Phosphofructose Kinase Research Articles

Interplay of CDKs and Cyclins with glycolytic regulatory enzymes PFK and PK.

In plants, as in all eukaryotes, the cell cycle is regulated by the heterodimer formed by cyclins (Cycs) and cyclin-dependent kinases (CDKs), that phosphorylate serine/threonine residues in target proteins. The extensive involvement of these heterodimers in nuclear cell cycle-related processes has been demonstrated. However, recent findings have linked Cyc-CDK complexes to the regulation of cytosolic processes, including various metabolic pathways, suggesting close coordination between the cell cycle and catabolic/anabolic processes to maintain cellular energy homeostasis.This study extends the analysis of Cyc-CDK complex regulation in maize to two key regulators of glycolysis: phosphofructose kinase (PFK) and pyruvate kinase (PK). Both are cytosolic enzymes, highly regulated positively and negatively by different metabolites, showing a similar activation pattern in their homotetrameric form and low activity when as dimers/monomers. Each enzyme exhibits two putative minimal phosphorylation motives for Cyc-CDKs, conserved in some plant species and in four (PFK) and three (PK) isoforms in maize. This work demonstrates that both enzymes are active with fluctuating levels of activity along maize germination; also, that they associate with different maize Cycs and CDKs as demonstrated by pull-down assays, as well as their in vitro phosphorylation by recombinant CycD;2-CDKA or CycD2;2-CDKB complexes. Additionally, the inhibition of PFK and PK activity following phosphorylation by active Cycs-CDKB complexes obtained by immunoprecipitation from imbibed embryonic axis protein extracts suggests a narrow and negative regulation of glycolysis as the cell cycle progresses. A decreased carbon flow through this pathway is proposed to divert carbon from sugars towards the oxidative pentose phosphate pathway, thereby promoting de novo nucleic acid synthesis precursors to stimulate cell cycle progression.

Taurine reduces glycolysis of pig skeletal muscle by inhibiting HIF-1α signaling.

The aim of this study was to investigate the effect of taurine on skeletal muscle glycolysis in pigs. The results showed that dietary supplementation of taurine significantly reduced the activities of hexokinase (HK), phosphofructose kinase (PFK), and pyruvate kinase (PK) in finishing pigs. Meanwhile, taurine reduced the protein and mRNA expression levels of hypoxia inducible factor 1α (HIF-1α) and the mRNA expression of glycolytic enzyme related genes (such as HK type II, HK Ⅱ; pyruvate kinase M2, PKM2; lactate dehydrogenase A, LDHA). In addition, taurine reduced the expression of HIF-1α, lactate content, and the expression of glycolysis related genes in porcine myotubes. These results suggest that taurine may regulate glycolysis in skeletal muscle of finishing pigs through the HIF-1α signaling pathway. To further investigate the mechanism by which taurine affects skeletal glycolysis, HIF-1α activator dimethyloxalyl glycine (DMOG) was used to treat porcine myotubes, our results showed that DMOG significantly increased the protein and mRNA expression levels of HIF-1α, lactate content, and glycolytic enzyme (HK, PFK, PK, and LDH) activity, but taurine treatment significantly inhibited this effect. Taken together, these results of in vivo and in vitro experiments revealed that taurine reduces skeletal muscle glycolysis by inhibiting HIF-1α signaling.

Acute Myeloid Leukemia Cells Rely on the Glycolytic Enzyme PGK1 to Support Energy Production and Amino Acid Metabolism

Acute myeloid leukemia (AML) cells frequently display an increased dependence on glucose compared to healthy hematopoietic stem and progenitors (HSPCs). Furthermore, increased glucose metabolism is associated with worse outcomes in AML. However, the upstream regulators that drive glucose metabolism as well as how glucose is utilized in AML remains largely unknown. Here, we have discovered that the oncogenic transcription factor (TF), JUN supports AML, in part, by regulating glycolytic enzymes that utilize glucose to support both cellular energetics and amino acid metabolism. Having previously reported that certain aggressive AML sub-types depend on JUN, we next set out to identify JUN-regulated transcriptional programs that support AML. To do so, we engineered the human AML cell line, THP-1 to express either control (shNT) or JUN-targeting shRNAs and then performed RNA-seq on cells from each condition. Next, we carried out a functional enrichment analyses and discovered that genes encoding numerous enzymatic regulators of glycolysis were significantly down-regulated by JUN-inhibition. Using qPCR validation, we confirmed that JUN inhibition results in reduced mRNA expression of Hexokinase 1 & 2 (HK1 & HK2), Glucose-Phosphate Isomerase (GPI), Phosphofructose Kinase (PFKP), Aldolase A (ALDOA), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Phosphoglycerate Kinase (PGK1), Enolase 1 (ENO1) and Pyruvate Kinase Muscle (PKM). ChIP assays confirmed that JUN directly localizes to the promoters of each of these genes except for ALDOA and PKM. To assess whether the JUN-dependent changes in glycolytic gene expression translated to defects in the ability of AML cells to catabolize glucose, we performed a glycolysis stress test on shNT- and shJUN-expressing human AML cell lines (e.g. THP-1, NOMO-1 and MOLM-14). From this assessment, we found that JUN inhibition resulted in a complete loss of glucose catabolism in all cell lines tested. We postulated that JUN activates glycolytic gene expression to support AML and thus, inhibition of JUN-regulated glycolytic genes will impede leukemia cell function. To test this hypothesis, we generated shRNAs targeting each of the JUN-regulated glycolytic enzymes. We then engineered mouse leukemia cells expressing the human leukemogenic allele, MLL-AF9 - found in both childhood and adult AML - to express these shRNAs and then assessed their colony forming capacity (CFC) in cytokine-enriched semisolid culture. In parallel, we also assessed how knockdown of each glycolytic enzyme impacted the CFC of healthy HSPCs. Notably, we observed that inhibition of each enzyme significantly impaired the CFC of MLL-AF9 leukemia cells but that inhibition of Pfkp, Aldoa or Pgk1 did not impact the CFC of healthy HSPC, suggesting that these enzymes were selectively important for AML. We next evaluated how knockdown of Pfkp, Aldoa or Pgk1 impacted leukemia propagation in vivo. Interestingly, inhibition of Pfkp or Pgk1, but not Aldoa, significantly delayed the onset of MLL-AF9 driven leukemia in vivo. Moreover, we found that a chemical inhibitor of PGK1, CBR-470-1, reduces the CFC of patient-derived AML cells in vitro. Since Pgk1 had not previously been studied in AML, we focused our attention on the its role in leukemia cell metabolism. We thus performed untargeted metabolomics of shNT- and shPgk1-expressing mouse MLL-AF9 cells using hydrophilic liquid chromatography mass spectrometry (HILIC-MS). PGK1 utilizes ADP to convert 1,3-bisphosphoglycerate into 3-phosphoglycerate (3-PG) and ATP. 3-PG can subsequently be used to generate phosphoenolpyruvate (PEP) or in the de novo synthesis of serine (Ser). As expected, we found that Pgk1 inhibition resulted in a significant decrease in 3-PG, PEP and Ser. We also observed that Pgk1 inhibition resulted in the decrease of several tricarboxylic acid cycle (TCA) intermediates that support oxidative phosphorylation energy production. Surprisingly, we also found that Pgk1 inhibition resulted in a marked decrease in several amino acids, including Alanine, Arginine and Histidine. We are currently investigating whether the decrease in these amino acids mediated by Pgk1 inhibition is due to an impediment of their biosynthesis or their consumption in gluconeogenic pathways. Collectively, our studies identify JUN as a previously unrecognized regulator of AML cell metabolism and shed new into how glucose is catabolized in AML cells.

Heart rot of Australian pineapples caused by Dickeya zeae

Pineapple plants (hybrid MD2) with bacterial heart rot were detected in a commercial plantation at Glasshouse Mountains, Queensland, in November 2015. The bacterial strain BRIP64263 isolated from infected tissue was shown to be a Gram negative soft-rotting bacterium capable of growth at 41 ºC, and based on its culture properties was provisionally identified as Dickeya. This strain was compared with other putative Dickeya strains affecting banana (BRIP64262) and potato (BRIP29490). Sequence analysis of the recombinase A genes of the pineapple strain placed it in phylotype I of D. zeae, whereas the banana strain was placed in phylotype II. This was confirmed by sequence comparisons for the phosphofructose kinase, RNA polymerase and aconitase genes which showed that the pineapple strain BRIP64263 is distinct from other strains that infect pineapples and other hosts in Australia and overseas. Furthermore, phylogenetic analysis of the replication initiation factor gene showed that strains affecting pineapples were distributed among both phylotypes of D. zeae, indicating multiple acquisitions or opportunistic infections of pineapple from this group of pathogens. The potato isolate, BRIP29490, was shown to be Rahnella aquatica, and is not likely to be pathogenic. It is not known whether the new isolate represents an incursion or whether it has long been associated with pineapples in Australia. Further study is required to determine the epidemiological characteristics of this strain, and what threat it poses to Australian pineapple production.

Enabling the biosynthesis of malic acid in Lactococcus lactis by establishing the reductive TCA pathway and promoter engineering

Lactococcus lactis, a homofermentative lactic acid bacterium, is widely applied in fermentation industry. In this study, a reductive TCA pathway was constructed in L. lactis for the biosynthesis of malic acid, a C4-dicarboxylic acid platform molecule. First, genes encoding malate dehydrogenase (mdh) from Actinobacillus succinogenes and pyruvate carboxylase (pycAY715T) from L. lactis were expressed in L. lactis NZ9000 to establish the reductive TCA pathway. The gene encoding lactate dehydrogenase (ldh) was simultaneously deleted. Malic acid was accumulated in the resulting strain NZ02-1 with a titer of 1.92 g/L. Subsequently, genes including ldhB, ldhX, als, and pfl, were deleted, increasing the titer to 3.13 g/L in strain NZ03. A promoter library was developed based on the core region of the phosphofructose kinase promoter Ppfk, and a stronger, constitutive promoter was screened for the enhanced expression of pycAY715T, leading to an increased titer of 5.75 g/L in strain NZ20. Through fed-batch fermentation, the titer in strain NZ20 was boosted to 14.1 g/L with a yield of 0.19 g/g glucose. Through a dual-phase fermentation, the titer was improved to 20.52 g/L with a yield of 0.29 g/g. Our study enabled the biosynthesis of malic acid in L. lactis, expanding the application of this strain as a chassis strain in fermentation industry.

Sex Modulates Lactobacillus johnsonii N6.2 and Phytophenol Effectiveness in Reducing High Fat Diet Induced mTOR Activation in Sprague-Dawley Rats.

Metabolic syndrome (MetS) is the underlying cause of some devastating diseases, including type 2 diabetes and cardiovascular disease. These diseases have been associated with over-activation of the mechanistic Target of Rapamycin (mTOR) pathway. This study utilizes a high fat diet (HFD) to induce MetS and to dissect the effects of a beneficial bacterium, L. johnsonii N6.2, and natural phenolics on mTOR complex 1 (mTORC1) expression compared to a reduced energy density diet (REDD). HFD significantly elevated MetS markers in males, as noted through an increase in weight, glucose levels, and triglyceride levels. Treatments were effective in reducing mTORC1-activating phosphorylation of pAKT-T308 and pAKT-S473 (p = 0.0012 and 0.0049, respectively) in HFD-fed females, with the combined treatments of L. johnsonii and phytophenols reducing phosphorylation below REDD-fed control levels, and significantly below HFD-fed control levels. Meanwhile, diet was the significant factor influencing male mTORC1-activating phosphorylation (p < 0.0001), as treatments were only effective in reducing phosphorylation in REDD-fed animals. Downstream analysis of mTORC1 activated genes phosphogluconate dehydrogenase (pgd) and phosphofructose kinase (pfk) followed this similar trend, enforcing the significant effect sex has on a treatments’ ability to modulate diet induced abnormalities. Analyzing mTORC1 stimulators such as insulin, inflammatory cytokines, and tryptophan, revealed no significant differences among groups. These results indicate that the effects observed on mTORC1 are a direct consequence of the treatments, and not exerted indirectly via the modulation of stimuli. This study highlights the potential use of commensal microorganisms and natural compounds in reducing the onset of metabolic diseases through mTORC1.

L-Asparaginase More Effectively Targets Leukemic Cells with Low Glycolytic Activity

Since O. Warburg first proposed aerobic glycolysis to be the main bioenergetic pathway activated in cancer cells, there has been near silence on the differences of normal and cancer metabolism. Our previous findings revealed that L-asparaginase (ASNase) triggers metabolic reprogramming of leukemic cells which is part of the adaptation process to stress caused by amino acid depletion (Hermanova et al, Leukemia 2015). We were interested if metabolic predisposition could influence the treatment response to ASNase and daunorubicine (DNR).We studied metabolic profile of 19 leukemic cell lines covering both lymphoid (B and T cell precursor acute lymphoblastic leukemia - BCP-ALL, T-ALL) and myeloid (acute and chronic myeloid leukemia - AML, CML) lineages. First we determined the level of glycolytic activity (GA) using Seahorse Bioanalyser XF24. GA was measured as extracellular acidification rate (ECAR) and divided into four stages: basal acidification (non-glycolytic acidification), glycolysis, glycolytic capacity (maximal capacity after inhibition of oxidative phosphorylation (OXPHOS)) and glycolytic reserve (the difference between glycolytic capacity and glycolysis). GA clustered together myeloid leukemic cell lines and T-ALL with overall higher GA in comparison to BCP-ALL cell lines which gathered in the second cluster. To find the marker of GA we measured expression of genes involved in GA and showed GLUT1 (glucose transporter 1) and MCT-4 (monocarboxylate transporter 4) to correlate most significantly (r=0.634, p=0.0072; r=0.532, p=0.024) with increased glycolysis. PFKM (phospho-fructose kinase muscle) gene expression was negatively associated with basal acidification (r=-0.568, p=0.01) and glycolytic reserve (r=-0.540, p=0.02) of leukemic cells.Another bioenergetic process, OXPHOS was measured as oxygen consumption rate (OCR). We divided OXPHOS analysis into four energetic states: basal respiration, ATP production (ATP synthesis is inhibited), maximal capacity of respiration (transport of electrons to oxygen is not limited) and spare capacity (the difference between maximal capacity and basal respiration). The OCR data in cluster analysis separated myeloid and lymphoid leukemias. Nevertheless, activity of OXPHOS positively correlated with GA in all studied cell lines showing myeloid cells to have higher oxidative metabolism compared to lymphoid cells.Next we correlated GA and OXPHOS with IC50 of ASNase and DNR determined using MTS cytotoxic assay, representing the level of sensitivity to the drug. In general, higher GA correlated with higher IC50 of ASNase (thus lower sensitivity to ASNase): basal acidification r=0.641, p=0.003; glycolysis r=0.645, p=0.003; glycolytic capacity r=0.680, p=0.001 and glycolytic reserve r=0.790, p<0.0001. Analysing lymphoid and myeloid lineage independently we observed striking difference. While low GA correlated with higher sensitivity to ASNase in lymphoid cells (basal acidification: r=0.704, p=0.02; glycolytic capacity r=0.693, p=0.024 and glycolytic reserve r=0.648, p=0.04), in myeloid cells correlation with higher sensitivity to ASNase was observed only for the glycolytic reserve (r=0.775; p=0.0211). There was no such correlation between IC50 of ASNase and OXPHOS. Sensitivity to DNR correlated neither with GA nor OXPHOS across the entire sample set. However, in lymphoid cell lines there was a correlation of lower sensitivity to DNR with enhanced GA and OXPHOS (basal acidification r=0.848, p=0.001; glycolytic capacity r=0.796, p=0.004 and glycolytic reserve r=0.897, p=0.0001; basal respiration r=0.747, p=0.01; ATP production r=0.716, p=0.02; maximal capacity r=0.742, p=0.01).In conclusion, we found significant difference between the key metabolic activities in myeloid and BCP and T lymphoid malignant cells. Our data suggest association between basal glycolytic state of leukemic cells and response to ASNase and DNR. Lower GA of lymphoid cells (in particular BCP) is associated with higher sensitivity to ASNase and DNR. In myeloid cells GA does not correlate with sensitivity to ASNase and DNR probably due to overall high GA. We assume that activation of glycolytic reserve under metabolic stress can affect the sensitivity to ASNase in myeloid cells. The effect of ASNase and DNR seems to be related to metabolic state of leukemic cells only in lymphoid leukemic cells.Supported by IGA NT1249, 15-28848A, UNCE204012. DisclosuresNo relevant conflicts of interest to declare.

Inhibitory effect of over-expression of Lin28A on glycosis in human gastric cancer cells and the underlying mechanism

Objective To explore the effects of over-expression of Lin28A on glycolysis of gastric cancer cells and the underlying mechanism. Methods Human gastric cancer BGC-823 cells in vitro were stably transfected with lentivirus-mediated over-expression of Lin28A. Lactic acid levels of cell culture medium were detected with biochemical method, phosphofructose kinase(PFK) levels in cells with ELISA, the expression levels of Lin28A, hypoxia-inducible factor (HIF)-1α, glucose transporter (GLUT)-1, and pyruvate kinase M2 (PKM2) were detected using Western blotting, and let-7a RNA using reverse transcription-PCR. Annexin V-allophycocyanin (APC) staining method was used to assess cell apoptosis. Results Compared with the normal control(CON) group and the negative control(NC) group, Lin28A protein level in the over-expression (OE) group was significantly higher (both P<0.001). RNA expression level of let-7a was significantly lower in the OE group than in the NC group (0.602±0.017 vs. 1.001±0.063, P=0.005 2). The levels of the HIF-1α, GLUT-1, PKM2 proteins were all significantly lower in the OE group than in the CON and the NC groups (all P<0.001). The lactic acid levels of cell culture medium in the CON, the NC, and the OE groups were (1.71±0.13), (1.53±0.11), (1.24±0.04)mmol/L, which was significantly lower in the OE group than in the CON group and the NC group (P=0.017 0, 0.031 0). Intracellular PFK level in the OE group was also significantly lower than in the CON group and the NC group [(1.79±0.05)mmol/L vs. (3.71±0.13)mmol/L, P=0.014 0; (1.79±0.05)mmol/L vs. (3.49±0.14)mmol/L, P=0.036 0]. Apoptosis rates of the CON, the NC, and the OE groups were 5.02%±0.14%, 7.16%±0.21%, 10.39%±0.37%, respectively, which was significantly higher in the OE group than in the CON group and the NC group (P=0.000 5, 0.000 7). Conclusion Over-expression of Lin28A can inhibit the expression of let-7a, and induce apoptosis of human gastric cancer cells by suppressing glycolysis of the cells. Key words: Lin28A; Gastric cancer; Glycolysis; Apoptosis

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10−10 cm2 s−1) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10−7 cm2 s−1 (parvalbumin) to 0.20 × 10−7 cm2 s−1 (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.

Efflux Time Courses of Cytosolic Proteins from Rabbit Skinned Muscle Fibers Reflect Dissociation of Enzyme Complexes

In many multi-system metabolic diseases characterized by mitochondrial dysfunction (e.g. Myalgic Encephalomyelitis), glycolysis appears to be an important compensatory pathway for generating ATP. It is postulated that glycolytic enzymes form a complex that enhances the rate of ATP production through substrate channeling of metabolic product intermediates. To search for evidence of a reduced diffusion coefficient indicative of supramolecular complexes, we examined the efflux of endogenous glycolytic enzymes from rabbit psoas muscle fibers. Single fiber segments were skinned in oil and transferred to physiological salt solution. Cytosolic proteins that diffused into the solution were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and cytomatrix binding. Optimization of model to data returned estimates of apparent diffusion coefficients that were very low at the onset of diffusion (∼10−10 cm2 s−1) but increased with time as cytosolic protein density decreased. The initial values are consistent with the presence of complexes in situ; higher later values (e.g., 0.2 × 10−7 cm2 s−1 for phosphofructose kinase), with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of ATP at rates beyond that possible with randomly distributed enzymes, thereby matching supply with demand. Metabolic channeling may allow glycolysis to better compensate for reduced ATP production in aerobic metabolic diseases.

FRI0024 Multiplatform metabolomic study of cd4+ cells after methotrexate and infliximab treatment: focus on safety and tolerability profiles

BackgroundActivated CD4+ cells comprise a large proportion of inflammatory cells involved in the development of rheumatoid arthritis (RA). Inflammatory cytokines, as tumor necrosis factor-a (TNFa), have also been implicated in...

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

Mitochondrial and sarcoplasmic protein changes in hearts from copper-deficient rats: up-regulation of PGC-1α transcript and protein as a cause for mitochondrial biogenesis in copper deficiency

Changes in mitochondrial and sarcoplasmic proteins using proteinomics and Western blotting in hearts from copper-deficient rats were explored in this study. Also, key enzymes that are involved in cardiac energy metabolism via glycolysis and fatty acid oxidation and related transcription factors were determined. Rats were fed one of two diets: a copper-adequate diet containing 6 mg Cu/kg diet or a diet with less than 1 mg Cu/kg diet for 5 weeks. Copper deficiency was confirmed by low liver copper levels, decreased hematocrit levels and cardiac hypertrophy. Proteinomic data revealed that of the more than 50 proteins identified from the mitochondrial fraction of heart tissue, six were significantly down-regulated and nine were up-regulated. The proteins that were decreased were beta enolase 3, carbonic anhydrase 2, aldose reductase 1, glutathione peroxidase, muscle creatine kinase and mitochondrial aconitase 2. The proteins that were up-regulated were isocitrate dehydrogenase, dihydrolipoamide dehydrogenase, transferrin, subunit d of ATP synthase, transthyretin, preproapolipoprotein A-1, GRP 75, alpha-B crystalline and heat shock protein alpha. Follow-up Western blots on rate-limiting enzymes in glycolysis (phosphofructose kinase), fatty acid oxidation (medium chain acyl dehydrogenase, peroxisome proliferator-actvator receptor-α or PPARα) and gluconeogenesis (phosphoenolpyruvate carboxykinase) did not reveal changes in metabolic enzymes. However, a significant increase in peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α protein, as well as the transcript, which increased 2.5-fold, was observed. It would appear that increased mitochondrial biogenesis known to occur in copper deficiency hearts is caused by an increased expression in the master regulator of mitochondrial biogenesis, PGC-1α.

The halothane gene, energy metabolism, adenosine monophosphate-activated protein kinase, and glycolysis in postmortem pig longissimus dorsi muscle1

The presence of the halothane gene results in PSE meat. However, the exact mechanisms linking the halothane gene and the incidence of PSE meat remain unclear. We hypothesize that the presence of the halothane gene accelerates energy consumption in postmortem muscle, which activates adenosine monophosphate-activated protein kinase (AMPK), leading to enhanced glycolysis and PSE meat. To test our hypothesis, energy status, AMPK activity, and glycolysis in the postmortem LM of the halothane gene carrier and halothane-negative pigs were compared. The results showed that the presence of the halothane gene accelerated energy depletion in postmortem muscle immediately after exsanguination, leading to rapid and early depletion of ATP, as shown by an increase in the (adenosine monophosphate + inosine monophosphate):ATP ratio in postmortem LM. In addition, an early AMPK activation was observed in LM from halothane carriers. The fructose-2,6-diphosphate concentration in postmortem LM was well correlated with AMPK activation. To be a potent stimulator of phosphofructose kinase, the increase in fructose-2,6-diphosphate is expected to activate phosphofructose kinase, a key enzyme controlling glycolysis, leading to enhanced glycolysis and early accumulation of lactic acid. In summary, this study showed that the presence of the halothane gene induced early energy depletion, which could be a primary reason causing AMPK activation, leading to accelerated glycolysis and an increased incidence of PSE meat. However, AMPK might also be activated by other mechanisms besides energy depletion, which warrants further studies.

Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations.

Metabolic flux analysis based on 13C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF- mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF- mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.

An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei

The glycosomes of in vitro grown procyclic trypomastigote forms of Trypanosoma brucei were purified by three different procedures and the results compared by electron microscopy, enzyme assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Centrifugation on a self-forming Percoll gradient followed by a sucrose gradient centrifugation resulted in the least enriched glycosomal preparation. Centrifugation on a pre-formed Nycodenz gradient gave an improved preparation but the most homogeneous preparation of intact glycosomes was obtained after centrifugation on two successive sucrose gradients. Glycosomes purified by both the Nycodenz and double sucrose gradient procedures appeared larger than in situ glycosomes presumably due to an osmotic effect resulting from disruption of the granular matrix of the organelles. Nevertheless, there appears to be no loss of cisternal contents due to the swelling of the organelles. The glycosomes of the bloodstream from trypomastigotes purified by the same procedures show, however, no sign of swelling. A comparison of glycosomes purified from procyclic trypomastigotes and bloodstream form trypomastigotes prepared by the same double sucrose procedure demonstrated that in the glycosome of procyclic trypomastigotes: (1) activities of hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase and phosphoglycerate kinase and diminished by 80–100%; (2) activities of glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and glycerol-3-phosphate dehydrogenase remain unchanged or are only slightly reduced; (3) there is an appearance of four major new proteins, among which could be phosphoenol pyruvate carboxykinase and malate dehydrogenase. These observations are in basic agreement with those by Hart et al. (Mol. Biochem. Parasitol. 12, 25–35, 1984).

The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei.

Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei.

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.

Time-course of enzyme adaptation. I. Effects of substituting dietary glucose and fructose at constant concentrations of dietary protein.

The effect of dietary fructose on liver, kidney, and adrenal enzymes was studied in male Sprague–Dawley rats. Dietary fructose increased relative liver and kidney sizes, liver glycogen, and liver protein. The percentage increases in relative liver and kidney sizes were independent of dietary protein, but relative liver sizes were smaller in the absence of protein.The activities of all the liver enzymes studied, except glucokinase, were increased by a 65% fructose diet containing 25% casein. The rates of increases differed between enzymes; the activities of L-α-glycerophosphate dehydrogenase and phosphoenolpyruvate carboxykinase (PEP-carboxykinase) reached almost maximum in 1 day, glucose 6-phosphatase, 6-phosphogluconate dehydrogenase, and pyruvate kinase activities reached maximum in about 2 days, and glucose 6-phosphate dehydrogenase, malic enzyme, dihydroxyacetone kinase, phosphofructose kinase, and phosphohexose isomerase required at least 3 days to reach maximum activity after the dietary change. The increases in the activities of liver fructose 1,6-diphosphatase, glucose 6-phosphate dehydrogenase, pyruvate kinase, and phosphohexose isomerase did not occur in the absence of dietary protein. The activities of liver phosphofructokinase and malic enzyme were increased equally well whether the fructose diet contained protein or not, while the increases in the activities of other enzymes were less in the absence of dietary protein.The half-life of liver malic enzyme was estimated as 3 days in the glucose to fructose dietary change and 1 day in the fructose to glucose dietary change. Since malic enzyme activity was increased by fructose feeding about threefold, the data suggest that under the conditions of the experiment fructose increased malic enzyme activity by decreasing the rate of breakdown of the enzyme.In general, kidney enzymes were affected by fructose to a lesser extent than the corresponding enzymes in liver. This was particularly significant in the case of kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme, the activities of which were only slightly increased in kidney. The activities of these three enzymes in the adrenal glands were not increased by fructose feeding.