Regulation of glucose 6-phosphate dehydrogenase in blue-green algae

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) has been partially purified from Anacystis nidulans and Anabaena flos-aquae by means of ammonium sulfate fractionation and exclusion gel chromatography and the kinetic properties determined.     

Identification of HeLa cell glucose 6-phosphate dehydrogenase

Although the electrophoretic mobility of HeLa G6PD is similar to that of the common Negro variant G6PD A+, several reports have suggested slight differences between HeLa G6PD and G6PD A+. This study, carried out using the pure homogeneous B+, A+, and HeLa G6PD, showed that (1) the electrophoretic mobility of HeLa G6PD is identical to that of G6PD A+, (2) the enzymatic properties and thermostability of HeLa G6PD are indistinguishable from those of G6PD A+, and (3) the peptide map of the tryptic digest of HeLa G6PD is identical to that of G6PD A+, with one peptide spot of HeLa G6PD different from the corresponding spot of G6PD B+. These results indicate that the structure of HeLa G6PD is identical to that of G6PD A+, and that the amino acid substitution in HeLa G6PD is from one asparagine residue in the wild-type G6PD B+ to an aspartic acid residue in HeLa G6PD.This research was supported by research grant GM 15253 from the National Institutes of Health.     

Regulation of glucose 6-phosphate dehydrogenase in Zymomonas mobilis CP4

Abstract Glucose 6-phosphate dehydrogenase was purified 29-fold from Zymomonas mobilis . The enzyme was active with both NAD and NADP. Phosphoenolpyruvate was found to be a negative allosteric effector and ATP inhibited the enzyme non-allosterically, at physiological concentrations.     

The ontogeny of phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in Japanese quails and chicken-quail hybrids

The ontogeny of phosphogluconate dehydrogenase in Japanese quails and chicken-quail hybrids was determined by vertical starch gel electrophoresis of pooled early embryos. The enzyme of maternal origin was found to be present at all stages of development. Paternal phosphogluconate dehydrogenase in pure quails was detected as early as the fourth hour of incubation, whereas that in chicken-quail hybrids was not observed until the eighth hour. Since embryonic development up to the day of hatching was generally retarded in chicken-quail hybrids when compared to pure quails, it was concluded that the embryonic gene coding for phosphogluconate dehydrogenase was expressed at the same stage of development in both quails and hybrids. The ontogeny of glucose 6-phosphate dehydrogenase in chicken-quail hybrids was compared to that of phosphogluconate dehydrogenase. The paternal form of both enzymes first appeared at the eighth hour of incubation, indicating that these two functionally related genes coding for the corresponding enzymes were probably activated at the same time in development.     

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.     

Isoforms of glucose 6-phosphate dehydrogenase in Deinococcus radiophilus

Glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in Deinococcus radiophilus, an extraordinarily UV-resistant bacterium, was investigated to gain insight into its resistance as it was shown to be involved in a scavenging system of superoxide (O2-1) and peroxide (O2-2) generated by UV and oxidative stresses. D. radiophilus possesses two G6PDH isoforms: G6PDH-1 and G6PDH-2, both showing dual coenzyme specificity for NAD and NADP. Both enzymes were detected throughout the growth phase; however, the substantial increase in G6PDH-1 observed at stationary phase or as the results of external oxidative stress indicates that this enzyme is inducible under stressful environmental conditions. The G6PDH-1 and G6PDH-2 were purified 122- and 44-fold (using NADP as cofactor), respectively. The purified G6PDH-1 and G6PDH-2 had the specific activity of 2,890 and 1,033 U/mg protein (using NADP as cofactor) and 3,078 and 1,076 U/mg protein (using NAD as cofactor), respectively. The isoforms also evidenced distinct structures; G6PDH-1 was a tetramer of 35 kDa subunits, whereas G6PDH-2 was a dimer of 60 kDa subunits. The pIs of G6PDH-1 and G6PDH-2 were 6.4 and 5.7, respectively. Both G6PDH-1 and G6PDH-2 were inhibited by both ATP and oleic acid, but G6PDH-1 was found to be more susceptible to oleic acid than G6PDH-2. The profound inhibition of both enzymes by beta-naphthoquinone-4-sulfonic acid suggests the involvement of lysine at their active sites. Cu2+ was a potent inhibitor to G6PDH-2, but a lesser degree to G6PDH-1. Both G6PDH-1 and G6PDH-2 showed an optimum activity at pH 8.0 and 30 degrees .     

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.     

Regulation of cytoplasmic microtubule complex organisation in somatic cell hybrids

Regulation of cytoplasmic microtubule complex (CMTC) organization was studied in cultured human fibroblasts and mouse macrophages by somatic cell fusion. The heterokaryons stained with antitubulin antibody had fibroblast-like CMTC even 72 hours after fusion. There was no change in CMTC pattern when more than one macrophage had fused with one fibroblast. However, the macrophage CMTC was expressed in heterokaryons when the former were located at the periphery of the heterokaryon. To evaluate the role of existing CMTC in determining the CMTC of heterokaryons, the heterokaryons were treated with nocodazole to depolymerize the CMTC and then allowed to recover. The resultant CMTC was fibroblast like.     

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.     

Purification and properties of Penicillium glucose 6-phosphate dehydrogenase

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000+/-10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP(+)- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP(+), protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The K(m) values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3x10(-5)m-NADP(+) and 1.6x10(-4)m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2x10(-5)m-NADP(+) and 2.5x10(-4)m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP(+) and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme-NADP(+)-6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ.mol(-1) (9.6 and 9.9kcal.mol(-1)) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5x10(-6)m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.