Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: Effects of nerve growth factor and 6-aminonicotinamide

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of¹⁴CO₂ generated from 1-[¹⁴CO₂]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[¹⁴CO₂]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.Special Issue dedicated to Dr. O. H. Lowry.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions and related enzymes of the cycle in adipose tissue

1. Measurements were made of the activities of the enzymes of the pentose phosphate pathway concerned in both the oxidative (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and the non-oxidative (ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) reactions of this pathway, together with hexokinase and phosphoglucose isomerase, in adipose tissue in a variety of nutritional and hormonal conditions. 2. Starvation for 2 days caused a significant decrease in the activities of all the enzymes of the pentose phosphate pathway, with the exception of glucose 6-phosphate dehydrogenase, when expressed as activity/2 fat-pads; only the activities of ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase were significantly decreased on the basis of activity/mg. of protein. Re-feeding with a high-carbohydrate or high-fat diet for 3 days restored the activity of all the enzymes of the pentose phosphate pathway to the range of the control values, with the exception of transketolase, which showed a marked ;overshoot' in rats re-fed with carbohydrate. Starvation for 3 days caused a marked decrease in the activities of glucose 6-phosphate dehydrogenase and transketolase. 3. On the basis of activity/two fat-pads, alloxan-diabetes caused a marked decrease, to about half the control value, in the activities of all the enzymes concerned in the pentose phosphate pathway, transketolase showing the smallest decrease; hexokinase and phosphoglucose isomerase activities were also decreased. Treatment with insulin for 3 and 7 days raised the activities to normal or supranormal values, transketolase showing the most marked ;overshoot' effect. On the basis of activity/mg. of protein the activity of none of the enzymes was significantly decreased in alloxan-diabetes; transketolase and transaldolase activities were raised above the control values. With insulin treatment for 3 or 7 days the activities of all the enzymes were significantly increased, except that of ribulose 5-phosphate epimerase at the shorter time-interval. Glucagon treatment did not alter any of the enzyme activities expressed on either basis. 4. Thyroidectomy caused a decrease of 30-40% in the activities of enzymes of the pentose phosphate pathway, except for transketolase activity, which fell to 50% of the control value. Little change occurred in adipose-tissue weight or protein content. 5. Adrenalectomy caused a decrease of 40% in the activity of glucose 6-phosphate dehydrogenase and of 20-30% in the activities of the remaining enzymes of the pentose phosphate pathway; hexokinase activity was also decreased. Treatment with cortisone for 3 days did not significantly raise the activity from that found in adrenalectomized rats. Treatment of normal rats with high doses of cortisone had no significant effect on the activities of the enzymes of the pentose phosphate pathway in adipose tissue. 6. The changes in enzyme activities are discussed in relation to: (a) the concept of constant-proportion groups of enzymes; (b) the known changes in the flux of glucose through alternative metabolic pathways; (c) the pattern of change found in liver with similar hormonal and dietary conditions.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等.葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中.本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用.     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

Physiological and biochemical changes associated with cotton fibre development

The relationship between growth and some enzymes of carbohydrate metabolism in developing cotton fibre were studied. Two respiratory pathways of glucose oxidation i.e. oxidative pentose phosphate pathway (OPPP) and glycolysis operates in the elongating cotton fibres and the extent of their operation varies with the demand for respiratory products. In this respect, hexokinase, G-6-PDH, 6PGDH, and MDH show increased activities during the period of rapid cell elongation and decreased activities when rate slows down. The conversion of PEP to malate and/or via a transhydrogenase system consisting of enzymes PEPC, MDH and NADP-MDH(d) may play a significant role in carbohydrate compartmentation of developing cotton fibre. As the rate of fibre growth slows down, a decline in enzyme activities, points to a shift in metabolic priorities.     

Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Nonoxidative Pentose Phosphate Pathway in Veillonella alcalescens

Crude cell-free extracts of Veillonella alcalescens C1, an anaerobe unable to ferment glucose, were assayed for individual enzymes of the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were not detectable. Constituent enzymes of the nonoxidative limb of the pentose phosphate pathway were demonstrable. The presence of transaldolase, transketolase, phosphoribose isomerase, and phosphoribulose epimerase in this organism suggests a primarily biosynthetic role for these enzymes. It is postulated that ribose is synthesized from lactate in V. alcalescens C1 via a modified reversal of glycolysis and the nonoxidative limb of the pentose phosphate pathway.     

The NADPH consumption regulates the NADPH-producing pathways (pentose phosphate cycle and malic enzyme) in rat adipocytes

The changes in the activity of the pentose phosphate cycle and the malic enzyme produced by the activation or inhibition of different NADPH-consuming pathways have been studied. The inhibition of the fatty acid synthesis by kynurenate produced a decrease in the flux through the pentose phosphate cycle and a diminution in the malic enzyme pathway. The incubation of the adipocytes in the presence of ter-butyl-hydroperoxide, a compound which is metabolized via a NADPH-consuming pathway, produced a big increase in the pentose phosphate cycle and the malic enzyme activities. The regulation of these NADPH-producing pathways by the NADPH/NADP ratio is discussed.     

Mutants of the pentose phosphate pathway in Aspergillus nidulans

Mutants of the pentose phosphate pathway have been isolated in Aspergillus nidulans. These fail to grow on a variety of carbohydrates that are catabolized through the pentose phosphate pathway. They also grow poorly on nitrate and nitrite as sole nitrogen sources. The pentose phosphate pathway mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the pentose phosphate pathway, of glucose-phosphate isomerase, and of mannitol-1-phosphate dehydrogenase. pppA(-) mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. Both classes of mutants accumulate sedoheptulose-7-phosphate to an extent that is increased considerably when nitrate is present in the medium. Nitrate does not cause an increase in accumulation of sedoheptulose-7-phosphate in double mutants which, in addition to the pppA1 mutation, carry a mutation that leads to the lack of nitrate reductase activity. These last results suggest that nitrate stimulates the flux through the oxidative pentose phosphate pathway, but that this stimulation depends upon the metabolism of nitrate.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

d-Xylose catabolism in Bacteroides xylanolyticus X5-1

The xylose metabolism of Bacteroides xylanolyticus X5-1 was studied by determining specific enzyme activities in cell free extracts, by following ¹³C-label distribution patterns in growing cultures and by mass balance calculations. Enzyme activities of the pentose phosphate pathway and the Embden-Meyerhof-Parnas pathway were sufficiently high to account for in vivo xylose fermentation to pyruvate via a combination of these two pathways. Pyruvate was mainly oxidized to acetyl-CoA, CO₂ and a reduced cofactor (ferredoxin). Part of the pyruvate was converted to acetyl-CoA and formate by means of a pyruvate-formate lyase. Acetyl-CoA was either converted to acetate by a combined action of phosphotransacetylase and acetate kinase or reduced to ethanol by an acetaldehyde dehydrogenase and an ethanol dehydrogenase. The latter two enzymes displayed both a NADH- and a NADPH-linked activity. Cofactor regeneration proceeded via a reduction of intermediates of the metabolism (i.e. acetyl-CoA and acetaldehyde) and via proton reduction. According to the deduced pathway about 2.5 mol ATP are generated per mol of xylose degraded.Abbreviations PPP Pentose phosphate pathway - PKP phosphoketolase pathway     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

转酮酶在癌症中的作用及其机制研究进展

肿瘤细胞的一大重要特征是代谢水平的改变。戊糖磷酸途径作为细胞产生NADPH和五碳糖的主要通路,在肿瘤发生发展过程中发挥着重要功能。转酮酶是戊糖磷酸途径中的关键酶之一,越来越多的研究表明其与癌症病人预后具有显著相关性。作为肿瘤诊断、治疗的潜在靶标,转酮酶具有重要的研究价值。我们就目前癌症研究中对转酮酶的研究进展做简要综述。     

Flavonoids influence growth and saccharide metabolism ofRhizobium meliloti

Supplementation of the growth media with flavonoids,viz. naringenin, flavone, quercetin and syringaldehyde, a phenolic compound and alfalfa seed exudate, differently affected the growth and saccharide metabolism ofRhizobium meliloti. Quercetin increased while syringaldehyde, flavone and alfalfa exudate decreased the growth and protein content ofR. meliloti cells. Naringenin had no effect on growth and protein content though it increased the exopolysaccharide production. Application of naringenin increased the activities of enzymes of the citrate cycle and the 6-phosphogluconate pathway, the major pathways of sugar metabolism in fast growing rhizobia, and decreased activities of glycolytic enzymes. The activities of enzymes of the pentose phosphate pathway remained unaffected by various treatments.     

Pentose phosphate pathway and nucleic acid metabolism in red and white muscles of fish

The activities of the pentose phosphate cycle enzymes, the content of nucleic acids and the activities of acid and alkaline RNAses in the heart and red and white muscles of cartilaginous and teleost fish were determined. It was found that the rates of the dehydrogenase and transferase reactions of the pentose phosphate pathway and the nucleic acid metabolism in the red muscles and heart are much higher than those in the white muscles of the species under study.     

Studies on the Growth in Culture of Plant Cells: XIV. CARBOHYDRATE OXIDATION DURING THE GROWTH OF ACER PSEUDOPLATANUS L. CELLS IN SUSPENSION CULTURE

Marked changes in the activities of the Embden–Meyerhof–Parnas(EMP) pathway and the pentose phosphate pathway occur duringthe progress of growth of sycamore cells in batch suspensionculture. The activities of the two pathways were investigatedby measuring the activities of six EMP pathway and four pentosephosphate pathway enzymes, and by determining the contributionsof ¹⁴C from (1-¹⁴C)- and (6-¹⁴C)-glucose to CO₂. During theearly stages of culture both the EMP pathway and the pentosephosphate pathway make appreciable contributions to carbohydrateoxidation, but following the initiation of cell division, carbohydrateoxidation is predominantly via the EMP pathway. All the enzymesassayed were found to be associated with the soluble fractionof the cells. The regulation of carbohydrate oxidation in sycamorecells and the possible role of the pentose phosphate pathwayin supplying NADPH for biosynthesis are discussed.