Accumulation Of Glucose 6-phosphate Research Articles

Glucose 6-phosphate transport in membrane vesicles isolated from Escherichia coli: effect of imposed electrical potential and pH gradient.

Imposition of a membrane potential (delta psi, interior negative) or a pH gradient (delta pH, interior alkaline) across the membrane of Escherichia coli DF2000 leads to a marked, transient increase in glucose 6-phosphate transport that varies systematically with pH. Outwardly directed potassium diffusion gradients in the presence of valinomycin (i.e., generation of delta psi, interior negative) drive glucose 6-phosphate transport at pH 7.5 but much less effectively at pH 5.5, although the magnitudes of the transient delta psi generated are comparable at both pH values. Similarly, imposition of delta psi (interior negative) retards the rate of passive, carrier-mediated glucose 6-phosphate efflux down a concentration gradient at pH 7.5 but not at pH 5.5. In contrast, imposition of delta pH (interior alkaline) by means of outwardly directed acetate diffusion gradients drives glucose 6-phosphate accumulation at pH 5.5 but is relatively ineffective at pH 7.5. The results are independent of the pK of glucose 6-phosphate and provide strong support for the argument that the glucose 6-phosphate porter catalyzes an electrically neutral reaction at acid pH and an electrogenic reaction at alkaline pH. In addition, they are entirely consistent with the hypothesis that the proton/glucose 6-phosphate stoichiometry increases at alkaline pH [Rottenberg, H. (1976) FEBS Lett. 66, 159; Ramos, S., & Kaback, H.R. (1977) Biochemistry 16, 854, 4271].

Computer simulation of metabolism in pyruvate-perfused rat heart. IV. Model behavior.

The behavior of a computer model of energy metabolism was determined for perfused rat hearts utilizing pyruvate as sole exogenous fuel and subjected to a rapid increase in work load. Computer-generated metabolite profiles, which are solutions of the differential equations for 1 min elapsed time, closely match 12 experimental curves (involving 120 concentration measurements) and exhibit the following properties. The computed cytosolic pyruvate level oscillates due to large changes in the rates of the processes that produce and consume this metabolite. Cytosolic Mg2+ seems to act as a coordinated controller of glycolytic enzymes; its transient increase permits a transient increase of glycolysis without an accumulation of glucose 6-phosphate. Lactate is exported to the interstitium by a lactate permease and then reimported and oxidized. As a result, the malate-aspartate shuttle reverses direction, and the Krebs cycle is "unspanned."

Genetical and Biochemical Studies of Glucosephosphate Isomerase Deficient Mutants in Saccharomyces cerevisiae

A number of glucose-negative mutants of Saccharomyces cerevisiae were isolated and shown to contain very low activities of glucosephosphate isomerase. Mutants almost totally lacking this enzyme (less than 1% of wild-type activity) grew on fructose if provided with a small quantity of glucose. Larger amounts of glucose led to the accumulation of glucose 6-phosphate and growth inhibition. These mutants did not grow on galactose. Other mutants with low enzyme activities (about 1% of wild type) grew on fructose alone and also on galactose. The mutant characters were determined in both cases by single gene mutations which were mapped on chromosome II and, presumably, identify a structural gene locus for the enzyme.

Inhibition of glycolysis in brain by a phospholipid effect on interconversion of fructose phosphates. A possible regulatory control on utilization of glucose 6-phosphate.

Glucose 6-phosphate accumulation in 10,000 X g supernatant of rat brain was enhanced up to 16-fold by the addition of phosphatidylcholine, other common phospholipids, or linoleate. This glucose 6-phosphate is of endogenous origin via UDP-glucose and glucose 1-phosphate but not glucose. The accumulation is the result of inhibition of glycolysis by an effect of phospholipid on the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate. Brain is therefore capable of gluconeogenesis from fructose 1,6-bisphosphate. A regulatory function for phospholipid which coordinates glycolysis and other major routes of utilization of glucose 6-phosphate in brain, e.g. inositol synthesis, is proposed.

Time Course of Early Events in the Action of Glucocorticoids on Rat Thymus Cells in Vitro: SYNTHESIS AND TURNOVER OF A HYPOTHETICAL CORTISOL-INDUCED PROTEIN INHIBITOR OF GLUCOSE METABOLISM AND OF A PRESUMED RIBONUCLEIC ACID

Abstract Previous work has shown that physiological amounts of cortisol added to rat thymus cells in vitro begin to inhibit glucose transport (as measured by the accumulation of glucose 6-phosphate during a 5-min glucose pulse) by 15 to 20 min; the effect of cortisol is blocked by actinomycin D present from 0 to 5 min, suggesting a rapid stimulation by cortisol of RNA synthesis. It is now shown that during the period from 15 to 30 min, but not prior to 15 min, the hormone effect is blocked by cycloheximide or puromycin. The coincidence in time of sensitivity to these inhibitors of protein synthesis with the development of inhibition of glucose transport, together with our earlier results, has led us to postulate that the hormone effect is expressed through induction of a protein that rapidly inhibits glucose transport. The preceding RNA-synthetic step possibly represents synthesis of mRNA for this protein. The previously demonstrated temperature-sensitive step is shown to lie between the RNA- and protein-synthetic steps. It perhaps corresponds to transfer of mRNA from nucleus to cytoplasm. Decay rates of the hypothetical cortisol-induced RNA and protein have been measured following removal of hormone from specific binding sites or addition of inhibitors of RNA or protein synthesis. The protein appears to have a life span of not more than 2 hours at 37°, while the RNA has a life span of less than 40 min.

Studies on the Uptake of Hexose Phosphates: II. THE INDUCTION OF THE GLUCOSE 6-PHOSPHATE TRANSPORT SYSTEM BY EXOGENOUS BUT NOT BY ENDOGENOUSLY FORMED GLUCOSE 6-PHOSPHATE

Abstract An Escherichia coli mutant, DF2000, accumulates up to 0.06 m glucose 6-phosphate when supplied glucose. This extremely large concentration of intracellular glucose 6-phosphate does not serve to induce the active transport of this ester. However, if the medium is then supplemented with only 5 x 10-4 m glucose 6-phosphate the transport system is induced. Thus, it appears that exogenous glucose 6-phosphate acts as an inducer but the endogenously formed ester is unable to do so. The ester accumulated by glucose-treated cells was isolated and purified. It proved to be an effective inducer of glucose 6-phosphate transport. The transport system for glucose 6-phosphate allows this ester to accumulate until the intracellular concentration is 20-fold greater than the concentration in the medium. When glucose-treated cells containing 0.06 m glucose 6-phosphate are induced for transport with 5 x 10-4 m ester the initial concentration ratio is 120:1. Consequently, glucose 6-phosphate exits from the newly induced cells until a concentration ratio of 20:1 is established. Accumulation of glucose 6-phosphate by cells of the mutant given glucose results in growth stasis. This toxic effect is reduced or prevented by inducing levels of glucose 6-phosphate in the medium.

Evidence for Irreversible, Actinomycin D-sensitive, and Temperature-sensitive Steps following Binding of Cortisol to Glucocorticoid Receptors and Preceding Effects on Glucose Metabolism in Rat Thymus Cells

Following cortisol binding to the specific glucocorticoid receptors of thymus cells at 37° (a process which takes about 7 min), there is a 5- to 10-min lag period before a cortisol effect can be observed on glucose 6-phosphate accumulation after a glucose pulse. Evidence is presented that during this period the signal initiated by cortisol binding traverses an irreversible, an actinomycin D-sensitive, and a temperature-sensitive step. The irreversible step is shown by the fact that removal of cortisol from the glucocorticoid receptors (by displacement with cortexolone, a metabolically inactive glucocorticoid competitor, or by washing) well before any metabolic effect has appeared does not prevent the subsequent appearance of the cortisol effect. The actinomycin D-sensitive step is shown by the fact that, whereas addition of actinomycin D to thymus cells together with cortisol prevents the cortisol effect from developing subsequently, addition of actinomycin D 5 min after cortisol does not prevent the cortisol effect. To produce these actions, actinomycin D must be used at 100 µg per ml. This same concentration is necessary to inhibit RNA synthesis rapidly in thymus cells by about 80%. The temperature-sensitive step is shown by the fact that the duration of the lag period preceding the appearance of a cortisol effect increases markedly at temperatures below 37° to more than 120 min at 20°. The duration of the lag period at 37° can be shortened if cells are first incubated with cortisol at 20°. The temperature dependence of the irreversible step is such that it cannot be identical with, but must precede, the temperature-dependent step. The actinomycin D-sensitive step may or may not be identical with one of the other two steps.

The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells.

1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The K(m) values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and P(i) found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [(14)C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the (14)CO(2) yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.

Mechanism of the diabetogenic action of growth hormone I. Effect of polypeptides derived from growth hormone on glycolysis in muscle

Experiments with two previously described fractions isolated by hydrolysis of growth hormone have shown that the well-known inhibition of glucose uptake following the injection of growth hormone can be attributed to one of these fractions (In-G) and that inhibition of glyceraldehyde-3-phosphate dehydrogenase and α-glycerophosphate dehydrogenase by this fraction leads to accumulation of glucose 6-phosphate and hence inhibition of phosphorylation of glucose. The reversal of this inhibition by the other fraction (Ac-G) and the consequent increase of glucose uptake can account for the initial hypoglycemic effect of the hormone.

The Accumulation of Glucose 6-Phosphate from Glucose and Its Effect in an Escherichia coli Mutant Lacking Phosphoglucose Isomerase and Glucose 6-Phosphate Dehydrogenase

Abstract When presented with glucose, an Escherichia coli mutant lacking both phosphoglucose isomerase and glucose 6-phosphate dehydrogenase accumulated, internally, high concentrations (approximately 50 mm) of glucose 6-phosphate. It also accumulated glucose in polysaccharide form. Other metabolism of glucose, if any, was very restricted. The accumulation of glucose 6-phosphate was associated with inhibition of growth on those carbon sources the metabolism of which requires functional fructose disphosphatase activity; inhibition of this enzyme in vitro by high concentrations of glucose 6-phosphate was shown.

Effects of isoniazid on the composition of mycobacteria, with particular reference to soluble carbohydrates and related substances.

1. When growing Mycobacterium tuberculosis BCG was exposed to 0.5-10mug. of isoniazid/ml. there was intracellular accumulation of soluble carbohydrate, combined phosphate and substances absorbing at 260mmu. Yellow pigments were formed when modified Sauton medium was used, but not with Proskauer & Beck medium. These processes were apparent after 1hr. but were more marked after about 6hr. These effects were not found with an isoniazid-resistant strain. 2. After 6hr. exposure of the sensitive strain to 10mug./ml. there was little change in the amounts (per g. of insoluble nitrogen) of total lipid, glycolipid, RNA, DNA or of carbohydrate in the nucleic acid fractions. 3. The major accumulation was of alphaalpha'-trehalose. There was also accumulation of glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, trehalose 6-phosphate (tentatively identified), a polysaccharide containing only glucose, and an oligosaccharide containing glucose and glucose 6-phosphate, but not of glycerol and glycerol 3-phosphate. The u.v.-absorbing materials appeared to be nucleotide sugar derivatives. 4. In Mycobacterium smegmatis a similar accumulation of trehalose occurred on exposure to isoniazid, but there was little accumulation of other compounds. 5. No evidence could be found that isoniazid specifically affected the oxidation of glycerol or glycerol 3-phosphate. 6. It is suggested that the primary action of isoniazid on mycobacteria may be partial inhibition of a reaction in some central area of metabolism, such as glycolysis.

Induction of an active transport system for glucose 6-phosphate in Escherichia coli

Evidence is presented for the induction of an active transport system for the entry of glucose 6-phosphate into Escherichia coli B. This system was induced by growth on glucose 6-phosphate as sole carbon source and required active protein synthesis. The analogue, 2-deoxy- d-glucose 6-phosphate- 32P, was found to be accumulated by glucose 6-phosphate-grown cells. Energy was required for this process. The rate of accumulation of radioactivity in cells from added glucose 6-phosphate- 14C was found to be a useful measure of permease activity. A Km of 2.7 × 10 −4 m was estimated for glucose 6-phosphate entry into adapted cells. These studies also indicated that glucose-grown E. coli was completely impermeable to glucose 6-phosphate. Other hexose phosphates markedly inhibited the accumulation of glucose 6-phosphate, whereas l-α-glycerophosphate, fructose 1,6-diphosphate, and glucose had no effect. Glucose 1-phosphate- 14C was accumulated by glucose 6-phosphate-adapted cells. A microprocedure for the preparation of d-glucose 6-sulfate- 14C is presented. No accumulation of this analogue was found in glucose 6-phosphate-adapted cells.