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PubMed1980

Genetic heterogeneity of glucose 6-phosphate dehydrogenase deficiency in Sardinia.

Glucose 6-phosphate dehydrogenase (G6PD) activity was assayed quantitatively in red cells from 100 consecutive G6PD-deficient newborn male babies born in a city hospital in Sassari, Sardinia. In four cases G6PD activity was between 30% and 45% of normal: these appeared on electrophoresis to be identical with G6PD Seattle-like. In 65 cases G6PD activity ranged from 2% to 14% of normal, while in the remaining 31 samples no activity could be detected in crude hemolysates. G6PD was partiall purified from 39 samples having activity below 14% of normal (including 20 with zero activity). G6PD activity could now be determined in all, and it was fully characterized in nine samples. These were shown to belong to two distinct classes on grounds of the Michaelis constant for glucose 6-phosphate and the elution profile from DEAE-Sephadex columns. These properties were compared with those of G6PD-deficient samples from Greece and from Israel. We conclude that there are at least three polymorphic G6PD-deficient variants in Northern Sardinia: G6PD Seattle-like, G6PD Mediterranean, and a new variant, which we designate G6PD Sassari. The GdMediterranean and GdSassari genes have been shown to breed true in family studies. We also produce evidence that the definition of G6PD Mediterranean must be carefully reassessed.

Genes↗
PubMedDate not supplied

Distinct phenotypic expression of two de novo missense mutations affecting the dimer interface of glucose-6-phosphate dehydrogenase.

Mutations encoding class I glucose-6-phosphate dehydrogenase (G6PD) variants are associated with chronic nonspherocytic hemolytic anemia (CNSHA), the most severe phenotypic expression of G6PD deficiency. These mutations frequently affect the G6PD dimer interface that is essential for enzymatic activity. We detected two de novo missense mutations concerning residues located close together in the dimer interface in two patients with severe G6PD deficiency. A novel c.1225C>T missense mutation was identified in a male neonate who presented with hemolysis and severe hyperbilirubinemia and the predicted Pro409Ser substitution constituted a novel class I variant, designated G6PD Utrecht. G6PD deficiency in the second patient was due to the once previously reported class I variant G6PD Sumaré (Val431Gly). Structural analysis revealed that the mutated residues Pro409 and Val431, located on different subunits, interact directly across the subunit interface and perturb formation of the G6PD dimer upon mutation. Favism and mild chronic hemolysis characterized the phenotype of the patient with G6PD Sumaré which contrasts with the more severe clinical picture of the patient with G6PD Utrecht and, in addition, that of the patient originally described with G6PD Sumaré. We postulate that this G6PD variant is at the crossing between class I and class II G6PD deficiency and its ultimate phenotypic expression is either aggravated or ameliorated by other (extra)genetic factors.

Binding Sites↗
PubMed2004

An update on the prevalence of glucose-6-phosphate dehydrogenase deficiency and neonatal jaundice in Tehran neonates.

OBJECTIVES: The aim of this study was to screen newborns in Tehran for glucose-6-phosphate dehydrogenase (G6PD) deficiency in relation to hyperbilirubinemia and jaundice. DESIGN AND METHODS: We performed quantitative and qualitative red blood cell (RBC) G6PD assays in cord blood of 2000 male and female at-term neonates. Observations for jaundice and bilirubin determination were made in G6PD-deficient and normal groups. Those with severe jaundice were treated with phototherapy or exchange transfusion. RESULTS: Our results showed that 2.1% (3.6% of males and 0.6% of females) was G6PD-deficient. Those with severe jaundice and hyperbilirubinemia (160 normal and 17 G6PD-deficient) were hospitalized and treated with phototherapy or exchange transfusion. Bilirubin levels in G6PD-deficient neonates were somewhat higher compared to G6PD-normal babies (18.8 +/- 2.4 mg/dl [321.5 +/- 41 micromol/l] vs. 15.7 +/- 3.2 mg/dl [268.5 +/- 54.7 micromol/l]; P < 0.05). G6PD activity was significantly lower in G6PD-deficient group than in the normal group (2.1 +/- 0.7 vs. 12.5 +/- 5.0 U/g Hb; P < 0.001). CONCLUSION: This study shows that the incidence of G6PD deficiency in newborns of Tehran is 2.1%, which is relatively high, and also hyperbilirubinemia and jaundice are approximately 3-fold higher in G6PD-deficient group than in the G6PD-normal group (51% vs. 16%). This emphasizes the necessity of neonatal screening on cord blood samples of both sexes for G6PD deficiency and the need to watch closely for development of hyperbilirubinemia.

Chi-Square Distribution↗
PubMed1991

Regulation of glucose-6-phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes.

Conditions were identified which, for the first time, demonstrate that primary hepatocytes can express the same range of glucose-6-phosphate dehydrogenase (G6PD) synthesis and mRNA as in live rats. Primary hepatocytes were cultured without prior exposure to serum, hormones or carbohydrates. Five modulators implicated in G6PD induction in vivo were examined: insulin, dexamethasone, tri-iodothyronine (T3), glucose and fructose, T3 did not affect G6PD activity, and did not interact with carbohydrate to affect the activity of G6PD. Neither glucose nor fructose alone affected G6PD activity, and they did not interact with insulin to increase G6PD activity. Hepatocytes isolated from fasted rats and cultured in serum-free media with amino acids ad the only energy source how a 12-fold increase in G6PD synthesis and mRNA (measured by a solution-hybridization assay). This induction does not require added hormones or carbohydrate. The addition of insulin alone caused another increase in G6PD synthesis and mRNA. There are at least three distinct phases to G6PD induction under these conditions. The largest increase in G6PD synthesis (12-fold) occurs in the absence of any hormones and with amino acids as the only energy source. This phase is due to increased G6PD mRNA. Insulin causes an additional 2-3-fold increase in G6PD synthesis and mRNA. However, dexamethasone and insulin are both required before G6PD synthesis is equal to that in rats which are fasted and refed on a high-carbohydrate diet.

Animals↗
PubMed1988

Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-).

Glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) A(-) is a common variant in Blacks that causes sensitivity to drug-and infection-induced hemolytic anemia. A cDNA library was constructed from Epstein-Barr virus-transformed lymphoblastoid cells from a male who was G6PD A(-). One of four cDNA clones isolated contained a sequence not found in the other clones nor in the published cDNA sequence. Consisting of 138 bases and coding 46 amino acids, this segment of cDNA apparently is derived from the alternative splicing involving the 3' end of intron 7. Comparison of the remaining sequences of these clones with the published sequence revealed three nucleotide substitutions: C33----G, G202----A, and A376----G. Each change produces a new restriction site. Genomic DNA from five G6PD A(-) individuals was amplified by the polymerase chain reaction. The base substitution at position 376, identical to the substitution that has been reported in G6PD A(+), was present in all G6PD A(-) samples and none of the control G6PD B(+) samples examined. The substitution at position 202 was found in four of the five G6PD A(-) samples and no normal control sample. At position 33 guanine was found in all G6PD A(-) samples and seven G6PD B(+) control samples and is, presumably, the usual nucleotide found at this position. The finding of the same mutation in G6PD A(-) as is found in G6PD A(+) strongly suggests that the G6PD A(-) mutation arose in an individual with G6PD A(+), adding another mutation that causes the in vivo instability of this enzyme protein.

Amino Acid Sequence↗
PubMed2002

Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal.

Mouse chimeras from embryonic stem cells in which the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene had been targeted were crossed with normal females. First-generation (F(1)) G6PD(+/-) heterozygotes born from this cross were essentially normal; analysis of their tissues demonstrated strong selection for cells with the targeted G6PD allele on the inactive X chromosome. When these F(1) G6PD(+/-) females were bred to normal males, only normal G6PD mice were born, because: (i) hemizygous G6PD(-) male embryos died by E10.5 and their development was arrested from E7.5, the time of onset of blood circulation; (ii) heterozygous G6PD(+/-) females showed abnormalities from E8.5, and died by E11.5; and (iii) severe pathological changes were present in the placenta of both G6PD(-) and G6PD(+/-) embryos. Thus, G6PD is not indispensable for early embryo development; however, severe G6PD deficiency in the extraembryonic tissues (consequent on selective inactivation of the normal paternal G6PD allele) impairs the development of the placenta and causes death of the embryo. Most importantly, G6PD is indispensable for survival when the embryo is exposed to oxygen through its blood supply.

Alleles↗
PubMed1980

Interaction between the glucose-6-phosphate dehydrogenase deficiency and thalassaemia genes at phenotype level.

No significant differences were observed in the mean values of Hb A2 levels and red cell indices between G6PD- and G6PD+ beta thalassaemia carriers apart from the MCV, which was significantly higher in beta thalassaemia G6PD- subjects, but still in the thalassaemia carrier range. No difference was seen between G6PD+ and G6PD- alpha thalassaemia carriers. G6PD+ beta thalassaemia carriers show a significant increase in G6PD levels expressed as activity per g of Hb and to lesser extent as activity per number of red cells x 10(9); in G6PD+ alpha thalassaemia carriers this increase is statistically significant only when the enzyme levels are expressed as activity per g of Hb. G6PD- beta thalassaemia carriers had enzyme levels higher than non-thalassaemic G6PD- subjects only when the activity is expressed per g of Hb. G6PD activity was found to be increased in G6PD+ and G6PD- Hb H disease patients.

Erythrocyte Count↗
PubMed2006

Incidence and complete molecular characterization of glucose-6-phosphate dehydrogenase deficiency in the Guangxi Zhuang autonomous region of southern China: description of four novel mutations.

BACKGROUND AND OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human metabolic disorder in southern China. We investigated the incidence and distribution of mutations, the molecular pathology of affected females and the haplotype association with G6PD deficiency in patients from the Guangxi region. DESIGN AND METHODS: A population-based molecular analysis combining phenotypic screening and genotypic detection using both multiplex primer extension/denaturing high performance liquid chromatography assay and DNA sequence analysis were performed in a total of 4,704 individuals. RESULTS: The mutation frequency of male G6PD-deficient individuals was observed to be 7.43%. Twenty-seven genotypes from 361 individuals were found. Statistical analysis showed that there were significant differences in both the percentages of methemoglobin and the G6PD/6PGD ratio between heterozygote and hemizygote in males and between heterozygote and homozygote in females. However, no statistically significance was seen between hemizyotes and homozygotes. The mutation profile showed that five mutations, G6PD Kaiping(1388A), G6PD Canton(1376T), G6PD Gaohe(95G), Chinese-5(1024T)and G6PD Viangchan(817A), are the most common in the area, accounting for 85% of the G6PD-deficient alleles. Ten rare mutations were detected in approximately 4% of the mutant chromosomes. Four novel mutations were found: G6PD Liuzhou(442A), G6PD Nanning(703T), G6PD Laibin(1414C,) and G6PD Hechi(202A/817A). In addition, two other rare mutations, c.196T-->A and c.202 G-->A, were detected for the first time in Chinese patients. A single dominant haplotype (- - + - -) was observed in 94.0% of 182 deficient chromosomes. INTERPRETATION AND CONCLUSIONS: Our protocol could be used to extend the knowledge of molecular defects of G6PD gene in different geographical areas.

Adult↗
PubMed1991

Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity.

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.

Animals↗
PubMed1989

Molecular heterogeneity of glucose-6-phosphate dehydrogenase A-.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is probably the most common disease-producing genetic polymorphism of humans. Virtually all G6PD-deficient Africans show the G6PD A- phenotype, an electrophoretically rapid, deficient enzyme. The recently acquired ability to identify the point mutations producing the different variants has given us new insights into the population genetics of G6PD variants. Twenty-nine males with the G6PD A- phenotype were studied. They were of African, Mexican, Spanish, and US white ethnic origin. All had the A---G transition at nucleotide 376 characteristic of G6PD A. In each case, one of three additional mutations was present, at nucleotides 202, 680, or 968. That in this population second mutations producing G6PD deficiency occurred only on the genetic background of G6PD A suggests that G6PD A was at one time the most common type of G6PD in Africa. However, the nucleotide sequence of the chimpanzee (Pan troglodytes) G6PD indicates that the primordial human type of G6PD was G6PD B.

Base Sequence↗
PubMed1986

Pathways for the reduction of oxidized glutathione in the Plasmodium falciparum-infected erythrocyte: can parasite enzymes replace host red cell glucose-6-phosphate dehydrogenase?

Plasmodium falciparum-infected human red cells possess at least two pathways for the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH): (1) the glucose-6-phosphate dehydrogenase (G6PD) pathway and (2) the glutamate dehydrogenase (GD) pathway using glutamate as a substrate. Uninfected erythrocytes lack the GD pathway. The NADPH generated can be used to reduce oxidized glutathione (GSSG), which accumulates in the presence of an oxidative stress. In red cell G6PD deficiency, this pathway is reduced or absent, and the host cells as well as the parasites within them are vulnerable to oxidant stress. In view of the presence of the GD pathway in parasitized red cells and the recent description of a parasite-derived G6PD enzyme, we have asked whether the pathways for the reduction of GSSG provided by the parasite can substitute for the host G6PD in red cells deficient in G6PD activity. We have devised a functional assay in which the reduction rate of GSSG is monitored in the presence of buffered infected or control red cell lysates and substrates. Infected G6PD-deficient erythrocytes were obtained from in vitro cultures after a single prior growth cycle of the parasites in G6PD deficient cells to eliminate contaminating normal red cells. The results show that only parasitized red cells can reduce GSSG via the GD pathway. In parasitized G6PD Mediterranean red cells (completely G6PD-deficient), there is a detectable GSSG reduction via the G6PD pathway, not found in uninfected lysates from the same individual. In G6PD A- (African type, featuring partial deficiency), a small increment in the G6PD-dependent reduction of GSSG can also be detected. However, when compared to G6PD normal red cells, the activities from the parasite-derived pathways are small and could not be considered substitutes for normal host enzyme activity. It is concluded that while the plasmodium provides additional pathways for the generation of NADPH that may serve its own metabolic needs, the host red cells and hence the parasite itself remain vulnerable to oxidant stress.

Erythrocytes↗
PubMed1987

Glucose-6-phosphate dehydrogenase of malaria parasite Plasmodium falciparum.

Plasmodium falciparum growth is impaired in glucose-6-phosphate dehydrogenase (G6PD)-deficient red blood cells (RBCs), and malaria has been implicated in the spreading of deficient variants in malaria-endemic areas. Recent reports suggest that the malaria parasite can adapt itself to grow in these variant RBCs by producing its own G6PD, but studies on parasite G6PD are very limited. In this report, we define the properties of the parasite G6PD. G6PD was partially purified from infected and uninfected variant RBCs associated with severe G6PD deficiency. G6PD from infected RBCs contained two components separable by starch gel electrophoresis: a major component (approximately 90% activity) with a very slow anodal electrophoretic mobility and a minor component (approximately 10% activity) with the same mobility as the host G6PD. Parasite G6PD exhibited much higher affinity (low Km) to G6P and nicotinamide-adenine dinucleotide phosphate (NADP) than did human G6PD. Southern blot hybridization indicated that the parasite genome contained nucleotide sequences that were hybridizable with the human G6PD cDNA. These data indicate that the parasite is capable of adapting to G6PD-deficient RBCs by producing its own G6PD.

Collodion↗
PubMed1989

Role of glucose-6-phosphate dehydrogenase on enhanced proliferation of pre-neoplastic and neoplastic cells in rat liver induced by N-nitrosomorpholine.

The relationship between G6PD and the number and size of pre-neoplastic and neoplastic lesions in rat liver was examined. Male Sprague-Dawley rats were given drinking water containing 250 mg/l of NNM for 8 weeks. Pre-neoplastic and neoplastic lesions with positive staining of GT were classified histochemically into G6PD-positive and G6PD-negative lesions. At week 24, quantitative analysis showed that the incidence and number of G6PD-positive lesions were significantly higher than those of G6PD-negative lesions. Histologically, hepatocellular carcinomas were more frequent and larger in G6PD-positive lesions than in G6PD-negative ones. Statistical analysis of the quantitative data also showed that the number and volume of G6PD-positive hepatocellular carcinomas were significantly higher than in G6PD-negative carcinomas. The G6PD-positive pre-neoplastic lesions had a significantly higher labelling index than G6PD-negative ones. These findings revealed a considerable difference in the growth of these 2 populations of the lesions, and indicate that G6PD activity may be closely related to proliferation of the pre-neoplastic and neoplastic cells in the liver.

Animals↗
PubMed1997

Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse.

The X-chromosomal gene glucose-6-phosphate dehydrogenase (G6pd) is known to be expressed in most cell types of mammalian species. In the mouse, we have detected a novel gene, designated G6pd-2, encoding a G6PD isoenzyme. G6pd-2 does not contain introns and appears to represent a retroposed gene. This gene is uniquely transcribed in postmeiotic spermatogenic cells in which the X-encoded G6pd gene is not transcribed. Expression of the G6pd-2 sequence in a bacterial system showed that the encoded product is an active enzyme. Zymogramic analysis demonstrated that recombinant G6PD-2, but not recombinant G6PD-1 (the X-chromosome-encoded G6PD), formed tetramers under reducing conditions. Under the same conditions, G6PD tetramers were also found in extracts of spermatids and spermatozoa, indicating the presence of G6pd-2-encoded isoenzyme in these cell types. G6pd-2 is one of the very few known expressed retroposons encoding a functional protein, and the presence of this gene is probably related to X chromosome inactivation during spermatogenesis.

Amino Acid Sequence↗
PubMed1997

Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency in south-east Sicily.

In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in south-east Sicily, we have analysed the G6PD gene in 25 unrelated males with abnormal G6PD activity and/or electrophoretic mobility, by using the analysis of the appropriate PCR-amplified fragment of DNA and subsequent digestion by appropriate restriction-enzymes, looking for the presence of certain known G6PD mutations. We amplified the entire G6PD coding sequence into eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis and sequencing of those individual fragments that were found to be abnormal by SSCP. Through these methods we found a total of twelve G6PD Mediterranean variants with the association of a silent mutation 1311 (also known as polymorphic site Bcl I), one G6PD Mediterranean without this association, four G6PD A-Val 68 and two G6PD Santamaria and five G6PD Chatham. In a subject with normal activity a mutation was found in exon 5, designated as G6PD Sao Borja. This is the first report on the molecular analysis of G6PD mutations in Sicily and we have obtained evidence for four distinct classes of variants.

Female↗
PubMed1988

Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

Amino Acid Sequence↗
PubMed1999

Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappaB activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD.

Antioxidants↗
PubMed2001

The unique cyanobacterial protein OpcA is an allosteric effector of glucose-6-phosphate dehydrogenase in Nostoc punctiforme ATCC 29133.

Glucose-6-phosphate dehydrogenase (G6PD), encoded by zwf, is essential for nitrogen fixation and dark heterotrophic growth of the cyanobacterium Nostoc punctiforme ATCC 29133. In N. punctiforme, zwf is part of a four-gene operon transcribed in the order fbp-tal-zwf-opcA. Genetic analyses indicated that opcA is required for G6PD activity. To define the role of opcA, the synthesis, aggregation state, and activity of G6PD in N. punctiforme strains expressing different amounts of G6PD and/or OpcA were examined. A single tetrameric form of G6PD was consistently observed for all strains, as well as for recombinant N. punctiforme His-G6PD purified from Escherichia coli, regardless of the quantity of OpcA present. However, His-G6PD and the G6PD of strain UCD 351, which lacks OpcA, had low affinities for glucose 6-phosphate (G6P) substrate (K(m)(app) = 65 and 85 mm, respectively) relative to wild-type N. punctiforme G6PD (K(m)(app) = 0.5 mm). Near wild-type affinities for G6P were observed for these enzymes when saturating amounts of His-OpcA- or OpcA-containing extract were added. Kinetic studies were consistent with OpcA acting as an allosteric activator of G6PD. A role in redox modulation of G6PD activity was also indicated, because thioredoxin-mediated inactivation and reactivation of His-G6PD occurred only when His-OpcA was present.

Allosteric Regulation↗