Redox metabolism in glucose-6-phosphate dehydrogenase deficient erythrocytes and its relation to antimalarial chemotherapy

Experiments in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes parasitized by Plasmodium falciparum proved that depletion of glutathione increased fluxes of reactive oxygen species and was detrimental to the parasite at various sites and developmental stages. Chloroquine is also considered an inducer of oxidant damage due to its role in preventing heme polymerization. Recently it has been found that GSH prevents cellular damage by degrading the toxic heme. Consequently, we suggest that the use of combinations of chloroquine and depletors of GSH would be highly efficient for the chemotherapy of malaria.     

The level of sorbitol dehydrogenase activity in the cytoplasm of mammalian liver cells

A comparative study of sorbitol dehydrogenase activity in bovine, calf, and rat liver cell cytoplasm has been carried out. The level of activity of the enzyme is several times greater than that of marker enzymes (alcohol dehydrogenase, glucose-6-phosphate dehydrogenase). The data obtained suggest that the polyol (sorbitol) metabolism pathway of glucose functions actively in mammalian liver cells.     

Effectors of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

Summary The lower Vmax of 6PGDH with respect to G6PDH and its higher sensitivity to inhibition by NADPH, suggest the existence of an imbalance between the two dehydrogenases of the pentose phosphate pathway in rat liver. Possible modulators of these activities, particularly in relation with the inhibition by NADPH in physiological conditions, have been investigated. The results suggest that in both cases the inhibition by NADPH is strictly isosteric and that the relative affinities for the reduced and oxidized forms of the pyridine nucleotide are unaffected by glutathion, the intermediates of the pentose phosphate shunt or some divalent ions.Abbreviations G6PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - 6PGDH 6-phosphogluconate dehydrogenase (EC 1.1.1.44) On leave from the Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.     

High activity of glucose-6-phosphate dehydrogenase in Kupffer cells isolated from rat liver

Summary Sinusoidal cells in the rat liver react intensively for G6DPH activity after appropriate incubation (Rieder et al. 1978). After isolation and purification of the sinusoidal Kupffer and endothelial cells, it was demonstrated that Kupffer cells exhibit a 5–8 times higher G6PDH activity on a per cell basis by comparison with endothelial cells, while the specific G6PDH activity was 3–4 times higher in Kupffer cells. The Kupffer cells can be divided into two groups which differ significantly in G6PDH activity calculated on a per cell basis. In histochemical studies, G6PDH can be used as a marker for Kupffer cell identification.     

Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP⁺, with K_m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K_m of 0.31 mM. The NADP⁺ double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K_m and V_max values with increasing NADP⁺, denoting negative cooperativity. The two K_m's for high and low NADP⁺ concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP⁺ binding sites.     

The suitability of saliva for detection of glucose-6-phosphate dehydrogenase deficiency

Saliva was investigated for its suitability as a biopsy tissue for the determination of glucose-6-phosphate dehydrogenase deficiency. It appears that there is a significant difference between the activity of the enzyme in patients and controls. However, some controls have very low values making discrimination between patients and controls using a qualitative method impossible.Glucose-6-phosphate dehydrogenase deficiency is a relevant clinical problem in many rural areas in developing countries. Existing methods for determination of the deficiency in blood and hair follicles do not meet the criteria necessary for their large scale introduction in the areas of the world that are concerned by the problem. The present study shows that saliva is not a suitable alternative.Between the three biopsy tissues compared: blood, hair follicles and saliva, hair follicles remain most attractive since their isolation hardly involves the risk of infection. A simplified method for the detection of glucose-6-phosphate dehydrogenase activity in hair follicles that would allow health service workers in the field to determine the carrier status of pregnant women might form the basis for a future kernicterus prevention programme.     

Sex-linked changes in immunoreactive glucose-6-phosphate dehydrogenase in rat liver

The level of hepatic immunoreactive glucose-6-phosphate dehydrogenase protein was found to correlate well with the enzyme activity in adult rats fed the stock laboratory diet in a variety of hormonal conditions. The amount of immunoreactive protein and enzyme activity was 2-fold greater in sexually mature female rats compared with aged matched male animals. However, this difference was absent in diabetic animals, and furthermore although triiodothyronine administration to the diabetic male rat could restore the level of enzyme activity to that of the normoglycaemic animal, it was much less effective in the female animal. In contrast, administration of insulin to the normoglycaemic animal increased the level of glucose-6-phosphate dehydrogenase in the female, but was without effect in the male. These results are discussed in relation to the possible role of thyroid status and steroid sex hormones in the regulation of hepatic glucose-6-phosphate dehydrogenase.     

Dog liver glucose-6-phosphate dehydrogenase: purification and kinetic properties

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in liver. G6PD was purified from dog liver with a specific activity of 130 U x mg(-1) and a yield of 18%. PAGE showed two bands on protein staining; only the slower moving band had G6PD activity. The observation of one band on SDS/PAGE with M(r) of 52.5 kDa suggested the faster moving band on native protein staining was the monomeric form of the enzyme.Dog liver G6PD had a pH optimum of 7.8. The activation energy, activation enthalpy, and Q10, for the enzymatic reaction were calculated to be 8.96, 8.34 kcal x mol(-1), and 1.62, respectively.The enzyme obeyed "Rapid Equilibrium Random Bi Bi" kinetic model with Km values of 122 +/- 18 microM for glucose-6-phosphate (G6P) and 10 +/- 1 microM for NADP. G6P and 2-deoxyglucose-6-phosphate were used with catalytic efficiencies (kcat/Km) of 1.86 x 10(6) and 5.55 x 10(6) M(-1) x s(-1), respectively. The intrinsic Km value for 2-deoxyglucose-6-phosphate was 24 +/- 4mM. Deamino-NADP (d-NADP) could replace NADP as coenzyme. With G6P as cosubstrate, Km d-ANADP was 23 +/- 3mM; Km for G6P remained the same as with NADP as coenzyme (122 +/- 18 microM). The catalytic efficiencies of NADP and d-ANADP (G6P as substrate) were 2.28 x 10(7) and 6.76 x 10(6) M(-1) x s(-1), respectively. Dog liver G6PD was inhibited competitively by NADPH (K(i)=12.0 +/- 7.0 microM). Low K(i) indicates tight enzyme:NADPH binding and the importance of NADPH in the regulation of the pentose phosphate pathway.     

Physicochemical characteristics of sorbitol dehydrogenase from the cytoplasm of the liver cells of susliks

Some physico-chemical properties of sorbitol dehydrogenase from squirrel liver cell cytoplasm have been investigated. Non-linear dependence of enzyme activity upon media pH is shown. Activity manifests only in the presence of NAD that can't be replaced by NADP. The enzyme exhibits stereospecificity: it dehydrates polyol of any length, the second and the fourth carbon atoms have common L-configuration concerning the first carbon atom. A diffusion zone with Rf = 0,09 is revealed on the electrophoregram. Three thin zones of activity about pH 7,2 are exposed by isoelectrofocusing method.     

Preliminary crystallographic study of glucose-6-phosphate dehydrogenase from rat liver

Crystals of D-glucose-6-phosphate: NADP+ oxidoreductase were obtained with the hanging drop, vapor diffusion and batch methods from ammonium sulfate-containing solutions. X-ray diffraction photographs indicate that the crystals belong to the orthorhombic space groups I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 66.0 A, b = 140.8 A and c = 177.8 A. These data, together with results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crystal density experiments, indicate that there is one 116,000 Mr dimer per asymmetric unit. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination.     

Specific activities of 6-phosphogluconate dehydrogenase,glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase during Bufo bufo development

It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.