The increase in activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in skeletal muscles of rats after subcutaneous administration of N,N′-dimethyl-para-phenylenediamine

Summary After subcutaneous administration of N,N-dimethyl-para-phenylenediamine (DPPD) in rats, a myogenic myopathy was produced in the skeletal muscles. In this communication, the results of the application of various histochemical techniques for the localization of oxidoreductases, transferases, hydrolases and isomerases and biochemical techniques for the estimation of activities of oxidoreductases in the experimental skeletal muscles are presented. The most striking result was the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase which increased dramatically during the early phase of the muscle disease. The increase in activity of the pentose phosphate shunt enzymes was the first pathological alteration and was present as early as 8 h after a single injection of DPPD. Histochemical techniques for demonstration of activity of both enzymes are therefore highly suited for the detection of minor diseases and the early onset of major diseases of the neuromuscular system. Some glycolytic enzymes as well as some enzymes of the aerobic part of the metabolism showed an early decrease or increase in activity indicating a metabolic imbalance in the muscle fibres. There were more fibres with an intermediate pattern of the energy yielding enzymes in the experimental muscle specimens then in specimens from the control groups. The activity of the catabolic hydrolytic enzymes was strongly increased in pathological muscles. The aerobic muscles were more vulnerable to DPPD than the anaerobic muscles.     

Pentose phosphate metabolism during dormancy breakage in Corylus avellana L.

Commercially obtained fruits of Corylus avellana exhibit the characteristic loss of dormancy of this seed following chilling under moist conditions. The activities of cytosolic and organellar enzymes of pentose phosphate pathway in cotyledonary tissue were assayed throughout stratification and over a similar period in damp vermiculite at 20° C. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic acid dehydrogenase (6PGDH) were both found in cytosolic extracts in all treatments; only 6PGDH was present in the organellar fraction.The enzyme activities monitored in seeds at 20° C remained relatively constant over the course of the investigation except in the case of cytosolic 6PGDH where it is suggested an inhibitor of the enzyme accumulated. This inhibitor was removed by the partial purification procedure. Increases in the activities of the enzymes occurred during stratification, the major increase coinciding exactly with dormancy breakage but prior to the initiation of germination. The marked increase in G6PDH and 6PGDH concurrent with the change in germination potential of the chilled seed may have considerable biochemical significance in breaking down the dormant state.Abbreviations G6P glucose-6-phosphate - G6PDH glucose-6 phosphate dehydrogenase - NADP nicotinamide adenine dinucleotide phosphate - 6 PGDH 6-phosphogluconic acid dehydrogenase - PPP pentose phosphate pathway     

The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle

Summary Classification of human skeletal muscle into type I and type II fibres is frequently based on their weak or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in human muscle specimens with disease of the neuromuscular system.Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and gluconeogenesis respectively. Moreover the activities of enzymes of the pentose phosphate pathway are determined.A most important feature of the biochemical findings is that the constancy of activity ratios of the examined enzymes, as is found apparently normal human skeletal muscle, was frequently not present in diseased human skeletal muscle. From these results and from the histochemical results it can be concluded that for fibre classification in diseased human skeletal muscle the histochemical demonstration of myosin ATPase activity exclusively is not sufficient, but that it is necessary to apply other enzyme histochemical techniques too.Moreover it was found that in diseased human skeletal muscles the activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was strongly increased. A third observation was the relative decrease of the activity of the examined aerobic enzymes in affected muscle fibres of neurogenic muscle diseases.     

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.     

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.     

Purification and cloning of chloroplast 6-phosphogluconate dehydrogenase from spinach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes encoded in eukaryotic chromosomes.

Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.     

An enzyme histochemical study of isoproterenol-induced myocardial necroses in rats

Summary The effect of isoproterenol on myocardial metabolism in rats was studied using qualitative and quantitative histochemical techniques. The activity and location of 20 enzymes that play a role in the aerobic and anaerobic pathways of energy metabolism were qualitatively examined. The activity and location of some hydrolytic enzymes and the glycogen content were also qualitatively studied. For the quantitative study the activity of 10 enzymes was measured.The isoproterenol injections induced necrosis with inflammatory infiltrates. The muscle fibres in the necrotic regions were characterized by defective aerobic energy metabolism and increased glycolytic capacity. There was a depletion of the glycogen reserves in the necrotic fibres. These fibres showed a markedly increased activity of enzymes belonging to the oxidative branch of the pentose phosphate pathway. The implication of this increase for the metabolism of the myocardial cells is discussed. The activity of acid phosphatase in the pathological muscle fibres was strongly increased. The inflammatory cells in the necrotic areas were characterized by preponderantly anaerobic metabolism.Dedicated to Prof. H. G. Goslar in honour of his 70th birthday.     

高等植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个酶.在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位.结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生.讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料.     

高等植物葡萄糖-6- 磷酸脱氢酶与6- 磷酸葡萄糖酸脱氢酶基因的不同进化起源

葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶是植物戊糖磷酸途径中的两个关键酶。在克隆了水稻质体葡萄糖-6-磷酸脱氢酶基因OsG6PDH2和质体6-磷酸葡萄糖脱氢酶基因Os6PGDH2基础上,分析比较了水稻胞质和质体葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因的基因结构、表达特性和进化地位。结合双子叶模式植物拟南芥两种酶基因的分析结果,认为高等植物葡萄糖-6-磷酸脱氢酶基因和6-磷酸葡萄糖酸脱氢酶基因在进化方式上截然不同,葡萄糖-6-磷酸脱氢酶的胞质基因与动物和真菌等真核生物具有共同的祖先;6-磷酸葡萄糖酸脱氢酶的胞质酶和质体酶基因都起源于原核生物的内共生。讨论了植物葡萄糖-6-磷酸脱氢酶与6-磷酸葡萄糖酸脱氢酶基因可能的进化模式,为高等植物及质体的进化起源提供了新的资料。     

The Respiratory Metabolism of Strelitzia juncea Ait. Seeds: The Effect of Dormancy Release through Oxygen Incubation of the Seeds on the Activity of Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase

Incubation of Strelitzia juncea seeds in an oxygen atmosphereresulted in an increase in the G6PDH activity of crude embryoextracts on day one, while radicle protrusion started on dayfive. Similarly, 6PGDH activity increased over the first 4 dof incubation in oxygen. The ratio of 6PGDH/G6PDH was 3.0<x < 3.7 regardless of treatment or incubation period. Supplying oxygen per se to dormant seed and studying its effecton the activity of the two key pentose phosphate (PP) pathwayenzymes, appear to support Roberts' hypothesis that oxygen shortagerestricts PP pathway activity in dormant seeds. Key words: Dormancy, pentose phosphate pathway, Strelitzia juncea     

Early and late changes in the metabolic pattern of the working myocardial fibres and Purkinje fibres of the human heart under ischaemic and inflammatory conditions: An enzyme histochemical study

Summary In this communication, the results of an enzyme histochemical study on the working myocardial fibres and Purkinje fibres of the atrioventricular conducting system of the human heart under ischaemic and inflammatory conditions are presented. The material was selected from patients showing changes which could be classified in three major groups: (1) early changes due to acute ischaemia either in the myocardial fibres or in the conducting system or in both; (2) chronic ischaemic changes due to cardiovascular insufficiency, such as old infarction, or coronary arteriosclerosis or both; and (3) inflammatory conditions such as myocarditis.The activity and location of about 20 enzymes that play a role in the aerobic and anaerobic pathways of energy metabolism were examined. The activity and location of some hydrolytic enzymes and the glycogen and lipid content were also studied.The most important findings were an obvious depletion of the glycogen reserves under acute ischaemic changes in both types of fibre. This was associated with a transient or permanent reduction in activity of many enzymes that play a role in aerobic and anaerobic metabolism. Further, there was an instantaneous and persistent increase in the activity of the NADPH-regenerating enzymes of the pentose phosphate pathway and of glyceraldehyde-3-phosphate dehydrogenase, the rate-limiting enzyme of glycolysis under ischaemic conditions. Chronic ischaemic changes were characterized by a gradual long-term increase in the activity of many anaerobic glycolytic enzymes. Moreover, there was an absence of activity of acetylcholine esterase immediately after the onset of infarction in the fibres of the conducting system. Lastly, a slight increase in lipid content was found in the hypertrophic chronic ischaemic fibres and in old infarcted areas. Cardiac fibres in inflamed areas showed a marked increased activity of the pentose phosphate shunt enzymes and a less pronounced increased activity of most anaerobic and hydrolytic enzymes. In contrast to the cardiac fibres in infarcted areas, the fibres in inflamed areas did not reveal a decrease or absence of activity of aerobic enzymes such as succinate dehydrogenase.     

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.     

Long-term adaptive response to dietary protein of hexose monophosphate shunt dehydrogenases in rat kidney tubules

We have studied the effects of several different macronutrients on the kinetic behaviour of rat renal glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Rats were meal-fed with high-carbohydrate/low-protein, high-protein/low-carbohydrate and high-fat diets. High-protein increased renal G6PDH and 6PDGH activities by 66 per cent and 70 per cent respectively, without significantly changing the Km values of either and each Hexose monophosphate dehydrogenase activity increased steadily, reaching a significant difference on day 4. A rise in carbohydrate or fat in the diets, produced no significant change in either the activity or the kinetic parameters, Vmax and Km of the two dehydrogenases. In addition, the administration of a high-protein diet for 8 days significantly increased both the pentose phosphate pathway flux (92.6 per cent) and the kidney weigth (35 per cent), whereas no significant changes in these parameters were found when the animals were treated with the other diets. Our results suggest that an increase in the levels of dietary protein induces a rise in the intracellular levels of these enzymes. The possible role of this metabolic pathway in the kidneys under these nutritional conditions is also discussed.     

The role of N-quinones in the regulation of glucose-6-phosphate metabolism

In experimental (white rats, rabbits) and clinical (erythrocytes, blood plasma) studies on 29 healthy subjects and patients it has been demonstrated that primary or secondary n-quinone deficiency is accompanied by increased tissue activity of glycolysis enzymes (aldolase, PGmutase) and aerobic pentose phosphate shunt (6 GPDH). Parallel rise in the amount of glycolysis metabolites (pyruvate and lactate) in the blood and the decline in blood plasma glucose level were observed. The changes in glucose-6-phosphate metabolism are, probably, secondary and reflect tissue structure alterations in the development of K and E avitaminosis.     

细胞质雄性不育水稻幼穗和花药的呼吸酶活性研究

研究珍汕97A和珍汕97B的雌雄蕊原基形成期、花粉母细胞形成期和花粉母细胞减数分裂期的幼穗及单核期、二核期和三核期的花药中呼吸代谢三羧酸循环（TCA）的苹果酸脱氢酶（MDH）和异柠檬酸脱氢酶（IDH）及戊糖途径（PPP）的磷酸葡萄糖脱氢酶（G6PDH）、磷酸葡萄糖酸脱氢酶（6PGDH）和5一磷酸核糖异构酶（RSPI）的活性。结果表明：可育花药的5种酶活性皆高于同期不育花药;而幼穗中,TCA途径中的MDH和IDH在不育系与保持系之间无差异,PPP途径的G6PDH和6PGDH及R5PI则保持系高于不育系。这说明不育系中PPP发生的变化早于TCA途径,PPP途径的改变可能与小孢子败育有着更为直接的关系。     

Effect of vitamin C and folic acid on seed vigour response and phenolic-linked antioxidant activity

Folic acid and vitamin C were used in the concentration range of 0-500muM as exogenous growth enhancers to stimulate pea (Pisum sativum) seedling vigour. The results suggest that a concentration of 50muM folic acid and 500muM vitamin C were optimum in maximally enhancing seed vigour and potentially seedling performance according to both agronomic and biochemical seed vigour parameters. Results indicated that germination percentage, shoot weight, shoot height, and root length were enhanced in folic acid and vitamin C treated plants compared to control plants. The levels of enhanced phenolic content in response to folic acid and vitamin C treatments were highest on days 8 and 10. Evaluation of critical biochemical parameters indicated that the average glucose-6-phosphate dehydrogenase (G6PDH) activity and proline content in response to treatments were higher than control and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Key enzymes, guaiacol peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) were also higher in response to treatments and correlated to enhanced phenolic content and DPPH-based antioxidant activity. Taken together, these studies support the hypothesis that the proline-linked pentose phosphate pathway stimulates phenolic synthesis and related free-radical scavenging antioxidant activity. Further, this proline-linked pentose phosphate pathway stimulation in response to folic acid and vitamin C was also correlated to antioxidant enzyme response indicated by the stimulation of GPX, SOD, and CAT activities. Therefore, this study indicates the enhancement of seed vigour response by folic acid and vitamin C as reflected in both agronomic and biochemical responses, and this occurred through the stimulation of phenolic-linked antioxidant response that is likely positively modulated through the proline-linked pentose phosphate pathway.     

Effectors of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

Summary The lower Vmax of 6PGDH with respect to G6PDH and its higher sensitivity to inhibition by NADPH, suggest the existence of an imbalance between the two dehydrogenases of the pentose phosphate pathway in rat liver. Possible modulators of these activities, particularly in relation with the inhibition by NADPH in physiological conditions, have been investigated. The results suggest that in both cases the inhibition by NADPH is strictly isosteric and that the relative affinities for the reduced and oxidized forms of the pyridine nucleotide are unaffected by glutathion, the intermediates of the pentose phosphate shunt or some divalent ions.Abbreviations G6PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - 6PGDH 6-phosphogluconate dehydrogenase (EC 1.1.1.44) On leave from the Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.     

Crystal structure of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 6-phosphogluconate dehydrogenase Gnd1

Background  

As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH) is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG). Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications.