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PubMedDate not supplied

[The activity of citrate glyoxylate and pentosephosphate cycle enzymes during yeast growth on hexadecane and glucose].

The activity of key enzymes of the citrate (CC), glyoxylate (GC), and pentose phosphate cycles (PPC) was determined in the cells of Candida lipolytica and Candida tropicalis growing on hexadecane and glucose. The activity of isocitrate lyase and malate synthase (enzymes of GC) was very high in hexadecane grown cells but was almost absent in "glucose" grown. The activity of citrate synthase and aconitase was 2-4 times and 1.5-2 times, respectively, higher in the cells, grown on n-alkane. The activity of citrate synthase of "hexadecane" and "glucose" yeasts was higher than the activity of other enzymes of CC (aconitase and isocitrate dehydrogenase), especially in the "hexadecane" cells. The activity of the key enzymes of PPC was almost the same in the yeast cells, grown on "hexadecane" and "glucose". Possible factors causing differences in the activities of the enzymes of GC and CC are discussed. Yeast organisms incapable to grow on n-alkanes were found to be able to grow on acetate (43 strains belonging to 35 yeast species have been studied) and to have a high activity of isocitrate lyase. Therefore, the absence of growth of many yeast strains on n-alkanes is not caused by the absence of the activity of enzymes of GC in these strains.

Aconitate Hydratase↗

PubMed2003

Glucose conversion by multiple pathways in brain extract: theoretical and experimental analysis.

Experimental and model studies were performed to characterize the flux of glucose metabolism and the sharing of glucose-6-phosphate (Glu6P) by the upper parts of glycolytic and pentosephosphate pathways in the brain extract. A mathematical model based upon the kinetic equations of the individual enzymes was evaluated to fit the experimental data. Glucose is converted to glucose-6-phosphate by hexokinase that controls almost exclusively the glucose metabolism. Experiments showed that this crossroad-metabolite was shared between glycolysis and pentosephosphate pathway in the brain extract in a ratio of 1.5:1. This ratio was favorable to the pentosephosphate pathway by the addition of high excess of exogenous glucose-6-phosphate dehydrogenase, standardly used for the activity assay of hexokinase, but still a significant part (17+/-3%) of the common intermediate was converted into the direction of glycolysis. Stimulation of glucose-6-phosphate formation via moderate (30-50%) increase of hexokinase activity by adding exogenous hexokinase or tubulin resulted in the slight increase of the relative flux into direction of glycolysis. The model correctly described all of these observations. However, when the activity of hexokinase was doubled with exogenous enzyme, significantly less glucose-6-phosphate was converted into direction of glycolysis than predicted. This discrepancy shows that the system did not behave in this case as an ideal one, which could be due to the formation of distinct pools for the intermediate.

Animals↗

PubMed1999

Mass spectrometry for metabolic flux analysis.

Mass spectrometry in combination with tracer experiments based on 13C substrates can serve as a powerful tool for the modeling and analysis of intracellular fluxes and the investigation of biochemical networks. The theoretical background for the application of mass spectrometry to metabolic flux analysis is discussed. Mass spectrometry methods are especially useful to determine mass distribution of metabolites. Additional information gained from fragmentation of metabolites, e.g., by electron impact ionization, allows further localization of labeling positions, up to complete resolution of isotopomer pools. To effectively handle mass distributions in simulation experiments, a matrix based general methodology is formulated. The natural isotope distribution of carbon, oxygen, hydrogen and nitrogen in the target metabolites is considered by introduction of correction matrices. It is shown by simulation results for the central carbon metabolism that neglecting natural isotope distributions causes significant errors in intracellular flux distributions. By varying relative fluxes into pentosephosphate pathway and pyruvate carboxylation reaction, marked changes in the mass distributions of metabolites result, which are illustrated for pyruvate, oxaloacetate, and alpha-ketoglutarate. In addition mass distributions of metabolites are significantly influenced over a broad range by the degree of reversibility of transaldolase and transketolase reactions in the pentosephosphate pathway. The mass distribution of metabolites is very sensitive towards intracellular flux patterns and can be measured with high accuracy by routine mass spectrometry methods. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗

PubMedDate not supplied

[The characteristics of the development of an adaptation syndrome in severe gestosis].

Basic metabolic pathways were studied of formation of the adaptive syndrome in the organism of patients with grave gestoses: glycolysis, gluconeogenesis, and pentosephosphate pathway of production of nicotinamide coenzymes. It has been found out that a stressful character of reconstruction of metabolic homeostasis tends to change the processes of glycolysis and gluconeogenesis that had come to be formed by evolution. This warrants further study, its purpose being a specific correction of intracellular metabolism and prevention of complications. Ozonohemo- and antioxidant therapy in a complex of intensive treatment measures for patients with severe gestoses make for stimulation of pentosephosphate pathway and glycolysis.

Acute Disease↗

PubMedDate not supplied

[Effect of starvation and diabetes on the activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases and on the free fatty acid content of rat kidney cortex and medulla].

Activity of dehydrogenases related to pentosephosphate pathway was not distinctly altered in soluble fraction of kidney cortex and medulla after 48 and 72 hrs of starvation. In diabetes the activity of these enzymes in rat kidney, as distinct from liver tissue, was not decreased but it was elevated and within 72 hrs after administration of alloxan the activity of glucose-6-phosphate dehydrogenase was increased 2-fold and the activity of 6-phosphogluconate dehydrogenase was increased by 30% above the normal level. Content of free fatty acids was also increased in kidney cortex of diabetic rats within 72 hrs after administration of alloxan. Alterations in content of free fatty acids were not observed either in kidney of diabetic animals within other studied periods (6 and 14-16 days) of treatment or in the tissue of starved rats. The data obtained suggest that free fatty acids do not participate immediately in controlling effect on dehydrogenases of pentosephosphate pathway in kidney in vivo.

Animals↗

PubMedDate not supplied

[Protective action of 5-hydroxy-6-methyluracil and ferrocenes in nitrite-ion poisoning].

It is shown that ferrocenes and 5-oxy-6-methyluracil efficiently inhibit methemoglobin-formation in experiments in vitro and in vivo. Preliminary administration of ferrocene or 5-oxy-6-methyluracil to laboratory animals essentially increases life time in case of sodium nitrite intoxication. Ferrocene protection from sodium nitrite intoxication normalizes the level of methemoglobin and lipoperoxidation in erythrocytes as well as activity of superoxide dismutase of erythrocytes. Activity of catalase, glutathione reductase and dehydrogenases of the pentosephosphate way were not normalized during ferrocene protection from sodium nitrite intoxication. In case of ferrocene protection the catalase activity during sodium nitrite intoxication remains lowered, while activity of glutathione reductase and dehydrogenases of the pentosephosphate pathway grows sharply.

Animals↗

PubMed2001

Suppression of the accumulation of triosephosphates and increased formation of methylglyoxal in human red blood cells during hyperglycaemia by thiamine in vitro.

The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications. A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate. To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions. In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal. The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate. Biochemical changes implicated in the development of diabetic complications were thereby prevented. This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.

Deoxyglucose↗

PubMedDate not supplied

[Assimilation of carbon dioxide by Stibiobacter senarmontii].

Early products of 14CO2 assimilation by a new microorganism Stibiobacter senarmontii are phosphoglyceric acid, phosphorous esters of sugars and aspartic acid, as was shown by chromatography and radioautography. Extracts of the cells displayed the activity of ribulosediphosphate carboxylase and phosphoenolpyruvate carboxylase (1 mU and 0.24 mU per 1 mg of protein in the extract, respectively). Therefore, the microorganism is capable of autotrophic nutrition involving mechanisms of the reductive pentosephosphate cycle. The latter seems to operate even in conditions of deficiency of the energy substrate which is caused by low solubility of antimony trioxide.

Aspartic Acid↗

PubMed1985

[Erythrocyte enzymes in chronic kidney failure].

Forty four patients with various degrees of chronic renal insufficiency (CRI) were studied. The activity of both dehydrogenases and pentosephosphate pathway (PPhP) in erythrocyte--glucose-6-phosphate dehydrogenase (G6PD), and 6-phosphogluconate dehydrogenase (6-PGD), as well as the activity of the main generating ATP-enzyme of glycolysis--pyruvate kinase (PK) was studied. The activity of the erythrocyte enzymes studied grows with the intensification of renal insufficiency, particularly manifested in G6PD and PK.

Adult↗

PubMedDate not supplied

[Mechanism of action of hydroxycoumarin derivatives].

Experimental and clinical studies have demonstrated the effects of hydroxycoumarin derivatives (pelentan) on the composition and correlation of phospholipids (PL) in red cell biomembranes. PL such as cardiolipin, cholesterol and its esters phosphatidylethanolamine, and phosphatidylcholine experienced the greatest changes. The changes in the composition of biomembrane PL were accompanied by an increase in the total cholesterol content in red cell biomembranes and by an increase in the total and free non-esterified fatty acids in blood plasma (P less than 0.05). At the same time the changes were discovered in the activity of transketolase and glucose-6-phosphate dehydrogenase limiting the pentosephosphate shunt.

Animals↗

PubMedDate not supplied

[Interrelation of basic pathways of carbohydrate metabolism in the brain during hypothermia].

Main energy substrates of brain--glucose and glycogen, key enzymes of their conversion--hexokinase, amylase and phosphorylase as well as main pathways of the carbohydrate utilization--respiration, glycolysis and pentosephosphate pathway were studied in rat brain at various phases of cooling. Phase alterations of metabolic reactions were observed in hypothermia. The hypothermia was accompanied by quantitative and qualitative alterations in relations between main metabolic pathways.

Amylases↗

PubMedDate not supplied

[Enzyme activity of carbohydrate metabolism in rat skeletal muscle after space flight].

Space flight factors did not influence activity of glycogen phosphorylase and adenylate cyclase in skeletal muscles of rats. Activity of glucose-6-phosphate and 6-phosphogluconate dehydrogenases increased noticeably in the most active muscles (gastrocnemius and tibialis anterior muscles). Activation of enzymes involved in the pentosephosphate pathway of glucose conversion may be associated with compensatory processes induced by muscle changes due to diminished motor activity of animals in space flight.

Animals↗

PubMed1993

Metabolic and functional changes in lymphocytes and macrophages as induced by ageing.

Key enzyme activities of glycolysis, pentosephosphate pathway, Krebs cycle, and glutaminolysis were measured in lymphocytes and macrophages of 3- and 15-month-old rats from the control, thioglycollate-injected, and Walker 256 tumor-implanted groups. The percentage of phagocytosis, phagocytic index, and production of H2O2 in macrophages and the rates of [2-14C]-thymidine and [5-3H]-uridine incorporation in cultured lymphocytes were also determined. The results indicate that the percentage of phagocytosis was not affected but the phagocytic index increased by twofold as a consequence of ageing, whereas the production of H2O2 reduced. The rates of both [2-14C]-thymidine and [5-3H]-uridine incorporation in lymphocytes from aged rats were lower as compared to those of mature animals in the three groups. Taken as a whole, the results of enzyme activities suggest that ageing may reduce the capacity for glucose utilization in lymphocytes and macrophages under the three conditions. Lymphocyte and macrophage glutamine metabolism was not markedly affected by ageing. Therefore, an impaired glucose metabolism during ageing may be one important mechanism for the alteration in lymphocyte proliferation and macrophage phagocytosis observed and also for the modification of the response to inflammatory and tumor challenges.

Aging↗

PubMed1988

Reduction of nitroxide free radical by normal and G6PD deficient red blood cells.

The electron spin resonance signal of Tempol decays in the presence of red cells. The decay is due to reduction of oxidant, paramagnetic nitroxide group by the metabolic activity of the red cell. In normal red cells, GSH level was stable and Tempol reduction rate followed a first-order kinetics. In G6PD-deficient red cells, GSH dropped and Tempol reduction rate was slower and followed a second-order kinetics. In normal red cells, diamide reversibly oxidized GSH. First-order kinetics of Tempol reduction rate was attained after a delay time proportional to the diamide concentration and corresponding to the full regeneration of GSH. In diamide-treated G6PD deficient, and in NEM-treated, normal red cells, irreversible disappearance of GSH was followed by irreversible dose-dependent decrease in Tempol reduction rate. A correlation between GSH levels and Tempol reduction rate was observed. A correlation was also established between Tempol reduction rate and stimulation of pentosephosphate shunt activity.

Adult↗

PubMed1997

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants.

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Chloroplasts↗

PubMed1995

Oxidation of Imported or Endogenous Carbohydrates by Isolated Chloroplasts from Green Pepper Fruits.

Recently, we demonstrated that intact chloroplasts isolated from green pepper (Capsicum annum L.) fruits use exogenous glucose-6-phosphate (Glc-6-P) as the most efficient precursor for starch biosynthesis (O. Batz, R. Scheibe, H.E. Neuhaus [1995] Planta 196: 50-57). Here we demonstrate that these chloroplasts transport this hexose phosphate in counter-exchange for orthophosphate. By measuring the release of 14CO2 from [1-14C]Glc-6-P, we show that isolated fruit chloroplasts also use exogenous Glc-6-P as a substrate for the oxidative pentose-phosphate pathway. The rate of decarboxylation appears to be linear with time and is significantly reduced in the presence of Triton X-100, indicating that the reaction is dependent on plastid integrity. Pyruvate has been identified as a positive effector for flux through the oxidative pentose-phosphate pathway. However, the highest rates of Glc-6-P-driven oxidative pentosephosphate pathway activity are achieved in the presence of nitrite, 2-oxoglutarate, and glutamine, indicating a strong interaction between nitrogen metabolism and this pathway. In addition, we show that carbohydrates liberated during starch mobilization are used as substrates for the oxidative pentose-phosphate pathway. Orthophosphate was found to act as an activator for the observed 14CO2 release from carbohydrates formerly bound as starch. In this context, we demonstrate that exogenous Glc-6-P competes with endogenous carbohydrates. A possible interaction between exogenous and endogenous carbohydrates is discussed with respect to altered levels of carbohydrates during fruit development.

Journal Article↗

PubMed1984

In Vitro Inhibition of the Plastid and Cytosolic Isozymess of 6-Phosphogluconate Dehydrogenase from Developing Endosperm of Ricinus communis by Fructose 2,6-bisphosphate.

Activities of the cytosolic and plastid isozymes of 6-phosphogluconate dehydrogenase from developing endosperm of Ricinus communis L. seeds were inhibited in vitro by hexosebisphosphates. Inhibition constants for glucose 1,6-bisphosphate were 221 and 209 micromolar for the cytosolic and plastid isozymes, respectively, and corresponding values for fructose 2,6-bisphosphate were 10.5 and 8.6 micromolar. In each case inhibition was of a mixed noncompetitive nature relative to 6-phosphogluconate. While the levels and distribution of fructose 2,6-bisphosphate in castor oil seed endosperm cells are not yet known, the levels reported to occur in leaf cytosol would be high enough to significantly inhibit carbon flux through the pentosephosphate pathway due to inhibition of 6-phosphogluconate dehydrogenase activity.

Journal Article↗

PubMed1985

Enzymic capacities of purified cauliflower bud plastids for lipid synthesis and carbohydrate metabolism.

Isolated cauliflower (Brassica oleracea) bud plastids, purified by isopycnic centrifugation in density gradients of Percoll, were found to be highly intact, to be practically devoid of extraplastidial contaminations, and to retain all the enzymes involved in fatty acid, phosphatidic acid, and monogalactosyldiacylglycerol synthesis. Purified plastids possess all the enzymes needed to convert triose phosphate to starch and vice versa, and are capable of conversion of glycerate 3-phosphate to pyruvate for fatty acid synthesis. They are also capable of oxidation of hexose phosphate and conversion to triose phosphate via the oxidative pentosephosphate pathway. Cauliflower bud plastids prove to be, therefore, biochemically very flexible organelles.

Journal Article↗