency in human is acute hemolytic anemia in response to oxidizing stimuli including microbial infection.…”

“…31 Since RBCs are not equipped with other NADPH-producing systems, when G6PD becomes dysfunctional, these RBCs become highly susceptible to oxidative stress. 31 Therefore, the most common complications of G6PD deficiency in human is acute hemolytic anemia in response to oxidizing stimuli including microbial infection. 32 Neurons are another cell type that is intrinsically vulnerable to oxidative stress and their antioxidant system is significantly alleviated in response to G6PD deficiency.…”

A new colorimetric assay for sensitive detection of glucose-6-phosphate dehydrogenase deficiency based on silver nanoparticles

“…Both products are essential for synthesizing various biological molecules, such as nucleic and fatty acids. NADPH serves as a cofactor in different enzymatic reactions and preserving antioxidant defenses with keep glutathione in its reduced form (GSH), protecting the RBCs against oxidative stress [4][5][6][7][8]. NADPH also works as a pro-oxidant to produce reactive oxygen species and reactive nitrogen species as signal molecules for elevating cellular processes, such as cell growth [9].…”