Studies on cell-free metabolism: Ethanol production by a yeast glycolytic system reconstituted from purified enzymes

A reconstituted glycolytic system has been established from individually purified enzymes to simulate the conversion of glucose to ethanol plus CO₂ by yeast. Sustained and extensive conversion occurred provided that input of glucose matched the rate of ATP degradation appropriately.ATPase activity could be replaced by arsenate, which uncoupled ATP synthesis from glycolysis. The mode of uncoupling was investigated, and it was concluded that the artificial intermediate, 1-arseno-3-phosphoglycerate, has a half-life of no more than a few milliseconds. Arsenate at 4 mM concentration could simulate the equivalent of 10 μmol ml^?1 min^?1 of ATPase activity.The reconstituted enzyme system was capable of totally degrading 1 M (18% w/v) glucose in 8 h giving 9% (w/v) ethanol. The levels of metabolites during metabolism were measured to detect rate-limiting steps.The successful operation of the reconstituted enzyme system demonstrates that it is possible to carry out complex chemical transformations with multiple enzyme systems in vitro.     

Physiology of pyruvate metabolism in Lactococcus lactis

Lactococcus lactis, a homofermentative lactic acid bacterium, has been studied extensively over several decades to obtain sometimes conflicting concepts relating to the growth behaviour. In this review some of the data will be examined with respect to pyruvate metabolism. It will be demonstrated that the metabolic transformation of pyruvate can be predicted if the growth-limiting constraints are adequately established. In general lactate remains the major product under conditions in which sugar metabolism via a homolactic fermentation can satisfy the energy requirements necessary to assimilate anabolic substrates from the medium. In contrast, alternative pathways are involved when this energy supply becomes limiting or when the normal pathways can no longer maintain balanced carbon flux. Pyruvate occupies an important position within the metabolic network of L. lactis and the control of pyruvate distribution within the various pathways is subject to co-ordinated regulation by both gene expression mechanisms and allosteric modulation of enzyme activity.     

Revisiting the Crabtree/Warburg effect in a dynamic perspective: a fitness advantage against sugar-induced cell death

The mechanisms behind the Warburg effect in mammalian cells, as well as for the similar Crabtree effect in the yeast Saccharomyces cerevisiae, are still a matter of debate: why do cells shift from the energy-efficient respiration to the energy-inefficient fermentation at high sugar concentration?

This review reports on the strong similarities of these phenomena in both cell types, discusses the current ideas, and provides a novel interpretation of their common functional mechanism in a dynamic perspective. This is achieved by analysing another phenomenon, the sugar-induced-cell-death (SICD) occurring in yeast at high sugar concentration, to highlight the link between ATP depletion and cell death.

The integration between SICD and the dynamic functioning of the glycolytic process, suggests that the Crabtree/Warburg effect may be interpreted as the avoidance of ATP depletion in those conditions where glucose uptake is higher than the downstream processing capability of the second phase of glycolysis. It follows that the down-regulation of respiration is strategic for cell survival allowing the allocation of more resources to the fermentation pathway, thus maintaining the cell energetic homeostasis.     

Inhibition of the glycolytic pathway by methylglyoxal in human platelets

The incubation of human platelets with methylglyoxal and glucose produces a rapid transformation of the ketoaldehyde to D-lactate by the glyoxalase system and a partial reduction in GSH. Glucose utilization is affected at the level of the glycolytic pathway. No effect of the ketoaldehyde on glycogenolysis and glucose oxidation through the hexose monophosphate shunt was demonstrated. Phosphofructokinase, fructose 1,6 diphosphate (F1, 6DP) aldolase, glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate mutase were mostly inhibited by methylglyoxal. A decrease in lactate and pyruvate formation and an accumulation of some glycolytic intermediates (fructose 1,6 diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate) was observed. Moreover methylglyoxal induced a fall in the metabolic ATP concentration. Since methylglyoxal is an intermediate of the glycolytic bypass system from dihydroxyacetone phosphate to D-lactate, it may be assumed that ketoaldehyde exerts a regulating effect on triose metabolism.     

Biochemistry of Entamoeba: A review

Entamoeba histolytica, the protozoan parasite causing amoebiasis, is carried by approximately 10% of the world's population although only a minority of carriers of the organism present active clinical symptoms. Although Entamoeba are classified as eukaryotes, the biochemistry of these organisms has a number of unusual facets which are reminiscent of prokaryotes. It has indeed been suggested that these cells represent early evolutionary forms that have been successful in surviving unchanged in the protected environment in which they reproduce (the intestine). Glycolysis in Entamoeba lacks several of the typical eukaryotic glycolytic enzymes. The pentose phosphate shunt enzymes are missing completely. Another unusual feature of the glycolytic path is the utilization of PP_i instead of ATP in a number of enzyme reactions e.g. phosphofructokinase. Entamoeba is the only eukaryote in which one of these PP_i utilizing enzymes, phosphoenolpyruvate carboxytransferase, has been found. The respiratory pathway is also an unusual one. Entamoeba are classified as anaerobes but the cells do have an affinity for oxygen. The oxygen is reduced to water at the end of a respiratory chain which is not well understood but which operates withough cytochromes or mitochondria. The nucleic acid, protein and lipid metabolic pathways have not been well studied and interest has mainly focused on the proteolytic processes of the amoeba which have been implicated in the pathogenic, histolytic behaviour of the parasite. Despite these efforts the mechanism of attack of the parasite and the stimuli that cause it to invade the host are not yet clear. This understandably is the goal of much of the present studies concerning E. histolytica but the organism also deserves study in its own right as an example of an organism that has an unusual biochemistry and may represent an early stage in the evolution of eukaryotic cells.     

探讨髓系白血病细胞株糖酵解表型特征

探讨髓系白血病细胞株的糖酵解表型特征及其潜在的调控机制。葡萄糖试剂盒和乳酸试剂盒分别检测5株白血病细胞培养上清液中的葡萄糖消耗（G）和乳酸生成含量（L）,计算L／G比值来评估糖酵解水平：定量PCR检测糖酵解相关基因GLUT、MCTlmRNA表达;CCK8法检测细胞体外增殖能力;Western blot检测NAKT蛋白磷酸化水平。结果显示,KG1和K562细胞体外培养24h后的L／G比值分别为1．78和1．71,接近糖酵解表型时L／G为2的比值,同时这两株细胞高表达糖酵解相关基因GLUTl和MCT1mRNA。低糖（0．5mmol／L）、中糖（5mmol／L）、高糖（10mmol／L）处理KGla和K562细胞40h后,两株细胞的增殖能力、葡萄糖消耗和乳酸生成随葡萄糖浓度增加而增强,高糖组增加更为显著（P〈0．05）。相反,若糖酵解抑制剂2-DG（0,5,10mmol／L）处理白血病细胞40h后,两株细胞的增殖能力及糖酵解代谢水平随2．DG浓度增加而降低,高浓度2．DG组（10mmol／L）降低更为显著（P〈0．05）。此外,AKT抑制剂低浓度（5gmol／L）短时间（12h）处理后能抑制白血病细胞AKT蛋白磷酸化水平,同时降低细胞的葡萄糖消耗和乳酸生成（P〈0．05）。该研究提示髓系白血病细胞具有高糖酵解表型,AKT可能参与调控白血病的糖代谢过程,这有助于阐明白血病的能量代谢特征以及为白血病的靶向抗代谢治疗奠定基础。     

Identification and quantitation of fatty acid ethyl esters in biological specimens

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.     

PKM2, cancer metabolism,and the road ahead

A major metabolic aberration associated with cancer is a change in glucose metabolism. Isoform selection of the glycolytic enzyme pyruvate kinase has been implicated in the metabolic phenotype of cancer cells, and specific pyruvate kinase isoforms have been suggested to support divergent energetic and biosynthetic requirements of cells in tumors and normal tissues. PKM2 isoform expression has been closely linked to embryogenesis, tissue repair, and cancer. In contrast, forced expression of the PKM1 isoform has been associated with reduced tumor cell proliferation. Here, we discuss the role that PKM2 plays in cells and provide a historical perspective for how the study of PKM2 has contributed to understanding cancer metabolism. We also review recent studies that raise important questions with regard to the role of PKM2 in both normal and cancer cell metabolism.     

Uptake and metabolism of pyruvate and glucose by individual sheep preattachment embryos developed in vivo

The uptake of pyruvate and glucose by individual sheep oocytes and preattachment sheep embryos at each state of development up to the hatching blastocyst was determined using a microfluorescence technique. After an initial increase at fertilization, pyruvate uptake was relatively constant (?15 pmol/embryo/h) from the zygote through to the morula. Upon blastocyst formation and hatching, there were significant increases in uptake (39 pmol/embryo/h, P < 0.001; and 53 pmol/embryo/h, P < 0.001, respectively). In contrast to that of pyruvate, glucose uptake was very low (?1 pmol/embryo/h) up to the time of genome activation (eight- to 16 cell stage), after which there were significant increases in uptake at each successive stage of development. By the hatching blastocyst stage, glucose uptake had reached 54 pmol/embryo/h. The ability of day-7 hatching blastocysts to oxidize pyruvate and glucose was determined indirectly by measuring the production of lactate when either substrate was present as the sole energy source. Unlike the mouse blastocyst, which has a considerable oxidative capacity for both pyruvate and glucose, the day-7 sheep blastocyst showed limited ability to oxidise either substrate. Rather, in the sheep blastocyst, 65% of pyruvate and 98% of glucose taken up could be accounted for as lactate. Such low levels of substrate oxidation appear to be inconsistent with the energy requirements of the proliferating preattachment ruminant blastocyst. The utilization of alternative substrates at the blastocyst, such as amino acids, is proposed. © 1993 Wiley-Liss, Inc.     

Regulation of pyruvate metabolism in Lactococcus lactis depends on the imbalance between catabolism and anabolism

Two strains of Lactococcus lactis ssp. cremoris, MG 1820 and MG 1363, which differed by the presence or absence of the lactose plasmid, respectively, were cultivated in batch-mode fermentation on lactose as carbon substrate. A correlation between the rate of sugar consumption, the growth rate, and the type of metabolism was observed. The MG 1820 strain grew rapidly on lactose and homolactic fermentation occurred. The major regulating factor was the NADH/NAD(+) ratio proportional to the catabolic flux, which inhibited glyceraldehyde-3-phosphate dehydrogenase activity. This control led to an increase in metabolite concentration upstream of this enzyme, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, and inhibition of pyruvate formate lyase activity, while lactate dehydrogenase was strongly activated by the high coenzyme ratio. The contrary was observed during growth of the MG 1363 strain. Further investigation during growth of L. lactis ssp. lactis NCDO 2118 on galactose as carbon substrate and on various culture media enabling the growth rate to proceed at various rates demonstrated that the relative flux between catabolism and anabolism was the critical regulating parameter rather than the rate of glycolysis itself. In a minimal medium, where anabolism was strongly limited, the rate of sugar consumption was reduced to a low value to avoid carbon and energy waste. Despite this low sugar consumption rate, the catabolic flux was in excess relative to the anabolic capability and the NADH/NAD+ ratio was high, typical of a situation of nonlimiting catabolism leading to a homolactic metabolism.     

Effect of exogenous adenosine and monensin on glycolytic flux in isolated perfused normoxic rat hearts: Role of pyruvate kinase

We studied the effect of exogenous adenosine in isolated perfused normoxic rat hearts on glycolytic flux through pyruvate kinase (PK). We compared its effect with that of myxothiazol, an inhibitor of mitochondrial ATP production. Moreover, we tested whether an increase of membrane ionic flux with monensin is linked to a stimulation of glycolytic flux through PK. After a 20-min stabilization period adenosine, myxothiazol or monensin were administrated to the perfusate continuously at various concentrations during 10 min. The contraction was monitored and the lactate production in coronary effluents evaluated. The amount of adenine nucleotides and phosphoenolpyruvate was measured in the frozen hearts. Myxothiazol induced a decrease of the left ventricular developed pressure (LVDP : −40%) together with a stimulation of glycolytic flux secondary to PK activation. In contrast, adenosine primarily reduced heart rate (HR: −30%) with only marginal effects on LVDP. This was associated with an inhibition of glycolysis at the level of PK. The Na⁺ ionophore monensin affected HR (+14%) and LVDP (+25%). This effect was associated with a stimulation of glycolysis secondary to the stimulation of PK. These results provide new information of action of adenosine in the heart and support the concept of a direct coupling between glycolysis and process regulating sarcolemmal ionic fluxes.