Glucose 6-phosphate dehydrogenase in Drosophila: A sex-influenced electrophoretic variant

A variant of glucose 6-phosphate dehydrogenase (G6PD) in Drosophila melanogaster shows different electrophoretic migration in males and females. In heterozygotes, the variant influences the migration of G6PD produced by both chromosomes. Mixing of homogenates of males and females changes migration of the female-produced enzyme, suggesting that a protein produced in males is capable of altering the variant G6PD molecule. The hypothetical protein is also present in pseudomales and intersexes produced by sex transformation genes.This research was supported by NIH grants # 5-T1-GM 216-06 and GM 12768-01 and NSF grants GB 4587 and GB 4824.     

Glucose 6-phosphate dehydrogenase in rainbow trout

Electrophoretic analysis of glucose 6-phosphate dehydrogenase from liver and blood of rainbow trout revealed a complex series of bands, which could differ between fish. The partial interconvertible nature of these bands was demonstrated with enzyme that had been incompletely inactivated at pH 8.4. In a single population of 40 fish, a homozygote and a heterozygote for an electrophoretic variant allele were found. We suggest that G6PD in rainbow trout liver and blood is determined by two alleles at a single locus, with posttranslational modification responsible for the complex electrophoretic patterns seen. The basis for this variation appears to be NADH binding to the protein molecule. Another variant and other properties of the enzyme are described.Supported in part by the State of California Department of Mental Hygiene and by USPHS Grants GM-15253, HD-04612, HD-00315, HD-05615, and HL-15125. One of us (S.D.C.) was a Special Postdoctoral Fellow of NIAMD (No. 1F03AM-40, 329-01) during part of this work.     

Glucose 6-phosphate dehydrogenase from sweet potato

Glucose 6-phosphate dehydrogenase was purified about 290-foldfrom sweet potato root tissue. The molecular weight was estimatedto be 110,000 by Bio-Gel 300 column chromatography. A LINEWEAVER-BURKplot of the reciprocal rate against reciprocal glucose 6-phosphateconcentration was concave downwards. A HILL coefficient lessthan 1 was obtained at lower concentrations of glucose 6-phosphate(below 0.5 mM). These results suggest that binding of glucose6-phosphate to the enzyme occurs with negative cooperativity. (Received April 30, 1970; )     

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from Lactobacillus casei: responses with different modulators

Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were separated and partially purified from glucose-grown cells of Lactobacillus casei. The enzymes had similar pH optima, thermosensitivity and molecular weights. They had different net charges and their pI values were 5.38 and 4.52, respectively. Histidine, arginine, lysine and cysteine residues were essential for the activity of G6PD, and all the above amino acids with the exception of lysine were required for 6PGD activity. Mg2+ activated 6PGD up to 15 mM concentration, above which it was inhibitory. It had no effect on G6PD activity. G6PD was specific for NADP+, but 6PGD showed some activity with NAD+ as the cofactor, although it was essentially NADP(+)-preferring. Both the enzymes, were inhibited by NADPH. 6PGD was also inhibited by its product, ribulose 5-phosphate. ATP inhibited 6PGD only at subsaturating concentrations of NADP+. The inhibition was sigmoidal in the absence of Mg2+ and hyperbolic in its presence.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

     

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.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in Drosophila melanogaster

Studies on two variants of X-linked enzyme, G6PD, in several inbred and outbred strains of Drosophila melanogaster suggest that (1) there is dosage compensation at this locus; (2) males have 20–33% more activity than females, due to enzyme-deficient eggs in the latter; (3) outcrossing Drosophila strains results in a significant rise in G6PD specific activity in such a way as to suggest the presence of two or more nonlinked loci specific in their effect on G6PD activity (the effect is twice as great in males as it is in females); (4) there is less A enzyme than B enzyme activity/mg protein in males, but they are equal in females; (5) the presence or absence of X-linked regulators for G6PD could not be ascertained.Aided by National Institutes of Health grants HD 00004, HD00486, and GM 14155.     

Glucose 6-phosphate dehydrogenase activity in the early rabbit and mouse embryo

Glucose 6-phosphate dehydrogenase (G6PD) activity was measured in individual preimplantation rabbit and mouse embryos. Substrate turnover by the enzyme is at least 30 times greater than glucose oxidation by the pentose shunt in the early rabbit embryo. There was no evidence during the preimplantation period of the embryos in either species of a bimodal distribution of G6PD activities among the embryos. Since cytological studies have not shown that inactivation of the X chromosome occurs during the early cleavage period and G6PD activity is sex-linked and gene-dose dependent in most higher animals, the evidence from the enzyme studies suggests that there is little or no synthesis of G6PD during the early preimplantation period. It is suggested that the enzyme is synthesized during oocyte development and the high levels of the enzyme found during the preimplantation period reflect the requirement of an earlier stage in oocyte development rather than the requirements of cleavage.Financial support for this work was obtained from National Institutes of Health Grants HD 03071 and HD 02315.     

Glucose 6-phosphate dehydrogenase deficiency and incidence of hematologic malignancy.

We have evaluated the hypothesis of a negative association between glucose 6-phosphate dehydrogenase (G6PD) deficiency and cancer in a cohort of 481 Sardinian males with hematological malignancies. The frequency of G6PD deficiency in the patients was not different from the incidence in a group of 16,219 controls. The same conclusion resulted from the comparison of the frequency of expression of the GdB gene in 23 heterozygous women having a clonal hematologic disease and a control group of 37 healthy heterozygotes. Therefore at present there is no evidence that G6PD deficiency has a protective effect against development of hematologic neoplasms.     

Glucose 6-phosphate dehydrogenase inhibition sensitizes melanoma cells to metformin treatment

Most cancer cells exacerbate the pentose phosphate pathway (PPP) to enhance biosynthetic precursors and antioxidant defenses. Metformin, which is used as a first-line oral drug for the treatment of type 2 diabetes, has been proposed to inhibit the malignant progression of different types of cancers. However, metformin has shown poor efficacy as single agent in several clinical trials. Thus, the aim of the present work was to investigate whether the pharmacological inhibition of G6PDH, the first and rate-limiting enzyme of the PPP, by 6-amino nicotinamide (6-AN) potentiates the antitumoral activity of metformin on different human melanoma cell lines. Our results showed that 6-AN has sensitizing properties to metformin cytotoxicity. The combination of metformin and 6-AN decreased glucose consumption and lactate production, altered the mitochondrial potential and redox balance, and thereby blocked melanoma cell progression, directing cells to apoptosis and necrosis. To our knowledge, this is the first study describing the effect of this combination. Future preclinical studies should be performed to reveal the biological relevance of this finding.     

Glucose 6-phosphate dehydrogenase isoenzymes in cultured human cell lines: Separation by isoelectric focusing

Isoelectric focusing in acrylamide gel slabs has been used to separate glucose 6-phosphate dehydrogenase isoenzymes in human cell lines. The pattern of four to six bands has been found to vary both in band position (isoelectric point) and in relative intensity. These differences can be used to characterize and distinguish different cell lines, both from different donors and from different tissues from the same donor. The method should provide a much-needed supplement to the limited number of techniques currently available for monitoring changes in cell cultures.