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1.
After grinding Trypanosoma brucei with alumina or silicon carbide, it is possible to prepare a multienzyme complex which catalyses the breakdown of glucose to l-glycerol-3-phosphate and 3-phosphoglycerate. The complex sediments with the postnuclear large granule fraction which pellets at 14,500g; it is also eluted in the void volume during Bio-Gel A-5m column chromatography of a cell homogenate. During isopycnic sucrose gradient centrifugation, the multienzyme complex bands at a density of 1.22 g/ml. Because this is the density of T. brucei microbodies, and because Triton X-100 treatment of the material greatly enhances the activities of its component enzymes, we conclude that the multienzyme complex is probably located in the microbodies of the bloodstream long slender forms of T. brucei.  相似文献   

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Adult pairs of Schistosoma mansoni convert glucose to lactate rapidly and almost quantitatively under aerobic and anaerobic conditions E. Bueding, 1950, Journal of General Physiology33, 475–495). Glycolysis is the principal source of energy of schistosomes and its inhibition by trivalent organic antimonials, at the phosphofructokinase step [EC 2.7.1.11], may be the basis for the chemotherapeutic effects of these agents E. Bueding and J. M. Mansour, 1957, British Journal of Pharmacology and Chemotherapy12, 159–165). We have developed standardized conditions for the comparison of rates of glucose consumption and lactate production by intact schistosomes in vitro and by centrifuged homogenates of worms. The rates of glycolysis of homogenates prepared from freshly isolated worms, and from worms that have been lyophilized immediately after harvesting and stored for prolonged periods at ?80 C were identical, when measured in media containing appropriate concentrations of glucose, NAD, ATP, MgCl2, KCl, and phosphate. The specific activities of the 11 glycolytic enzymes and of 3 related enzymes (fructose-biphosphatase [EC 3.1.3.11], glycerol-3-phosphate dehydrogenase [EC 1.1.1.8], and malate dehydrogenase [EC 1.1.1.37]) were measured in homogenates under optimal conditions. The profile of the relative activities of glycolytic enzymes of S. mansoni resembles closely that of Ehrlich ascites tumor cells, and differs markedly from that observed in erythrocytes or skeletal muscle. As is the case in many animal tissues, hexokinase [EC 2.7.1.1] was the enzyme of lowest specific activity, and the rate of glycolysis of homogenates was almost the same as the hexokinase activity. Several other lines of evidence support the view that the hexokinase reaction is the rate-limiting step in the glycolysis of worm homogenates. Hexokinase activity was not particulate in schistosome homogenates, and there was no detectable high Km glucokinase-like activity. The rate of glycolysis by homogenates exceeded that of intact worms by a factor of nearly 5. The contributions of glucose transport, availability of ADP and inorganic phosphate, regulatory enzymes, and a substrate cycle catalyzed by fructose-bisphosphatase are considered as possible mechanisms for the restraint of glycolysis in intact worms. The mechanisms contributing to the rapid rates of glycolysis of adult S. mansoni have not been identified, although several can be excluded (unusually high capacity of the glycolytic enzymes, the presence of mitochondrial hexokinase, the occurrence of glycosomes, and the operation of defective mitochondrial shuttles). In view of the regulatory role of hexokinase in the glycolysis of S. mansoni, inhibition of this enzyme is a potentially important target for the development of new antischistosomal drugs.  相似文献   

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In vitro, 4-amino-6-trichloroethenyl-1,3-benzenedisulfonamide, a potent fasciolicide, causes a potent concentration-dependent inhibition of glucose uptake by mature Fasciola hepatica. In F. hepatica treated with the disulfonamide and then fed [U-14C]glucose, there was a 60% inhibition of glucose utilization and a corresponding inhibition of acetate and propionate formation. Treated fluke parasites possessed much lower levels of adenosine triphosphate, phosphoenolpyruvate, glucose 6-phosphate, and fructose 6-phosphate than untreated parasites and contained higher levels of glycerol and the free sugars fructose and mannose. Direct measurement of the effect of the disulfonamide on the glycolytic enzymes of F. hepatica demonstrated that 3-phosphoglycerate kinase (EC 2.7.2.3) and phosphoglyceromutase (EC 2.7.5.3) were inhibited. It is therefore suggested that the fasciolicidal activity of 4-amino-6-trichloroethenyl-1, 3-benzenedisulfonamide is due to inhibition of the enzymes 3-phosphoglycerate kinase and phosphoglyceromutase which effectively blocks the Embden-Myerhof glycolytic pathway.  相似文献   

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Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme’s catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology.  相似文献   

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B. Liedvogel  R. Bäuerle 《Planta》1986,169(4):481-489
Chloroplasts from the cotyledons of mustard (Sinapis alba L.) seedlings were isolated on Percoll gradients, and showed a high degree of intactness (92%) and purity as judged by electron microscopy and marker-enzyme analysis (cytoplasmic contamination lower than 0.4% on a protein basis). The chloroplasts synthesized longchain fatty acids from both precursors [1-14C] acetate and [2-14C]pyruvate; maximum incorporation rates were 96 nmol·(mg Chl)-1·h-1 for acetate and 213 nmol·(mg Chl)-1·h-1 for pyruvate. Acetyl-CoA-producing enzymatic activities, namely acetyl-CoA synthetase (EC 6.2.1.1.) and a pyruvate dehydrogenase complex, showed specific activities of 14.8 nmol·(mg protein)-1·min-1 and 18.2 nmol·(mg protein)-1·min-1, respectively. The glycolytic enzymes phosphoglyceromutase (EC 2.7.5.3) phosphopyruvate hydratase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were all found to be components of these chloroplasts, thus indicating a possible pathway for intraplastid acetyl-CoA formation.Abbreviations ACS acetyl coenzyme A synthetase - Chl chlorophyll - DTE 1,4-dithioerythritol - PDHC pyruvate dehydrogenase complex - 3-PGA 3-phosphoglyceric acid  相似文献   

9.
To localise the controlling point of the glycolytic system, the temporal changes of concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) step; a stationary state with all glycolytic intermediates having concentrations oscillating at nearly stationary mean values; and a depletion state at which the intermediates before the GAPDH step are being depleted due to the exhaustion of glycogen. In all these states, the mean ethanol production rate and the concentration of ATP and the intermediates beyond the GAPDH-step are maintained fairly constant, while the glycogen consumption rate and intermediate concentrations of the upper part of the glycolytic system change considerably: the glycogen consumption rate varies 4-fold and fructose-bis-phosphate concentration more than 10-fold. Doubling of the initial glycogen concentration and the addition of a great excess of fructose-bis-phosphate do not affect the ethanol production rate and the mean glycerate-3-phosphate (3-PGA) and pyruvate levels. By contrast, ethanol production was accelerated by an increase of the net ATP consumption rate resulting from either the addition of apyrase or by substitution of trehalose for glycogen. Neither the mean absolute ATP level nor the adenylate energy charge were measurably affected, however all this data can be interpreted in terms of a very strong stoichiometric regulation and stabilization of the lower part of the glycolytic system.  相似文献   

10.
The changes in the partial pressures of oxygen and carbon dioxide (PO2 and PCO2) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581–596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO2 and HbCO2 dissociation curves at varied O2, CO2, pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676–1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing PO2 and PCO2 to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of PO2 and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg2+ binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations – notably ATP, 2,3-BPG, and Mg2+. The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature – warranting a special consideration for relevant systems.  相似文献   

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