Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.     

Glucose metabolism by trout (Salmo trutta) red blood cells

Summary Glucose metabolism has been studied in Salmo trutta red blood cells. From non-metabolizable analogue (3-O-methyl glucose and l-glucose) uptake experiments it is concluded that there is no counterpart to the membrane transport system for glucose found in mammalian red blood cells. Once within the cells, glucose is directed to CO₂ and lactate formation through both the Embden-Meyerhoff and hexose monophosphate shunts; lactate appears as the most important endproduct of glucose metabolism in these cells. From experiments under anaerobic conditions, and in the presence of an inhibitor of pyruvate transfer to mitochondria, most of the CO₂ formed appears to derive from the hexose monophosphate pathway. Appreciable O₂ consumption has been detected, but there is no clear relationship between this and substrate metabolism. Key enzymes of glucose metabolism hexokinase, fructose-6-phosphate kinase and, probably, pyruvate kinase are out of equilibrium, confirming their regulatory activity in Salmo trutta red blood cells. The presence of isoproterenol, a catecholamine analogue, induces important changes in glucose metabolism under both aerobic and anaerobic conditions, and increases the production of both CO₂ and lactate. From the data presented, glucose appears to be the major fuel for Salmo trutta red blood cells, showing a slightly different pattern of glucose metabolism from rainbow trout red blood cells.Abbreviations EM Embden-Meyerhoff pathway - G6D glucose-6-phosphate dehydrogenase - GOT glutamate oxalacetate transaminase - GPI glucose phosphate isomerase - HK hexokinase - HMS hexose monophosphate shunt - IP isoproterenol - LDH lactate dehydrogenase - MCB modified Cortland buffer - OMG 3-O-methyl glucose - PFK fructose-6-phosphate kinase - PK pyruvate kinase - RBC red blood cells - TAC tricarboxylic acid cycle     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

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.     

Activity rhythms of enzymes in human red blood cell suspensions

Abstract

We have established the presence of a rhythm in the activity of 4 enzymes in in‐vitro cell suspensions of human red blood cells. Glucose 6‐phosphate dehydrogenase and glutamate oxaloacetate transaminase demonstrated semicircadian patterns of activity, while acid phosphatese and acetylcholine esterase exhibited circadian activity rhythms. The ratios between the highest to lowest activities varied from 2:1 to 10:1 among the various enzymes. The affinity of glucose 6 phosphate dehydrogenase to its substrate and coenzyme remained constant throughout the cycle. No evidence was obtained for the presence of a soluble inhibitor at the lower levels of the activity. Sonication of hemolysates with low glucose 6 phosphate dehydrogense activity yielded additional activity comparable to that of the peak activity. Sonication of hemolysates from the time of the peak activity did not change the original activity. The observations point to a role of the cell membrane in the biological clock.     

Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite

Selected aspects of the metabolism of Plasmodium falciparum are reviewed, but conclusions based on the study of other species of plasmodia are intentionally not included since these may not be applicable. The parasites increase glucose consumption 50-100 fold as compared to uninfected red cells; most of the glucose is metabolized to lactic acid. The parasite contains a complete set of glycolytic enzymes. Some enzymes such a hexokinase, enolase and pyruvate kinase are vastly increased over corresponding levels in uninfected red cells. However, the pathway for synthesizing 2,3-diphosphoglycerate (2,3-DPG) is absent. Parasitized red cells show a decline in the concentration of 2,3-DPG which may function as an inhibitor for certain essential enzyme pathways. Pentose shunt activity is increased in absolute terms, but as a percent of total glucose consumption, there is a decrease during parasite infection of the red cell. The parasite contains a gene for G6PD and can produce a small quantity of parasite-encoded enzyme. It is not clear if the production of this enzyme can be up-regulated in G6PG deficient host red cells. The NADPH normally produced by the pentose shunt can be obtained from other parasite pathways (such as glutamate dehydrogenase). NADPH may subserve additional needs in the infected red cell such as driving diribonucleotide reductase activity--a rate limiting enzyme in DNA synthesis. The role of NADPH in protecting the parasite-red cell system against oxidative stress (via glutathione reduction) remains controversial. Parasitized red cells contain about 10 times more NAD(H) than uninfected red cells, but the NADP(H) content is unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

Organization of enzymes of glycolysis and of glutathione metabolism in human red cell membranes.

1) The activities of 16 enzymes of glycolysis and of glutathione metabolism were determined in intact human red cell membranes (ghosts) which were prepared by hypotonic hemolysis. 2) Enzymes and hemoglobin of the ghosts were resolved by two toluene extractions. Only the four enzymes hexokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and pyruvate kinase could not be released completely from the ghosts. 3) The residual membrane fraction, which was obtained after the toluene extraction of ghosts prepared at 30 imOsM, contained 0.02% of the original hemoglobin content of the red cell. Between 6.5 and 23% of the hemolysate activities of glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase and fructose-bisphosphate aldolase were detected in this fraction after mechanical disruption. 4) Sonication of intact ghosts increased the activities of fructose-bisphosphate aldolase, pyruvate kinase and phosphoglycerate kinase. 5) In "white" ghosts prepared at 5 imOsM phosphate buffer which contained 0.5% of the original hemoglobin the activities of fructose-bisphosphate aldolase and glyceraldehyde-phosphate dehydrogenase were detected at high levels. The activities of pyruvate kinase and phosphoglycerate kinase were low in these preparations. 6) The results indicate that one part of all enzymes is loosely attached to the inner surface of the membrane as is hemoglobin. A second part, the "cryptic enzyme activity", is available after resolving by toluene. A residual part of four enzymes is firmly bound to the membrane. Two of them (fructose-bisphosphate aldolase and glyceraldehyde-phosphate dehydrogenase) are oriented toward the inner surface of the membrane, whereas pyruvate kinase and phosphoglycerate kinase are hidden in the lipid core of the membrane.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Improving the environment for immobilized dehydrogenase enzymes by modifying Nafion with tetraalkylammonium bromides

Recent research in our group has shown that mixture-casting Nafion with quaternary ammonium bromides can increase the electrochemical flux of redox couples through the membrane and allow for larger redox species to diffuse to the electrode surface. The research has also suggested that when these salts are cast with Nafion micellar pore size is changing. Therefore, it was proposed that the quaternary ammonium salts could be employed to tailor the structure of the Nafion membrane for immobilizing enzymes in the polymer. For cations with a high affinity for the sulfonic acid groups of Nafion, the modified structure of Nafion can also help to stabilize the enzyme and increase activity by providing a protective outer shell and an ideal chemical environment that resists a decrease in pH within the pore structure. This research examines the ability to immobilize dehydrogenase enzymes in Nafion that has been modified with quaternary ammonium bromides. Fluorescence assays, fluorescence microscopy, and cyclic voltammetric studies were employed to analyze the ability to immobilize an enzyme within the membrane, to determine the activity of the immobilized enzyme and to examine the transport of coenzyme within the membrane. Dehydrogenase enzymes immobilized in tetrabutylammonium bromide/Nafion membranes have shown high catalytic activity and enzyme active lifetimes of greater than 45 days. A variety of dehydrogenase enzymes have been successfully immobilized in the membrane, including: alcohol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, and lactic dehydrogenase.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Characterisation of G418-induced metabolic load in recombinant CHO and BHK cells: effect on the activity and expression of central metabolic enzymes

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo ^r protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of diabetes and insulin-induced hypoglycemia on hexokinase and glucose-6-phosphate dehydrogenase in red blood cells

Activities of hexokinase and glucose-6-phosphate dehydrogenase have been measured in red blood cells from control, diabetic and insulin treated rats. After an initial decrease, the enzyme activities increased, but remained lower than control levels. A reversal of the diabetes effect was seen with insulin administration. Insulin induced hypoglycemia increased both enzymes. An overall control of glucose metabolism by insulin in red blood cells was observed.     

Coordination of photosynthetic and respiratory metabolism in Chlorella vulgaris UAM 101 in the light

In Chlorella vulgaris UAM 101, the presence of glucose altered the photosynthetic and respiratory metabolism in the light. When glucose was added to the growth medium, an increase in the cellular level of enzymes involved in glucose oxidation, namely glucose-6-P dehydrogenase (EC 1.1.1.49) and NAD⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), was observed. Glucose also enhanced respiratory O₂ consumption. In addition, CO₂ released by glucose oxidation was refixed in photosynthesis. The presence of glucose also affected photosynthesis. Phosphoribulokinase (EC 2.7.1.19) and NADP⁺-dependent glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13), two regulatory enzymes of the reductive pentose phosphate cycle, were increased by glucose. However, Rubisco (EC 4.1.1.39) activity of these cells was lower than that of autotrophic cells. Despite these alterations, the photosynthetic O₂ evolution was not significantly inhibited by glucose. On the other hand, an increase in the cytosolic NADP⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.9) that is involved in obtaining reducing power for anabolic processes was observed. The CO₂ levels in the growth medium did not significantly affect the cellular level of enzymes measured in this work, except those involved in biosynthetic pathways. These data suggest that the effect of glucose on photosynthesis and respiration can be explained by alteration of the cellular level of photosynthetic enzymes and respiratory substrates, respectively.     

Activity of pentose phosphate pathway enzymes in alkane-oxidizing yeast cells]

The activity of the key enzymes of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase) was determined in cell-free homogenates of Candida lipolytica 695 and Candida tropicalis 303 growing on different carbon sources. The activity of these enzymes remained almost the same in the course of growth of both cultures. The activity of the enzymes differed only slightly in the cells metabolizing hexadecane and glucose. The activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the cell-free homogenates of C. tropicalis 303 was twice as high as in the cells of C. lipolytica 695. The activity of transketolase was the same in both cultures. The main role of the pentose phosphate pathway is presumed to consist not in catabolism of the carbon source, but in biosynthesis of pentoses and other important intermediates.     

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.     

低氧环境对三阴性乳腺癌细胞糖代谢途径的重编程

代谢改变是癌细胞的特征之一。研究表明,低氧会使癌细胞的糖代谢发生改变,但是更详细的分子机制仍有待进一步研究。本研究利用转录物组测序技术(RNA-sequencing,RNA-seq)和生物信息学分析发现,低氧导致BT549细胞中334个基因和MDA-MB-231细胞中215个基因在转录水平的表达改变。这些表达变化的基因多与糖代谢相关。进一步分析RNA-seq数据并应用Western 印迹、酶活性检测和代谢产物定量测定的结果显示,低氧通过升高BT549细胞中葡萄糖转运蛋白1(GLUT1)和MDA-MB-231细胞中GLUT1和GLUT3的表达以增加葡萄糖的摄入;低氧使催化糖的无氧氧化途径几乎全部反应的酶都至少有一种同工酶或酶蛋白亚基,以及调节酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)和4(PFKFB4)同工酶的表达增加来促进了糖的无氧氧化;低氧还通过增加调节丙酮酸脱氢酶激酶1(PDK1)和3(PDK3)同工酶基因的表达,以及降低关键酶异柠檬酸脱氢酶3(IDH3)同工酶、琥珀酸脱氢酶B亚基和D亚基的表达来减少糖的有氧氧化途径进行;低氧可能还增加磷酸戊糖途径的关键酶葡糖-6-磷酸脱氢酶、糖原合成途径的关键酶糖原合酶GYS1同工酶的表达以促进这2条途径的进行,而对糖异生和糖原分解代谢途径酶基因的表达影响较小。生物信息学分析乳腺癌组织样本在线数据库中糖代谢途径酶基因在转录水平表达结果与细胞研究结果基本一致。总之,该文系统分析了低氧对糖代谢6条代谢途径中全部酶以及2种重要调节酶的影响,可见低氧会通过改变这些酶的同工酶或亚基的基因表达使糖代谢途径进行重编程,这对进一步认识低氧环境下癌细胞糖代谢的分子机制具有一定的意义。     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.     

The relationship between red cell aging and enzyme activities in experimental animals.

1. The relationship between red cell aging and enzyme activities was studied in rabbit, guinea-pig, hamster, rats (F344/N and SD), and mice (BALB/c and DBA/2). 2. The activities of six enzymes: glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD), hexokinase (Hx), glutamate oxaloacetate transminase (GOT), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE), were measured in the red cells of different ages which were obtained either by centrifugation or experimental anaemia. 3. Hx, AChE and GOT activities were much higher in younger red cells than in older cells, hence the activities of these enzymes may be used as an indicator of age of the cells.