Structure and function of glucose-6-phosphate dehydrogenase-deficient variants in Chinese population

A systematic study on the structure and function of Glucose-6-phosphate dehydrogenase (G6PD) variations was carried out in China. A total of 155,879 participants were screened for G6PD deficiency by the G6PD/6PGD ratio method and 6,683 cases have been found. The prevalence of G6PD deficiency ranged from 0 to 17.4%. With informed consent, 1,004 cases from 11 ethnic-based groups were subjected to molecular analysis. Our results showed the followings: (1) The G6PD variants are consistent across traditional ethnic boundaries, but vary in frequencies across ethnic-based groups in Chinese population, (2) The G6PD variants in Chinese population are different from those in African, European, and Indian populations, (3) A novel G6PD-deficiency mutation, 274C→T, has been found, and (4) Denaturing high performance liquid chromatography is of great advantage to detecting G6PD-deficient mutations for diagnosis and genetic counseling. Moreover, functional analysis of the human G6PD variants showed the following: (1) The charge property, polarity, pK-radical and side-chain radical of the substituting amino acid have an effect on G6PD activity, (2) The G6PDArg459 and Arg463 play important roles in anchoring NADP⁺ to the catalytic domain to maintain the enzymatic activity, and (3) The sequence from codon 459 to the carboxyl terminal is essential for the enzymatic function.     

Three glucose 6-phosphate dehydrogenase variants found in Japan

Summary Three Japanese glucose 6-phosphate dehydrogenase (G6PD) variants were investigated. G6PD Mediterranean-like had markedly decreased activity, normal electrophoretic mobility, low Km G6P, low Km NADP, increased utilization of all three substrate analogues (2-deoxy-G6P, Gal-6P, and deamino-NADP) and slightly decreased heat stability and slightly biphasic pH curve. G6PD Ogori had markedly decreased activity, but otherwise normal characteristics. G6PD Hofu had moderately decreased activity, normal electrophoretic mobility, slightly reduced Km G6P, normal Km NADP, normal utilization of 2-deoxy-G6P and Gal-6P, but increased utilization of deamino-NADP and normal heat stability as well as normal pH curve.     

Glucose-6-phosphate dehydrogenase (G6PD) Guantánamo and G6PD Caujerí: Two new glucose-6-phosphate dehydrogenase-deficient variants found in Cuba

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was identified in two children who were studied because of hemolytic episodes. The electrophoretic and kinetic properties of the mutant enzymes allowed us to conclude that both of them were new variants. They were named G6PD Guantánamo and G6PD Caujerí.     

Further studies on the properties of glucose 6-phosphate dehydrogenase from the coccidium Eimeria stiedai (Protozoa).

1. Glucose 6-phosphate dehydrogenase from Eimeria stiedai does not reduce NAD or any of its analogs tested. It does reduce NADP and its thionicotinamide and 3-acetylpyridine analogs. 2. It will accept D-glucose as substrate, but not 2-deoxy-D-glucose, glucose 1-phosphate, or 2-deoxy-D-glucose 6-phosphate. 3. Its response to a number of compounds that activate or inhibit the enzyme from other organisms has been determined. 4. The molecular weight is ca. 240,000 by gel chromatography, and only one isoenzyme could be detected by disc electrophoresis. 5. The enzyme resists conditions that commonly cause dissociation to lighter weight active forms.     

Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes

Historically, it has been theorized that the oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular gluthione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired to catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H₂O₂-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H₂O₂ challenge in the G6PD-deficient cell whiel only decrease was observed in normal cells. Supplmentation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P < 0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP⁺ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ration in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.     

The same extra FokI cleavage site exists in glucose-6-phosphate dehydrogenase variants A(+) and A(-).

Two common variants of glucose-6-phosphate dehydrogenase (G6PD), i.e., A(+) and A(-), exist in blacks in high frequencies. The mutation of the A(+) gene is a single nucleotide transition, A/G in equilibrium Asp) in the G6PD protein and produces an additional FokI cleavage site of the mutation site. Thus, the FokI fragment types detected by the genomic clone that contain the mutation site differ in the normal B(+) DNA and the variant A(+) DNA. The FokI fragment type of the variant A(-) is the same as that of the A(+). Since A(+) and A(-) enzymes differ at the protein level, the A(-) gene was presumably evolved by stepwise mutations through the A(+) gene.     

Mutation analysis of glucose-6-phosphate dehydrogenase (G6PD) variants in Costa Rica

Summary Glucose-6-phosphate dehydrogenase (G6PD) deficiency has previously been reported among both the black and white populations of Costa Rica. All 28 G6PD A — samples were found to be of the common G6PD A-^376G/202Atype. A previously described mutation associated with nonspherocytic hemolytic anemia, G6PD Puerto Limón, was found to be due to a GA transition at nucleotide (nt) 1192, causing a glulys substitution. Mutations in this region of the G6PD molecule seem invariably to be associated with chronic hemolytic anemia. G6PD Santamaria had been described previously in two unrelated white subjects. We found that both did, indeed, have the same mutations. In this variant the AG substitution at nt 376 that is characteristic of G6PD A was present, but an AT mutation at nt 542, apparently superimposed on the ancient G6PD A mutation, resulted in an aspval substitution. Thus, the gain of a negative charge at amino acid 126 was counterbalanced by the loss of a charge at amino acid 181, giving rise to a variant with the G6PD A mutation but with normal electrophoretic mobility.     

Linkage analyses using biochemical variants in mice. II. Levulinate dehydratase and autosomal glucose 6-phosphate dehydrogenase

The level of hepatic -aminolevulinate dehydratase varies among inbred strains of mice and is regulated by codominant alleles at the Lv locus. Twenty-two inbred strains have been classified with respect to this locus. Lv is 5±2 recombination units from brown, b, in linkage group VIII. The locus for autosomal glucose 6-phosphate dehydrogenase (Gpd-1) has also been assigned to linkage group VIII and is 32±5 units from brown. The order of the loci is Lv-b-Gpd-1. Incidental note is made of linkage between the malic dehydrogenase (Mdh-1) and dilute (d) loci, linkage group II, with 10±3 % recombination between the two.Supported by the Roche Institute of Molecular Biology, Nutley, New Jersey, and by Public Health Service Research Grant CA-05873 from the National Cancer Institute.     

On the involvement of a glucose 6-phosphate transport system in the function of microsomal glucose 6-phosphatase

A model for microsomal glucose 6-phosphatase (EC 3.1.3.9) is presented. Glucose 6-phosphatase is postulated to be resultant of the coupling of two components of the microsomal membrane: 1) a glucose 6-phosphate - specific transport system which functions to shuttle the sugar phosphate from the cytoplasm to the lumen of the endoplasmic reticulum; and 2) a catalytic component, glucose-6-P phosphohydrolase, bound to the luminal surface of the membrane. A large body of existing data was shown to be consistent with this hypothesis. In particular, the model reconciles well-documented differences in the kinetic properties of the enzyme of untreated and modified microsomal preparations. Characteristic responses of the enzyme to changes in nutritional and hormonal states may be attributed to adaptations which alter the relative capacities of the transport and catalytic components.     

Novel pronounced reversible inhibition of phosphofructokinase, glucose 6-phosphate dehydrogenase, and phosphoglucose isomerase by hexacyanoferrate (II).

At pH 6.8, pig kidney phosphofructokinase (PFK) is inhibited 90% by 1 mm hexacyanoferrate(II), in a reaction mixture containing 0.2 mm fructose 6-phosphate (F-6-P) and 1 mm ATP. Glucose 6-phosphate dehydrogenase and phosphoglucose isomerase are inhibited 70% by 5 mm hexacyanoferrate(II), at a 0.2 mm concentration of their respective substrates. Unlike all previously reported inhibitions of glycolytic enzymes by hexacyanoferrate, this inhibition seems not to involve an oxidation of enzyme, substrate, or enzyme-substrate complex. It appears to be due to reversible binding of the hexacyanoferrate at, or near, the hexose phosphate binding site of each enzyme. These inhibition studies were carried out in 50 mm 2-mercaptoethanol, and spectral studies showed that these conditions ensured that all the hexacyanoferrate was in the reduced (II) state. The inhibition of PFK was competitive with respect to the substrate F-6-P. Some reaction between hexacyanoferrate(II) and the substrate could not be definitely ruled out, but such reactions cannot be the major basis for the inhibitions observed. Increasing the magnesium concentration did not overcome the PFK inhibition. For all three enzymes, addition of a high concentration of hexose phosphate substrate to an assay mixture containing highly inhibited enzyme resulted in removal of the inhibition. The inhibition was instantaneous, and there was no increase in inhibition with time of incubation with hexacyanoferrate(II). These results may provide an approach to active-site labeling of these three enzymes at their hexose phosphate binding sites. These results should also be of interest to other workers, especially those involved in oxidative phosphorylation studies, who use ferro- and ferricyanide as research tools. The effects from such experiments may, in some cases, be due to binding of these compounds at, or near, hexose phosphate binding sites in the system.     

Glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants and the PvuII polymorphism in Southern African populations

Summary Southern African Bantu-speaking negroid and San populations were examined with regard to the glucose-6-phosphate dehydrogenase (G6PD) PvuII restriction fragment length polymorphism (RFLP) showing alleles of 4kb and 1.6 kb, called Type 1 and Type 2, respectively. The standardized disequilibrium coefficient for the electrophoretic G6PD types and PvuII alleles in the Southern African population was 0.28. The molecular lesion causing the Gd^A mutation is the same in the San and Southern African negroid populations. Gd^A chromosomes are found in association with both the Type 1 and Type 2 alleles, whereas none of the 62 Gd^B chromosomes from the Southern African populations had the Type 2 allele. Five of the 44 Gd^B chromosomes studied in the American Black population had the Type 2 allele, indicating that the Gd^B allele in the two populations may have different origins. The presence of all 3 A^– deficiency mutations in the G6PD A gene, in a region where the ancestral population was thought to have predominantly G6PD B, may be explained by their origin in Africa after the divergence of the races.     

A new glucose 6-phosphate dehydrogenase variant (G6PD Thessaloniki) in a patient with idiopathic myelofibrosis

A new deficient glucose 6-phosphate dehydrogenase (G6PD) variant, G6PD Thessaloniki, which was found in the red blood cells of a 70-year-old woman who had idiopathic myelofibrosis, is described. G6PD Thessaloniki had a low Michaelis constant (Km) for G6P (20 microM), high Km for NADP (10.1 microM), normal pH optimum, reduced heat stability, decreased electrophoretic mobility (96-98% of the normal), increased 2-deoxy-G6P and decreased galactose 6-phosphate utilization. Several other enzymatic activities measured in the patient's red blood cells were normal. Studies of red blood cell survival and glucose utilization gave evidence of haemolysis caused by defective glucose utilization by the pentose phosphate pathway. The only son of the patient had normal G6PD in his red blood cells. In an attempt to investigate the origin of G6PD Thessaloniki, heat stability tests of G6PD extracted from the patient's skin have been performed.