Maternal effect for genes encoding 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in Drosophila melanogaster

We studied the maternal effect for two enzymes of the pentose cycle, 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD), using a genetic system based on the interaction of Pgd^? and Zw^? alleles, which inactivate 6PGD and G6PD, respectively. The presence and formation of the enzymes was investigated in those individuals that had not received the corresponding genes from the mother. We revealed maternal forms of the enzymes, detectable up to the pupal stage. The activities of “maternal” 6PGD and G6PD per individual increased 20-fold to 30-fold from the egg stage to the 3rd larval instar even in the absence of normal Pgd and Zw genes. Immunologic studies have shown that the increase in 6PGD activity is due to an accumulation of the maternal form of the enzyme molecules. We revealed a hybrid isozyme resulting from an aggregation of the subunits of isozymes controlled by the genes of the mother and embryo itself. These results indicate that the maternal effect in the case of 6PGD is due to a long-lived stable mRNA transmitted with the egg cytoplasm and translated during the development of Drosophila melanogaster.     

A genetic and molecular analysis of P-induced mutations at the glucose-6-phosphate dehydrogenase locus in Drosophila melanogaster

Summary We examined P factor induced mutations of the Zw gene of Drosophila melanogaster in order to learn more about the site specificity of such mutations. Approximately 70000 chromosomes were screened using a powerful positive selection scheme. As only two mutants were discovered, Zw is a cold spot for transposable element insertion. One mutation involved a complex P element associated chromosomal rearrangement which was used to define the orientation of the gene with respect to the centromere of the X chromosome. The second mutation was either a simple, non-dysgenically induced point mutation or a very unstable insertion.     

X-linked glucose-6-phosphate dehydrogenase deficiency inMus musculus

A mouse with X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency has been recovered in offspring of 1-ethyl-1-nitrosourea-treated male mice. The activity alteration was detected in blood but can also be observed in other tissue extracts. Hemizygous, heterozygous, and homozygous mutants have, respectively, about 15, 60, and 15% G6PD remaining activity in the blood as compared to the wild type. Erythrocyte indices did not show differences between mutants and wild types. The mutation does not affect the electrophoretic migration, the isoelectric point, or the thermal stability. Kinetic properties, such as theK _m for glucose-6-phosphate or for NADP and the relative utilization of substrate analogues, showed no differences between wild types and mutants with the exception of the relative utilization of deamino-NADP which was significantly lower in mutants. This is presently the only animal model for X-linked G6PD deficiency in humans.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.     

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

Developmental variation in effects of the second and third chromosomes on the activities of the glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster

Developmental profiles of the second- and third-chromosome modifiers of the activities of glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in Drosophila melanogaster were investigated. Third-chromosome modifiers showed very strong effects on both enzyme activities at larval, pupal, and adult stages, whereas second-chromosome effects were detected mainly at larval and adult stages. For both enzyme activities and both chromosomes, the correlation over line means between larval and pupal stages was significantly positive, but the correlation between larval or pupal stage and adult stage was not significant. This result suggests that the actions of modifiers on G6PD and 6PGD activities are influenced by the change of developmental stages. Correlation between G6PD and 6PGD activities was positive and highly significant throughout the developmental stages for both sets of chromosomes, although third-chromosome correlations were slightly higher than second-chromosome correlations. The magnitude of the correlation between G6PD and 6PGD activities does not seem to be influenced by the change of development. Diallel crosses for both sets of chromosomes indicate that the action of activity modifiers is mainly additive for both sets of chromosomes, but dominance effects were detected in some cases in adult males. Significant maternal effects were detected for the third chromosome for both enzyme activities until the pupal stage. The change of the activity modifier action after emergence of the imago and the significant correlation between G6PD and 6PGD activities were also detected for diallel progeny.This work was supported by Public Health Service Grant NIH-GM11546.Paper No. 10211 of the journal series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695.     

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.     

6-Phosphogluconate dehydrogenase from Drosophila melanogaster. I. Purification and properties of the a isozyme

6-Phosphogluconate dehyrogenase is evident at all developmental stages of Drosophila melanogaster. The activity level is highest in early third instar larvae and declines to a lower, but relatively constant, level at all later stages of development. The enzyme is localized in the cytosolic portion of the cell. The A-isozymic form of 6-phosphogluconate dehydrogenase was purified to homogeneity and has a molecular weight of 105,000. The enzyme is a dimer consisting of subunits with molecular weights of 55,000 and 53,000. For the oxidative decarboxylation of 6-phosphogluconate the K_m for substrate is 81 µm while that for NADP⁺ is 22.3 µm. The optimum pH for activity is 7.8 while the optimum temperature is 37 C.This work was supported by National Research Council of Canada Grant A5860 and by the University of Calgary Research Policy and Grants Committee.     

Functional interpretation,cataloging, and analysis of 1,341 glucose-6-phosphate dehydrogenase variants

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Characterization of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase from hazel cotyledons

NADP reduction was shown to occur in a crude cytosolic extract from the cotyledonary material of hazel seed prior to the addition of erogenous dehydrogenase substrate. This activity interfered with the assay of glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities. The inherent NADP reduction was removed by ammonium sulphate fractionation. Subsequent de-salting of the resulting partially-purified fraction permitted assay of G6PDH and 6PGDH. Both enzymes were shown to be NADP specific. Typical Michaelis-Menten kinetics were shown for each enzyme, towards NADP and their respective substrate.     

Rabbit bone marrow glucose-6-phosphate dehydrogenase during erythroid cell development

Studies were carried out on glucose-6-phosphate dehydrogenase (G6P-DH) during the differentiation of rabbit bone marrow erythroid cells. It was found that G6P-DH, although displaying a 7-fold activity decrease, did not change the relative amounts of its three dimeric forms.Using homogeneous enzyme preparations, we observed that from dividing to non-dividing erythroblasts the following properties remained constant: V max dependence on pH and temperature, Km for G6P dependence on pH, heat stability, 2-deoxy glucose-6-phosphate utilization, molecular weight, while the Km for NADP significantly increased in non-dividing erythroblasts. These results indicate that no shift towards the oxidized form of the enzyme and no substantial modifications of the protein take place during cell differentiation.     

Effect of phenolic compounds on glucose-6-phosphate dehydrogenase isoenzymes

Effector studies with two isoenzymes (I and IV) of glucose-6-phosphate dehydrogenase (G6PDH) from tobacco suspension culture WR-132 revealed that chlorogenic acid, at 0.4 mM, inhibited both isoenzymes almost 100%, with the inhibition decreasing as the concentration of the acid was reduced. At 0.3 and 0.4 mM, the coumarin glucosides scopolin and esculin were inhibitory, whereas their aglucones scopoletin and esculetin were less inhibitory, and at low concentrations of glucose-6-phosphate (G6P), the latter two were actually stimulatory for G6PDH I. Of the possible effectors studied, only scopoletin and esculetin exhibited a significant activation of G6PDH I under these conditions. However, with G6PDH IV these two effectors do not show the same marked activation at the low G6P concentrations. The phenolic acids, caffeic and ferulic, were less inhibitory than the coumarins tested. The activation of G6PDH I by scopoletin, a compound which accumulates in tobacco under certain stress conditions, gives a possible clue as to the resulting enhanced activity of the hexose monophosphate pathway that has been reported for some plants subjected to stress conditions.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

Genetic variation in Cameroon: Thermostability variants of hemoglobin and of glucose-6-phosphate dehydrogenase

The technique of heat denaturation was used in addition to electrophoresis for the detection of thermostability variants of hemoglobin and glucose-6-phosphate dehydrogenase in an attempt to measure the amount of genetic variability present in villages in the United Republic of Cameroon, Equatorial Africa. A minimum of three to a maximum of 13 thermostability variants were estimated for HbA and HbS, and a minimum of two to a maximum of ten thermostability variants were estimated for GdA, GdB, and GdA —. It is suggested that hemoglobin and glucose-6-phosphate dehydrogenase thermostability variants are genetically determined and that the sites of these variants are at the hemoglobin and glucose-6-phosphate dehydrogenase structural loci. The evidence for the existence of these hidden variants and their importance in the neutralist v. selectionist controversy are discussed.This work was supported in part by National Institutes of Health Grant HL 16005. S. C. B. was an International Telephone and Telegraph International Fellow to Cameroon, was supported by Training Grant NIH-GM 07197, and is currently an Insurance Medical Scientist Scholar. This work is in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Genetics by S. C. B.     

Biochemical properties of esterase 6 in Drosophila melanogaster

Biochemical properties of esterase 6 in Drosophila melanogaster were investigated using partially purified preparations from three genotypes, 1/1, 1/2, and 2/2. The molecular weight of the enzyme is estimated to be about 90,000, and treatment with sodium dodecylsulfate cleaves the enzyme into four units with a molecular weight of about 22,000. The activity toward 28 naturally occurring esters was assayed and shown to vary considerably with substrate, the 1/1 preparation having in general higher activity than 1/2 and 2/2, which were very similar. Heat sensitivity, the effect of metal ions, and the effects of the presence or absence of an end product were also studied. The differences demonstrated between allozymes would allow considerable scope, under appropriate conditions, for differential selection to operate between genotypes.Supported in part by an SRC Research Studentship (N.D.D.).     

Eukaryotic glucose-6-phosphate dehydrogenases: Structural screening of related proteins

Rapid assessment of structural relationships between yeast glucose-6-phosphate dehydrogenases and other eukaryotic types of this enzyme is described. Separation and size estimation of large fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis, electroblotting onto disks, and sequencer analysis provide data that permit alignment of the segments thus characterized with the related proteins, and utilize existing structural knowledge to assess new enzyme structures. Affinity labeling allows further correlations. The results establish the overall structural arrangements of the new proteins, including the location of the active-site lysine residue, even though the yeast enzyme structures are found to differ markedly from the few previously characterized glucose-6-phosphate dehydrogenases.     

Cytochemical observations on glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and -glycerophosphate dehydrogenase in chick liver cell cultures infected with Trichomonas vaginalis

Cytochemical reactions specific for glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and α-glycero-phosphate dehydrogenase were observed in the epithelial cells and macrophages of chick liver cell cultures; α-glycerophosphate dehydrogenase activity was observed also in the fibroblasts. Distribution of three of the enzymes was limited to the cytoplasm, their activity being localized primarily in cytoplasmic inclusions. Weak staining of the nuclei and strong staining of the nucleoli occurred in addition to the cytoplasmic reaction in cells treated for glucose-6-phosphatase. In cell cultures inoculated with Trichomonas vaginalis, the activity of three of the enzymes decreased progressively in the course of infection, but that of α-glycerophosphate dehydrogenase increased.     

Regulation of specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in Penicillium chrysogenum

Abstract The specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase changed when Penicillium chrysogenum was grown on different carbon sources. In the presence of 2% lactose, the activities of these enzymes were approximately 25–35% lower than those in media containing 2% glucose or 2% fructose. We assume that an increase in cAMP concentration was responsible for the observed decreases in the enzyme activities, because a higher cAMP concentration could be detected when the mycelium was grown in a medium containing solely lactose as carbon source. The likely role played by cAMP in the regulation was also demonstrated by the addition of either cAMP or caffeine to the medium.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.