How to keep glycolytic metabolite concentrations constant when ATP/ADP and NADH/NAD+ change

Glycolytic flux may increase over 100 times in skeletal muscle during rest-to-work transition, whereas glycolytic metabolite concentrations remain relatively constant. This constancy cannot be explained by an identical direct activation of all glycolytic enzymes because the concentrations of ATP, ADP, AMP, P(i), NADH and NAD+, modulators of the activity of different glycolytic enzymes, change. It is demonstrated in the present in silico study that a perfect homeostasis of glycolytic metabolite concentrations can be achieved if glycolysis is divided into appropriate blocks of enzymes that are directly activated to a different extent in order to compensate the effect of the modulators.     

Glycosis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas. Rate-limiting steps and activation at fertilization.

The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas, before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.     

Therapeutic Potential of Riboflavin,Niacin and Ascorbic Acid on Carbohydrate Metabolizing Enzymes in Secondary Endometrial Carcinoma Bearing Rats

Curative potential of riboflavin, niacin and ascorbic acid against tamoxifen mediated endometrial carcinoma was established by studies on carbohydrate metabolizing enzymes. The enzymes investigated were glycolytic enzymes namely, hexokinase; aldolase; phosphoglucoisomerase and the gluconeogenic enzymes namely, glucose-6-phosphatase and fructose-1, 6-biphosphatase in endometrial carcinoma bearing rats. A significant increase in glycolytic enzymes and a subsequent decrease in gluconeogenic enzymes were observed in plasma, liver and kidney of endometrial carcinoma animals. The administration of riboflavin (45 mg/kg bw/day), niacin (100 mg/kg bw/day) and ascorbic acid (200 mg/kg bw/day) along with tamoxifen (45 mg/kg bw/day) caused a significant decrease in the activity of glycolytic enzymes and a significant increase in the activities of gluconeogenic enzymes to near normal levels in experimental animals. Our results suggest that riboflavin, niacin and ascorbic acid have potential combination therapy against tamoxifen mediated secondary endometrial carcinoma in experimental rats. However, there were no deleterious side effects observed in combinants alone treated animals.     

Parts of the sequence between the complete rRNA operons are repeated on either side of the extra 16 S rRNA gene in chloroplast DNA of Euglena gracilis strain Z

The maximum activity of the key glycolytic enzymes, hexokinase and 6-phosphofrustokinase, was measured in tissues of control and cold-acclimated rats. The only significant change in activity was seen in brown adipose tissue where the activity of these enzymes was increased 2-fold. This increase in glycolytic capacity along with the hypertrophy of BAT observed in cold acclimation suggests that this tissue could play an important role in glucose utilisation by the rat.     

Culture kinetics of glycolytic enzyme induction, glucose utilization, and thymidine incorporation of extended-exposure phytohemagglutinin-stimulated human lymphocytes

Glycolytic enzyme activity is significantly (P less than 0.05) induced between 24 and 48 hours of incubation in phytohemagglutinin-stimulated human lymphocytes. Nonstimulated cultured cells do not show this induction although these cells have an approximate daily doubling of thymidine incorporation. Maximal glycolytic enzyme activity is reached between 96 and 120 hours of culture in stimulated cells (3.5-fold increase) and maintained until at least 168 hours. There is no significant induction of the hexosemonophosphate shunt or the TCA cycle during seven-day transformation. Induction of glucose utilization becomes significantly (P less than 0.05) greater in stimulated as compared to nonstimulated cultures between 48 and 72 hours of culture and is significantly elevated for at least an additional 96 hours. There is a 17% increase in total protein in the stimulated cells after 24 hours of culture and higher levels of protein content are then maintained over the control. Thymidine incorporation is significantly greater in stimulated cells from 24-144 hours of culture but is not significantly different from the nonstimulated cells at 168 hours (P = 0.98) although glycolytic enzyme activity remains elevated in the stimulated cells. There is a greater enzyme induction of the latter phase of glycolysis during transformation and this phenomenon continues in extended cultures. Increases in glycolytic enzyme activity during mitogenesis appear to be an intrinsic phenomenon independent of cell proliferation and glucose transport. The mitogen-induced increase in the activity of the glycolytic enzymes accompanies blastogenesis and the sustained elevated activity of these enzymes to be related to the high metabolic rate of transformed cells.     

Development of the activities of oxidative, glycolytic and muscle enzymes during early larval life in three families of freshwater fish

The development of the activities of oxidative (COX, CS), glycolytic (PFK, PK, LDH) and muscle enzymes (CK, MK, Pase) was studied in representatives of the families Coregonidae, Salmonidae and Cyprinidae, from hatching to an age of approximately 100 days. In addition, the activities of two enzymes of amino acid metabolism (GOT, GPT) were followed in rainbow trout and in roach.
Water content of fresh body weight and protein content of dry body weight decrease during the early larval period. Specific activities of the two oxidative enzymes decline, whereas those of glycolytic and muscle enzymes increase in all species.
A family-specific event is the enormous increase in glycolytic and muscle enzymes from very low values in the early larva to very high levels in adult Coregonus sp. In rainbow trout, CS activity begins with a low-level period lasting throughout the yolk-sac period, whereas in the other species CS activity is high immediately after hatching.
Acclimation to either 15 or 20° C has no effect on the mass-specific activities of PFK, M K, CK and Pase in roach and chub, but the former three enzymes appear to be strongly dependent on rearing conditions during the early larval period, whereas Pase is not.     

Changes in concentrations of glycolysis and Krebs cycle metabolites in mosquitofish,Gambusia holbrooki,induced by mercuric chloride and starvation

Synopsis Concentrations of glycolysis and Krebs cycle metabolites in the tail tissues of mosquitofish,Gambusia holbrooki, were measured in response to starvation and to exposure to 0.62 mg Hg l^–1 (as HgCl₂) for 48 h. In control fish, starvation caused decreased concentrations of glucose-6-phosphate (GO, –58%) and fructose-6-phosphate (F6P, –79%). Mercury exposure resulted in decreased concentrations of G6P (– 56%) and F6P (– 79%), and to increased concentrations of pyruvate (+ 75%), -ketoglutarate (+ 41%), succinate (+ 39%), and malate (+ 47%). Krebs cycle activity increased in response to mercury exposure, perhaps in response to greater energy needs associated with maintaining homeostasis under stressful conditions. We conclude that glycolytic activity is reduced in fish exposed to mercury and that this response is similar to that caused by a cessation of feeding.     

Activities of glucose metabolic enzymes in human preantral follicles: in vitro modulation by follicle-stimulating hormone, luteinizing hormone, epidermal growth factor, insulin-like growth factor I, and transforming growth factor beta1

Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor beta1 (TGFbeta1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFbeta1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFbeta1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities.     

Association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of glioma cells

A latex phagocytosis technique was used to prepare relatively pure plasma membranes with inside-out orientation. This method was adapted through a number of modifications in order to evaluate the association of glycolytic enzymes with the cytoplasmic side of the plasma membrane of C6 glial cells. As phosphorylation is strictly coupled with transport in these cells, glycolytic enzymes, especially hexokinase, could metabolize glucose in close vicinity to its transporter. Of the enzymes tested, hexokinase is present in considerable quantities on these membranes (nearly 40% of homogenate specific activity), followed by D-glyceraldehyde-3-phosphate dehydrogenase (10%), pyruvate kinase (8%), and 3-phosphoglycerate kinase (1%). Except for hexokinase, the enzyme pattern presented here is different from that published for other membrane preparations.     

不同糖源及糖水平对大菱鲆糖代谢酶活性的影响

采用34双因素实验设计, 以初始质量为(8.060.08) g的大菱鲆幼鱼(Scophthalmus maximus L.)为对象, 研究在饲料中添加3种糖源(葡萄糖、蔗糖和糊精)及4个水平(0、5%、15%、28%)对大菱鲆肝脏糖酵解关键酶己糖激酶(HK)、葡萄糖激酶(GK)、磷酸果糖激酶(PFK)、丙酮酸激酶(PK)和糖异生关键酶磷酸烯醇式丙酮酸羧激酶(PEPCK)、1, 6-二磷酸果糖酶(FBPase)活性的影响。结果表明: 饲料糖添加量从0升高到15%时, 大菱鲆的糖酵解酶GK和PK活性随饲料葡萄糖或糊精含量的增加而增加; 当饲料中葡萄糖或糊精含量为28%时, GK和PK活性有下降的趋势。3种糖源的4个添加水平对HK和PFK活性均无显著影响(P 0.05)。添加不同水平的葡萄糖对大菱鲆糖异生途径的PEPCK活性无显著影响(P 0.05), 但在饲料中葡萄糖添加量为5%时显著促进了FBPase活性(P 0.05), 当葡萄糖添加量升高为15%或28%时, FBPase活性与对照组无显著差异(P 0.05)。糊精作为饲料糖源时抑制了大菱鲆肝脏FBPase和PEPCK的活性, 而添加不同水平的蔗糖对FBPase和PEPCK活性的影响均不显著(P 0.05)。总的来说, 从大菱鲆幼鱼肝脏糖代谢角度而言, 在饲料中添加15%的葡萄糖或糊精时, 可以有效促进大菱鲆肝脏糖酵解能力; 较添加葡萄糖, 糊精在促进大菱鲆肝脏糖酵解的同时对糖异生存在一定程度的抑制。蔗糖作为饲料糖源时, 仅在添加量为28%时显著促进糖酵解酶GK活性, 糖酵解其他酶活性以及糖异生酶活性均不受蔗糖水平的显著影响。       

Coordinate regulation of glycolysis by hypoxia in mammalian cells

The impact of hypoxic exposure on the activities of all 11 glycolytic enzymes was studied in cell culture into mammalian cells—mouse lung macrophages and L8 rat skeletal muscle cells. During hypoxic exposure, the measured activity of all glycolytic enzymes increased, establishing coordinate regulation. Three nonglycolytic cytoplasmic enzymes showed no change in activity under the same conditions, suggesting a specific mechanism. Hypoxia appears to increase the activities of all glycolytic enzymes whether rate-limiting or not, presumably increasing adenosine triphosphate availability despite decreased O₂ supply.     

Carbohydrate metabolism in two apple genotypes that differ in malate accumulation

In the apple variety 'Usterapfel', there are two known genotypes, which differ in malic acid content. One hundred days after full bloom, low-acid fruit (LA-fruit) contained 125 micromolg(-1) dry matter (DW) of malate, while the high-acid genotype (HA-fruit) reached levels up to 627 micromolg(-1) DW. There was no difference in the catalytic activity of enzymes involved in malate metabolism, such as PEPcarboxylase, malate dehydrogenase, and NADP malic enzyme. After [14C]glucose incorporation into the excised tissue of either genotype, the organic acid fraction was labeled to approximately the same extent. Furthermore, uptake of [14C]malate was significantly lower in excised tissue of LA-fruit. These findings suggest that low malate content in LA-fruit is the result of a restricted ability to accumulate malate in apple parenchyma cells. The different ability to accumulate malate had a pronounced effect on overall carbon partitioning. However, the rate of respiration and the rate of malate synthesis was similar in both genotypes. In HA-fruit, the glycolytic flux through pyruvate kinase was increased to compensate for the carbon that accumulated in the vacuole as malate. Since malate storage in the LA-fruit was restricted, it was more easily available for gluconeogenesis, and was correlated with a three-times higher activity of PEPcarboxykinase. LA-fruit showed higher concentrations of ATP, which stimulated Glc6P and fructose-6-phosphate formation. The elevated hexosephosphate content led to an enhanced partitioning of carbon into starch (+40%), hemicellulose (+104%), and sucrose (+40%) in more mature fruit. The activation of carbohydrate synthesis resulted in a significant drop in glucose-1-phosphate (Glc1P). To meet the increased demand for Glc1P, the activities of neutral and acid invertase, hexokinase, and phosphoglucomutase were higher in LA-fruit. Glucose was a more versatile substrate for this metabolic route than was fructose. It was also evident that glycolytic flux in apple was dependent on glucose level, and that the reaction catalysed by phosphoglucomutase contributed to the regulation of carbon partitioning between malate and carbohydrate polymers.     

Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation.

The rate at which Z. mobilis (Entner-Doudoroff pathway) converts high concentrations of glucose (20%) into ethanol plus CO2 changes as ethanol accumulates in the surrounding broth. This decline in glycolytic activity (per milligram of cell protein) does not result from inhibitory effects of ethanol, which can be reversed immediately by ethanol removal. The peak of fermentative activity (58 mumol of CO2 evolved per mg of cell protein per h) occurred after the accumulation of 1.1% ethanol (18 h) and declined to one-half this rate after 30 h (6.2% accumulated ethanol), although the cell number continued to increase. These times corresponded to the end of exponential growth and to the onset of the stationary phase (on the basis of measurement of cell protein), respectively. An examination of many of the requirements for fermentation (nucleotides, magnesium, enzyme levels, intracellular pH, delta pH) revealed three possible reasons for this early decline in activity: decreased abundance of nucleotides, a decrease in internal pH from 6.3 to 5.3, and a decrease in the specific activities of two glycolytic enzymes (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase). 31P nuclear magnetic resonance spectra of perchlorate extracts from cells fermenting in broth revealed very low levels of glycolytic intermediates (Entner-Doudoroff pathway) in cells examined at the peak of fermentative activity (18-h cells) in comparison with cells examined at a later stage (30-h cells), consistent with limitation of the fermentation rate by glycolytic enzymes near the end of the pathway. It is likely that cell death (loss of colony-forming ability) and the collapse of delta pH also contribute to the further decline in fermentative activity after 30 h.     

Cathepsin D and other hydrolases in the kidney of streptozotocin-diabetic mice. Possible relevance to microangiopathy

The lysosomal enzymes cathepsin D (E.C. 3.4.23.5), alpha-glucosidase (E.C. 3.2.1.20) and beta-galactosidase (E.C. 3.2.1.23), potentially involved in the breakdown of the peptide component and the disaccharide units of basement membrane glycoproteins, were studied in the kidney cortex and liver of streptozotocin-diabetic mice. In the liver of diabetic mice, as compared to controls, an increase was found for the total activity (measured in frozen-thawed homogenates) of cathepsin D (+135%, P less than 0.01) and beta-galactosidase (+32%, P less than 0.05). In the kidney a decrease was observed for both the free activity (measured in 12,000 g supernatant) and the total activity of these two enzymes (cathepsin D: -62% and -24%; beta-galactosidase: -29% and -23%; P less than 0.05 in all instances). Alpha-glucosidase did not show significant changes in either tissues. Total protein content of the two organs did not change significantly with diabetes and therefore cannot account for the enzyme alterations observed. These data indicate that the response of kidney to diabetes is opposite to that of liver (decrease versus increase in catabolic enzymes), and suggest decreased degradation of basement membrane in some tissues in diabetes, which may contribute to the thickening of basement membrane and therefore to the development of microangiopathy.     

Phorbol ester regulation of the human gamma-glutamyltransferase gene promoter

In the present study the molecular mechanisms underlying tetradecanoylphorbol-13-acetate (TPA) mediated regulation of the human gamma-glutamyltransferase (GGT) gene were examined. TPA challenge of HeLa cells resulted in an increase of GGT mRNA and enzyme activity. Deletion analysis of the promoter revealed that the -348 to +60 fragment was able to mediate TPA induced expression. Gel shift and supershift analyses showed that TPA treatment increased nuclear protein binding to a putative AP-1 site (-225 to -214) and that c-Jun was part of the complex. This AP-1 element, when cloned either in its native arrangement or as tandem repeat 5' of the minimal thymidine kinase promoter, mediated a significant increase of luciferase activity after TPA treatment of transfected HeLa cells, while its mutated counterpart abolished the induction. The same AP-1 element was able to mediate TPA induced expression in HepG2 cells. Collectively these results indicate that like other GSH metabolising enzymes, GGT too is a target for AP-1 mediated regulation.     

Bcl-2 inhibits tumor necrosis factor-alpha-mediated increase of glycolytic enzyme activities and enhances pyruvate carboxylase activity

To understand the effects of bcl-2 on glucose metabolism and tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity, the activities of glycolytic enzymes (hexokinase, 6-phosphofructo-1-kinase, and pyruvate kinase), lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were examined with or without TNF-alpha treatment in TNF-alpha sensitive L929 cells and TNF-alpha resistant bcl-2 transfected L929 cells. In TNF-alpha-treated L929 cells, the activities of the glycolytic enzymes and lactate dehydrogenase greatly increased, but there was no detectable change in phosphoenolpyruvate carboxykinase. Pyruvate carboxylase activity decreased by about 25% between 6 and 12 h after TNF-alpha treatment. The activities of the glycolytic enzymes and lactate dehydrogenase in bcl-2 transfected L929 cells were lower than in L929 cells upon TNF-alpha treatment. On the other hand, the activity of pyruvate carboxylase was 20-100% greater after 6 h of TNF-alpha treatment than in the L929 cells. The activity of phosphoenolpyruvate carboxykinase of bcl-2 trasfected L929 cells was lower by up to 25% than in L929 cells after 12 h. The increase of pyruvate carboxylase activity and decrease of phosphoenolpyruvate carboxykinase activity in bcl-2 transfected L929 cells may contribute to the protective effects of bcl-2 against TNF-alpha mediated cytotoxicity.     

T-leukemia cell lines CCRF-CEM, HPB-ALL, JM and MOLT-4: changes in isoenzyme profiles during induction of differentiation

Biochemical analysis has been used to monitor the induction of differentiation in cultured human T-leukemia cell lines (CCRF-CEM, HPB-ALL, JM and MOLT-4) by the phorbolester 12-0-tetradecanoylphorbol 13-acetate (TPA). The isoenzymes of carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase were separated by isoelectric focusing on horizontal thin-layer polyacrylamide gels and stained by histo-cytochemical methods. TPA inhibited the proliferative activity in all four cell lines and led to aggregation of cells seen as floating clusters. TPA induced an increase in number and staining intensity of isoenzymes of all four enzymes in the cell lines studied. This corresponds to an induced isoenzymatic maturation as the progressive increase in number and staining intensity of the isoenzymes parallels the differentiation along the T-cell pathway. However, regardless of the initial stage of arrested differentiation, the cell lines could be induced only to differentiate to a certain more mature stage, but could not be triggered to differentiate terminally with regard to expression of isoenzyme patterns.     

Effect of phorbol myristate acetate and concanavalin A on the glycolytic enzymes of human peripheral lymphocytes

The effects of Concanavalin A and the tumor promoting agent, phorbol 12-myristate 13-acetate (PMA), on glycolytic enzymes in human peripheral lymphocytes have been studied. A combination of Concanavalin A plus PMA stimulates DNA and protein synthesis to a significantly greater extent than when each are added individually. PMA and concanavalin A together, but not individually, also increase the levels of the activity of the glycolytic enzymes in peripheral lymphocytes treated for 48 h. The increase in hexokinase activity induced by PMA plus concanavalin A appeared to be due to the expression of the isoenzyme form, hexokinase II. The results suggest that the expression of glycolytic enzymes in stimulated lymphocytes is a late event (perhaps associated with the S phase) which is regulated by a cellular signal system controlled by the combined action of PMA plus concanavalin A.