Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Purification and characterization of mouse glucose 6-phosphate dehydrogenase

Summary Glucose-6-phosphate dehydrogenase was purified to homogeneity from testes and kidneys of the inbred strain of mice (DBA/2J) by a simple two-step affinity column procedure. This involved the sequential application of 8-(6-aminohexyl)-amino-AMP-and -2, 5-ADP-Sepharose columns and biospecific elution with NADP⁺ in both steps. The molecular and biochemical properties of the purified enzyme were studied in detail. These include the molecular weight determination, amino acid composition, steady-state kinetics, inactivation by high temperature, urea and iodoacetate, and immunology. The purified enzyme from mouse kidneys or testes was shown to be a tetramer with a molecular weight of 220,000. The enzyme is highly specific for glucose-6-phosphate, exhibits almost no activity with NAD⁺ as a coenzyme and is little inhibited by AMP or ATP. Michaelis constants for glucose-6-phosphate and NADP⁺ were determined to be 50 m and 10 m respectively. NADPH is a competitive inhibitor of NADP⁺ and has a K_i of 18 µm. Rabbit antisera against glucose-6-phosphate dehydrogenase were raised. The antisera also cross-react with the same enzyme from human and guinea pig.     

Sex differences in activity of glucose 6-phosphate dehydrogenase from cultured human fetal lung cells despite X-inactivation

In a previous report, it was noted that glucose 6-phosphate dehydrogenase (G6PD) specific activity was approximately 45% higher in fibroblasts cultured from female fetal lung than in fibroblasts from male fetal lung. This sex difference was nullified during the first postnatal weeks by an abrupt rise in G6PD activity in cultured male lung rather than by any changes in G6PD activity in cultured female lung. No sex differences for G6PD activity were found in fetal or postnatal cultured skin (Steele and Owens, 1973). In the present report, analysis of the G6PD phenotype of clones derived from skin and lung fibroblasts from a 14-week fetus heterozygous for the AB electrophoretic variants of G6PD indicates that in these fetal cells only one X chromosome is active. Therefore, the sex differences in the specific activity of G6PD in fetal lung cells cannot be attributed to lack of X-inactivation in the female but must result from yet undefined regulatory mechanisms operative in the male.This work was supported in part by HRSF of Pittsburgh Grant No. L-22, NIH GRS Grant No. 5-S01FR05507, National Cancer Institute Grant No. R01 CA12113, and NIH Grant No. HD 05465.     

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.     

Temporal relationship between glucose 6-phosphate dehydrogenase activity and DNA-synthesis

Summary Wounds have been inflicted in the skin of guinea-pigs. Measurements were made on the basal cells 39–54 h later, at different times of the day. It has been shown that there can be two peaks of mitosis, one about mid-day and the other about 22 h. Synthesis of DNA, measured by Feulgen microdensitometry, preceded mitotic activity. Marked changes were found in glucose 6-phosphate dehydrogenase activity, measured by quantitative cytochemistry and microdensitometry. The greatest activity preceded DNA-synthesis, indicating that pentose-shunt metabolic activity is involved in biosynthetic processes required for cell proliferation.     

Age-related changes of glucose-6-phosphate dehydrogenase activity in mouse oocytes

Summary Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured in follicular oocytes and in ovulated eggs of prepubertal, adult and aged mice. G6PDH activity in ovulated eggs was 60% of the activity in follicular oocytes in all age groups. The mean G6PDH activity was significantly higher in follicular oocytes of adult mice than in oocytes of both prepubertal and aged mice. In aged mice, the decreased mean activity in follicular oocytes as well as in ovulated eggs was mainly due to a high percentage of cells with extremely low activity (25 and 18%, respectively). The percentage of preovulatory oocytes with low activity in prepubertal mice was 9% and in adult mice 0.3%. For ovulated eggs these percentages were 0% for both prepubertal and adult mice. In every age group, all ovulated eggs showed a normal morphology. When ovulated eggs with extremely low G6PDH activity can still be fertilized, it can be questioned whether this loss of activity could cause disturbances in development of (preimplantation) embryos. Our findings emphasize the potentialities of investigating intact single oocytes for changes in enzyme activities, which could be applied as parameters for quality control of these cells.     

Effects of insulin on the adaptation of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in rat adipose tissue

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1. The effects of insulin on adipose tissue glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied.     

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.     

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.