Enzyme kinetics and molecular modeling studies of G6PDMahidol associated with acute hemolytic anemia.

G6PDMahidol enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PDMahidol enzyme was characterized by molecular modeling to understand its kinetics. G6PDMahidol, G6PDG487A and G6PDWT proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PDWT, the Km and Vmax of NADP+ with G6PDG487A were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PDG487A showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PDG487A was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PDG487A -helix d and -strand E, besides the conformation of -strand D. In conclusion, the biochemical and structural properties of G6PDG487A and G6PDWT enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.

[ role of 6-aminonicotinamide in the resistance of peritoneal macrophages against Leishmania major infection in BALB/c mouse]

The objective of this study was to investigate the relationship between glucose-6-phosphate dehydrogenase inhibition in macrophages treated with 6-Aminonicotinamide, the amount of nitric oxide [NO] production and the resistance of infected macrophages against Leishmania major infection. Peritoneal macrophages of BALB/c mice were isolated and treated with different concentrations [1.25, 2.5, 5, 10 mM] of 6-aminonicotinamide. After 24 hours, the viability of treated macrophages was measured by MTT assay at 540 nm. G6PD activity was measured in the cell extracts 24 hours later. Macrophages were then infected with leishmanial amastigotes and after 18 hours NO production was determined using Griess-reagent. In order to study the inhibition of macrophage activity, 5 mM concentration of 6-AN was used and number of leishmanial amastigotes was recorded in these cells from day 1 to7. Different concentrations of 6-AN were shown to cause a significant increase in cell death and decrease in G6PD activity and NO production in macrophages. Also, the number of amastigotes in macrophages was increased significantly [p < 0.05]. He concentration of 6-aminonicotinamide and G6PD activity affect the viability of BALB/c mice peritoneal macrophages through production of NO. Inhibition of G6PD activity leads to decreased leishmani-cidal activity of mouse peritoneal macrophages

effect of aluminum on human erythrocyte glucose 6-phosphate dehydrogenase

Glucose 6-phosphate dehydrogenase [G6PD], the first enzyme in initiating the pentose phosphate shunt, is an important component in generation of NADPH. Although innumerable studies have been performed on human erythrocyte G6PD, however, the effect of trace elements on the enzyme activity requires further investigations. To study the effect of aluminum on human erythrocyte G6PD. In this experimental research, following the purification of G6PD using chromatographic methods, the effect of different concentrations of Al[3+] [up to 100 micro-molar] on G6PD activity was studied. The enzyme activity was measured at different concentrations of glucose 6-phosphate and NADP[+] to determine the type of inhibitory action. Aluminum at the concentration of 100 microM showed a considerable inhibitory effect on G6PD activity [60%]. The type of inhibitory action, depending on the use of glucose-6-phosphate or NADP[+], was competitive and noncompetitive, respectively. Aluminum exerts an inhibitory action on human erythrocyte G6PD activity

Regulation and properties of purified glucose-6-phosphate dehydrogenase from rat brain.

Glucose-6-phosphate dehydrogenase from rat brain was purified 13,000 fold to a specific activity of 480 units/mg protein. The molecular weight was 121 kDa. The kinetics of brain glucose-6-phosphate dehydrogenase are compatible with a model involving two possible states of the enzyme with a low and high affinity for the substrate D-glucose-6-phosphate. NADP+ and ADP offered protection against p-chloromercuribenzoate inhibition. NADPH is a powerful competitive inhibitor with respect to NADP+. The apparent Ki for NADPH inhibition was lower than the Km for NADP+. ADP inhibited the enzyme competitively with respect to NADP+. ATP inhibited the enzyme non-competitively with respect to NADP+, whereas kinetics of mixed inhibition was observed with respect to substrate D-glucose-6-phosphate. The interplay between NADP+ and NADPH leading to enzyme activation or inhibition according to their relative or absolute concentrations as well as the control of enzyme activity by the adenine nucleotide system may contribute a refined mechanism for the regulation of glucose-6-phosphate dehydrogenase and therefore the pentose phosphate pathway in brain.

Inhibition of two HMP shunt pathway enzymes by fatty acids and their CoA esters in developing human brain: role of fatty acid binding protein.

Inhibitory effects of fatty acids and their CoA esters on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities of human fetal brain cytosol have been studied. Purified human fetal brain fatty acid binding protein reverses the inhibitory effects of palmitoyl-CoA and oleic acid on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in human fetal brain cytosol. This protein, when added alone, activates the enzymes. Levels of fatty acid binding proteins as well as the activities of these two HMP shunt pathway enzymes, which provide cofactors like NADPH for reductive biosynthesis, increase with gestation. These results indicate that a relationship exists between the high demand for fatty acids and synthesis of cofactors for lipid biosynthesis in developing brain.