Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Do non-equilibrium reactions in the glucose-to-triose phosphate pathway exist in the liver?

The circadian changes in the contents of intermediates of the initial reactions of the glycolytic pathway in pigeon liver were studied. the concentrations of glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate and triose phosphates were found to change synchronously, being maximal at the dark time and minimal during the light daytime. The glycogen content in the liver decreased steadily between 12.00 and 09.00. The diurnal variations in the concentrations of metabolite pairs (glucose and glucose-6-phosphate, glucose-6-phosphate and fructose-6-phosphate, fructose-6-phosphate and fructose-1.6-diphosphate, fructose-1.6-diphosphate and triose phosphates) appeared to correlate significantly. The results obtained suggest that in the liver at least there are no limiting i. e. physiologically non-equilibrium reactions in the carbohydrate metabolic pathway from glucose to triose phosphates.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

Liver dehydrogenase levels in rainbow trout, Salmo gairdneri, fed cyclopropenoid fatty acids and aflatoxin B1

Cyclopropenoid fatty acids in the diet of rainbow trout caused significant reductions in liver protein and activity of glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. Changes in total activity were usually accompanied by similar changes in specific activity. The activity of glucose-6-phosphate dehydrogenase appeared to be more sensitive to the ingestion of cyclopropenoid fatty acids than the other dehydrogenases studied. Feeding 20 ppb aflatoxin B(1) to rainbow trout did not significantly change the activity of the dehydrogenases except for a small increase in the activity of glucose-6-phosphate dehydrogenase after 21 days of feeding. Relationships of these changes to the cocarcinogenicity of cyclopropenoid fatty acids and the carcinogenicity of aflatoxin are discussed.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular cloning of DNA sequences complementary to rat liver glucose-6-phosphate dehydrogenase mRNA. Nutritional regulation of mRNA levels

The nutritional regulation of rat liver glucose-6-phosphate dehydrogenase was studied using a cloned DNA complementary to glucose-6-phosphate dehydrogenase mRNA. The recombinant cDNA clones were isolated from a double-stranded cDNA library constructed from poly(A+) RNA immunoenriched for glucose-6-phosphate dehydrogenase mRNA. Immunoenrichment was accomplished by adsorption of polysomes with antibodies directed against glucose-6-phosphate dehydrogenase in conjunction with protein A-Sepharose and oligo(dT)-cellulose chromatography. Poly(A+) RNA encoding glucose-6-phosphate dehydrogenase was enriched approximately 20,000-fold using these procedures. Double-stranded cDNA was synthesized from the immunoenriched poly(A+) RNA and inserted into pBR322 using poly(dC)-poly(dG) tailing. Escherichia coli MC1061 was transformed, and colonies were screened for glucose-6-phosphate dehydrogenase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from clones which gave positive signals, and the identity of the glucose-6-phosphate dehydrogenase clones was verified by hybrid-selected translation. A collection of glucose-6-phosphate dehydrogenase cDNA plasmids with overlapping restriction maps was obtained. Northern blot analysis of rat liver poly(A+) RNA using nick-translated, 32P-labeled cDNA inserts revealed that the glucose-6-phosphate dehydrogenase mRNA is 2.3 kilobases in length. RNA blot analysis showed that refeeding fasted rats a high carbohydrate diet results in a 13-fold increase in the amount of hybridizable hepatic glucose-6-phosphate dehydrogenase mRNA which parallels the increase in enzyme activity. These results suggest that the nutritional regulation of hepatic glucose-6-phosphate dehydrogenase occurs at a pretranslational level.     

Levels of glucose dehydrogenases with different substrate specificities in rat and beef livers

The relative substrate specificities of glucose dehydrogenases (E.C. 1.1.1.47) from beef liver and rat liver are very different. The beef enzyme oxidizes glucose more rapidly than either glucose-6-phosphate or galactose-6-phosphate. On the other hand, the dehydrogenase from rat liver prefers the hexose phosphates to glucose.A procedure for estimating the level of glucose dehydrogenase in rat and beef liver is described. The glucose-6-phosphate dehydrogenase activity attributed to glucose dehydrogenases is estimated to be about one-fifth and one-third that of cytoplasmic glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) in female and male rat liver respectively.A fluorometric adaptation of the less sensitive spectrophotometric assay for glucose dehydrogenase is described.     

A study of the in-vitro regulation of phosphoenolpyruvate carboxylase from the epidermis of Commelina communis by malate and glucose-6-phosphate

Phosphoenolpyruvate (PEP) carboxylase activity in epidermal extracts of Commelina communis has been compared in the presence of malate and glucose-6-phosphate. The activity of PEP carboxylase was inhibited by increasing malate concentrations at several substrate (PEP) concentrations and changes in both the apparent K _m (PEP) and V _max values in the presence of malate suggested the occurence of mixed-type inhibiton. In the presence of glucose-6-phosphate no increase in enzyme activity was observed, although there was a slight decrease in the K _m (PEP). However, glucose-6-phosphate appeared to alleviate the inhibition caused by malate. The possible implications of these properties in the control of malate production in guard cells is discussed.Abbreviations PEP phosphoenolpyruvate - Glc6P glucose-6-phosphate     

The effects of platinum complexes on seven enzymes.

The effects of K2PtCl4, cis-Pt(NH3)2Cl2, and trans-Pt(NH3)2Cl2 on the activities of glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, fructose-1,6-bisphosphate aldolase, catalase, tyrosinase, and peroxidase have been investigated. All of the enzymes which are thought to have essential sulfhydryl groups (glyceraldehyde-3-phosphate dehydrogenase, aldolase, and glucose-6-phosphate dehydrogenase) were significantly inhibited by K2PtCl4. The other four enzymes studied are not known to have essential sulfhydryl groups, and were not significantly affected by the Pt compounds under the conditions employed. Glyceraldehyde-3-phosphate dehydrogenase was the only enzyme inhibited by all three Pt compounds tested, with K2PtCl4 being the most effective and cis-Pt(NH3)2Cl2 the least effective inhibitor. Semilogarithmic plots of residual activity versus inhibition time indicated that the inhibition reactions were not simple first-order processes, except for the inhibition of glucose-6-phosphate dehydrogenase by K2PtCl4 which appeared to be first-order with respect to enzyme concentration.     

Energy coupling of the hexose phosphate transport system in Escherichia coli

The active transport of hexose phosphates in Escherichia coli was inhibited by many uncouplers or inhibitors of oxidative metabolism. Fluoride and the lipid soluble cation, triphenylmethylphosphonium, had little effect. The uninduced level of transport was sensitive to fluoride, but not to azide. After energy uncoupling of active transport, the cells could equilibrate their intracellular water with the glucose-6-phosphate in the medium and displayed exit counter-flow suggesting the existence of carrier-mediated transport in the energy-uncoupled cells. The uncoupled transport of glucose-6-phosphate was inhibited by fructose-6-phosphate; the uninduced level of glucose-6-phosphate transport was not inhibited by fructose-6-phosphate. After energy uncoupling, the influx had a low affinity suggesting that, unlike the transport of beta-galactosides, the energy coupling for the active transport of hexose phosphate involved a change in the affinity of influx.     

Enhanced proteolysis of glucose-6-phosphate dehydrogenase in the presence of palmitoyl coenzyme A

Palmitoyl coenzyme A at concentrations below its critical micelle concentration increases the rate of proteolysis of baker's yeast glucose-6-phosphate dehydrogenase by proteinase A in the pH range 4-5. both glucose-6-phosphate and NADP protect glucose-6-phosphate dehydrogenase against proteolysis, but these protective effects are diminished in the presence of palmitoyl coenzyme A. Since palmitoyl coenzyme A is known to dissociate glucose-6-phosphate dehydrogenase into dimers, the results imply that the in vivo half life of glucose-6-phosphate dehydrogenase may be controlled by a process based on the regulation of the oligomeric structure of the enzyme by the collective actions of various molecules, including palmitoyl coenzyme A.     

The effect of wounding on glucose-6-phosphate dehydrogenase of Solanum tuberosum tuber tissue

Two forms of glucose-6-phosphate dehydrogenase were separated by disc electrophoresis of potato tuber extracts. The slower moving enzyme has a MW of 260 000 the faster one of 130 000. Wounding of potato tubers enhances the relative activity of the slower moving enzyme. Addition of NADP⁺ to the cathode buffer during electrophoresis has the same effect as wounding, whereas addition of glucose-6-phosphate has an opposite effect. The role of the wound induced increase of the pyridine nucleotide level in the interconversion of the two forms of glucose-6-phosphate dehydrogenase is discussed.     

Screening and Mutagenesis of Aspergillus niger for the Improvement of Glucose-6-Phosphate Dehydrogenase Production

The Aspergillus niger strain ZBY-7 was selected as the original strain of glucose-6-phosphate dehydrogenase production. After mutagenesis of the strain by means of UV irradiation and nitrosoguanidine, mutants of Aspergillus niger resistant to a certain metabolic inhibitor were obtained. Five of the mutants showed increased glucose-6-phosphate dehydrogenase production. The mutant resistant to antimycin A (Aspergillus niger AM-23) produced the highest level of glucose-6-phosphate dehydrogenase (695.9% of that produced by the original strain).     

Reversal of oxidative phosphorylation in submitochondrial particles using glucose 6-phosphate and hexokinase as an ATP regenerating system.

During steady-state, the Pi released in the medium is derived from glucose-6-phosphate which continuously regenerates the ATP hydrolyzed. A membrane potential (delta psi) can be built up in submitochondrial particles using glucose-6-phosphate and hexokinase as an ATP-regenerating system. The energy derived from the membrane potential thus formed, can be used to promote the energy-dependent transhydrogenation from NADH to NADP+ and the uphill electron transfer from succinate to NAD+. In spite of the large differences in the energies of hydrolysis of ATP (delta G degrees = -7.0 to -9.0 kcal/mol) and of glucose-6-phosphate (delta G degrees = -2.5 kcal/mol), the same ratio between Pi production and either NADPH or NADH formation were measured regardless of whether millimolar concentrations of ATP or a mixture of ADP, glucose-6-phosphate and hexokinase were used. Rat liver mitochondria were able to accumulate Ca2+ when incubated in a medium containing hexokinase, ADP and glucose-6-phosphate. The different reaction measured with the use of glucose-6-phosphate and hexokinase were inhibited by glucose concentrations varying from 0.2 to 2 mM. Glucose shifts the equilibrium of the reaction towards glucose-6-phosphate formation thus leading to a decrease of the ATP concentration in the medium.     

CHANGES IN ACTIVITY OF DGLUCOSE-6-PHOSPHATE: NADP AND 6-PHOSPHO-D-GLUCONATE: NADP OXIDOREDUCTASES IN RELATION TO LIGNIFICATION OF BAMBOO

D-Glucose-6-phosphate: NADP oxidoreductase (glucose-6-phosphatedehydrogenase; EC 1.1.1.49 [EC] ) and 6-phospho-D-gluconate: NADPoxidoreductase (6-phosphogluconate dehydrogenase; EC 1.1.1.44 [EC] )were found to be present in immature bamboo. Optimal pHs ofthe glucose-6-phosphate- and 6-phosphogluconate dehydrogenaseswere found to be 8.0 and 8.5, respectively. Both enzymes were demonstrated to be NADP-specific and NADPcould not be replaced by NAD. Fructose-6-phosphate was indirectlyutilized after convrsion to glucose-6-phosphate by glucose-6-phosphateisomerase coexisting in the enzyme preparation. Pattern of enzyme activity and of respiratory breakdown of glucose-1-¹⁴Cand glucose-6-¹⁴C were investigated in connection with lignificationof bamboo and discussed in comparison with sugar metabolismof fungi-infected plant tissues. As for the changes in the enzymeactivity with growth of bamboo, it was recognized that therewas a tendency that the activity of both enzymes increased andwas maintained at a certain level even in the aged tissues.In addition there was a drop of the C₆/C₁ ratio toward the tissuesof lower parts containing considerable amount of lignin andthis phenomenon was the same as that observed in pentose phosphatemetabolism of fungi-infected plant tissues. (Received September 5, 1966; )     

Involvement of cyclic AMP-dependent protein kinase on the phosphorylase kinase inhibition by glucose-6-phosphate in adipose tissue extracts

In order to achieve further clarification of the regulation of glycogenolysis in adipose tissue, we studied the effect of glucose-6-phosphate on phosphorylase activation in Sephadex G-25 filtrate of adipose tissue. The activity of phosphorylase kinase was decreased by 50% and by 75% in the presence of 0.5 mM and 2 mM of glucose-6-phosphate, respectively. This inhibition could be partially prevented by 0.5 mM AMP. Furthermore, we investigated the influence of glucose-6-phosphate on the effect of cyclic-AMP-dependent protein kinase on the activation of phosphorylase. The addition of cyclic-AMP and cyclic-AMP-dependent protein kinase caused a decrease in the inhibition of the phosphorylase activation by glucose-6-phosphate. Also, the glucose-6-phosphate at physiological concentration, decreased adipose tissue cyclic-AMP-dependent protein kinase activity.     

Role of lipids in regulation of microsomal glucose-6-phosphatase activity after modification of mcirosomes in vivo and in vitro. Effect of phospholipid effectors on the activity of glucose-6-phosphatase]

Modification of microsomal membranes in vivo and in vitro results in changes of the glucose-6-phosphate and inorganic pyrophosphate phosphohydrolase activities of liver microsomal glucose-6-phosphate phosphohydrolase (EC 3.1.3.9). It was demonstrated that the glucose-6-phosphate phosphohydrolase activity of glucose-6-phosphatase depends on the content of phosphatidylethanolamine in the microsomal membranes, whereas the inorganic pyrophosphate phosphohydrolase activity seems to be dependent on the phosphatidylserine content. It is assumed that the regulation of the corresponding enzyme activities by these phospholipids is performed by the same allosteric mechanism in vitro and in vivo.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

Differential effects of glucagon on induction of rat liver glucose-6-phosphate dehydrogenase by liver injury and dietary change.

Glucagon effectively prevented the increase in glucose-6-phosphate dehodrogenase activity of rat liver following the administration of a glucose-casein mixture without altering the amount of the diet consumed. However, the increase of the enzyme level in carbon tetrachloride-injured rat liver was virtually insensitive to glucagon. The results obtained gave further evidence for the difference between these two induction mechanisms of glucose-6-phosphate dehydrogenase.     

Catalases protect cellular proteins from oxidative modification in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae cells had higher antioxidant enzyme activities under growth in ethanol than that in glucose as a carbon and energy source. The correlations between catalase activity and protein carbonyl level (r(2)=0.857), between catalase and glucose-6-phosphate dehydrogenase activities (r(2)=0.924) and between protein carbonyl levels and glucose-6-phosphate dehydrogenase activity (r(2)=0.988) under growth in ethanol were found. Growing in ethanol the strain deficient in cytosolic and peroxisomal catalases had 7.1-fold higher level of carbonyl proteins than that of wild-type strain. Our data suggest that in vivo catalases may protect glucose-6-phosphate dehydrogenase against oxidative inactivation.