Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD⁺/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.     

What controls glycolysis in bloodstream form Trypanosoma brucei?

On the basis of the experimentally determined kinetic properties of the trypanosomal enzymes, the question is addressed of which step limits the glycolytic flux in bloodstream form Trypanosoma brucei. There appeared to be no single answer; in the physiological range, control shifted between the glucose transporter on the one hand and aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) on the other hand. The other kinases, which are often thought to control glycolysis, exerted little control; so did the utilization of ATP. We identified potential targets for anti-trypanosomal drugs by calculating which steps need the least inhibition to achieve a certain inhibition of the glycolytic flux in these parasites. The glucose transporter appeared to be the most promising target, followed by ALD, GDH, GAPDH, and PGK. By contrast, in erythrocytes more than 95% deficiencies of PGK, GAPDH, or ALD did not cause any clinical symptoms (Schuster, R. and Holzhütter, H.-G. (1995) Eur. J. Biochem. 229, 403-418). Therefore, the selectivity of drugs inhibiting these enzymes may be much higher than expected from their molecular effects alone. Quite unexpectedly, trypanosomes seem to possess a substantial overcapacity of hexokinase, phosphofructokinase, and pyruvate kinase, making these "irreversible" enzymes mediocre drug targets.     

Experimental validation of metabolic pathway modeling

In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PP(i)-PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PP(i) was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PP(i) was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower V(max) values for hexose-6-phosphate isomerase, PP(i)-PFK and aldolase were induced by PP(i) or ATP inhibition. It should be noted that PP(i) and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed 'in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties.     

Trypanosoma brucei: activities and subcellular distribution of glycolytic enzymes from differently disrupted cells

The effect of disruption procedure on the subcellular distribution and the activities of 11 enzymes catalyzing the glycolytic pathway in Trypanosoma brucei has been studied. The activities of the enzymes varied with the lytic procedure used. Maximum specific enzyme activity values were obtained after treatment with saponin whereas digitonin treatment gave the lowest results. The intracellular location of the enzymes was examined by means of differential centrifugation following cell lysis with saponin, Triton X-100, digitonin, or by freezing and thawing. Irrespective of the method of cell lysis employed, the six enzymes, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, glycerol phosphate dehydrogenase, and glycerokinase, were particulate. Of the remaining 5 enzymes, digitonin liberates only phosphoglycerate mutase (partially); saponin or Triton X-100 liberates phosphoglucose isomerase, phosphoglycerate mutase, enolase, and pyruvate kinase but not glyceraldehyde 3-phosphate dehydrogenase; freezing and thawing acts like saponin or Triton X-100 except that it fails to liberate phosphoglucose isomerase, while cell grinding with silicon carbide liberates only glyceraldehyde phosphate dehydrogenase (partially), phosphoglycerate mutase, enolase, and pyruvate kinase. The relative maximal activities of the enzymes suggest that the rate-limiting steps in glycolysis in T. brucei are the reactions catalyzed by aldolase and phosphoglycerate mutase.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

The effect of enzyme-enzyme complexes on the overall glycolytic rate in vivo.

Recent studies have demonstrated that most glycolytic enzymes can reversibly associate to form heterogeneous enzyme-enzyme (binary) complexes in vitro. However, kinetic analysis of these complexes has shown that the individual enzymes have a varied response to complex formation: some enzymes are inhibited, some are activated and some are unaffected. In order to determine the potential role of binary complexes in regulating glycolytic flux, we have mathematically calculated enzyme distributions and activities using data from in vitro binding and kinetic studies. These calculations suggest that, overall, formation of binary complexes would lower flux through phosphofructokinase and aldolase, would increase flux through glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and would not affect flux through triosephosphate isomerase, phosphoglycerate kinase and pyruvate kinase. The implications of these results are discussed with respect to the effect of complex formation on overall glycolytic flux and on the flux through individual enzyme loci.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody-like organelle, the glycosome, which they are believed to reach post-translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain. Modelling of the three-dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two 'hot spots' about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three-dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.     

Effect of pH on the regulatory characteristics of energy metabolism in human erythrocytes

The glucose consumption rate versus ATP content in human red cells (regulatory patterns of glycolysis) and ATP concentration versus glucose uptake rate in red cell suspension (regulatory patterns of total ATPases), when the rate of glucose uptake is constant and lower than the rate of glucose consumption at physiological conditions, were measured at different pH values. The shape of both types of kinetic curves was found to be dependent on the pH of the incubation medium but the same for the red cells taken from different donors. It is supposed that at alkaline pH, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions become the rate-limiting steps of glycolysis instead of hexokinase and phosphofructokinase under physiological conditions.     

Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.     

Regulation of glycolytic enzyme phosphoglycerate mutase-1 by Sirt1 protein-mediated deacetylation

Emerging proteomic evidence suggests that acetylation of metabolic enzymes is a prevalent post-translational modification. In a few recent reports, acetylation down-regulated activity of specific enzymes in fatty acid oxidation, urea cycle, electron transport, and anti-oxidant pathways. Here, we reveal that the glycolytic enzyme phosphoglycerate mutase-1 (PGAM1) is negatively regulated by Sirt1, a member of the NAD(+)-dependent protein deacetylases. Acetylated PGAM1 displays enhanced activity, although Sirt1-mediated deacetylation reduces activity. Acetylation sites mapped to the C-terminal "cap," a region previously known to affect catalytic efficiency. Overexpression of a constitutively active variant (acetylated mimic) of PGAM1 stimulated flux through glycolysis. Under glucose restriction, Sirt1 levels dramatically increased, leading to PGAM1 deacetylation and attenuated activity. Previously, Sirt1 has been implicated in the adaptation from glucose to fat burning. This study (i) demonstrates that protein acetylation can stimulate metabolic enzymes, (ii) provides biochemical evidence that glycolysis is modulated by reversible acetylation, and (iii) demonstrates that PGAM1 deacetylation and activity are directly controlled by Sirt1.     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Kinetics of metabolic pathways. A system in vitro to study the control of flux.

A method for determining Control Coefficients is proposed for systems studied in vitro and applied to a model pathway. Rat liver extract, which converts glucose into glycerol 3-phosphate, was used with the addition to the incubation mixture of fructose-bisphosphate aldolase, triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase as 'auxiliary' enzymes, which leaves all the control on the first three enzymes. The flux of the metabolic pathway was recorded by assaying NADH decay. Flux Control Coefficients (CJE) of hexokinase, glucose-6-phosphate isomerase and phosphofructokinase were calculated by titration of the system with increasing quantities of extraneous enzymes. It is shown that the summation property is fulfilled. The applicability of this procedure to study the control in any metabolic pathway is discussed. Possible relevance of the method to conditions in vivo and its limitations are considered.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions and related enzymes of the cycle in adipose tissue

1. Measurements were made of the activities of the enzymes of the pentose phosphate pathway concerned in both the oxidative (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and the non-oxidative (ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) reactions of this pathway, together with hexokinase and phosphoglucose isomerase, in adipose tissue in a variety of nutritional and hormonal conditions. 2. Starvation for 2 days caused a significant decrease in the activities of all the enzymes of the pentose phosphate pathway, with the exception of glucose 6-phosphate dehydrogenase, when expressed as activity/2 fat-pads; only the activities of ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase were significantly decreased on the basis of activity/mg. of protein. Re-feeding with a high-carbohydrate or high-fat diet for 3 days restored the activity of all the enzymes of the pentose phosphate pathway to the range of the control values, with the exception of transketolase, which showed a marked ;overshoot' in rats re-fed with carbohydrate. Starvation for 3 days caused a marked decrease in the activities of glucose 6-phosphate dehydrogenase and transketolase. 3. On the basis of activity/two fat-pads, alloxan-diabetes caused a marked decrease, to about half the control value, in the activities of all the enzymes concerned in the pentose phosphate pathway, transketolase showing the smallest decrease; hexokinase and phosphoglucose isomerase activities were also decreased. Treatment with insulin for 3 and 7 days raised the activities to normal or supranormal values, transketolase showing the most marked ;overshoot' effect. On the basis of activity/mg. of protein the activity of none of the enzymes was significantly decreased in alloxan-diabetes; transketolase and transaldolase activities were raised above the control values. With insulin treatment for 3 or 7 days the activities of all the enzymes were significantly increased, except that of ribulose 5-phosphate epimerase at the shorter time-interval. Glucagon treatment did not alter any of the enzyme activities expressed on either basis. 4. Thyroidectomy caused a decrease of 30-40% in the activities of enzymes of the pentose phosphate pathway, except for transketolase activity, which fell to 50% of the control value. Little change occurred in adipose-tissue weight or protein content. 5. Adrenalectomy caused a decrease of 40% in the activity of glucose 6-phosphate dehydrogenase and of 20-30% in the activities of the remaining enzymes of the pentose phosphate pathway; hexokinase activity was also decreased. Treatment with cortisone for 3 days did not significantly raise the activity from that found in adrenalectomized rats. Treatment of normal rats with high doses of cortisone had no significant effect on the activities of the enzymes of the pentose phosphate pathway in adipose tissue. 6. The changes in enzyme activities are discussed in relation to: (a) the concept of constant-proportion groups of enzymes; (b) the known changes in the flux of glucose through alternative metabolic pathways; (c) the pattern of change found in liver with similar hormonal and dietary conditions.     

High performance purification of glycolytic enzymes and creatine kinase from chicken breast muscle and preparation of their specific immunological probes

We developed a novel procedure for isolation of the muscle isozymes of aldolase, triose phosphate isomerase (TPI), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase, pyruvate kinase (PK) and lactic dehydrogenase (LDH), and also creatine kinase (CK), at high purity, specific activity and yield. Protein was extracted from chicken breast muscle and glycolytic enzymes were purified by a three step procedure consisting of: Ammonium sulfate combined with pH fractionation. Phosphocellulose chromatography with performance of high pressure liquid chromatography, exploiting a pH gradient formed by a gradient of the buffering ion for protein elution. Affinity chromatography causing elution by substrate or pH. The enzymes, obtained at over 95% purity as judged by specific activity and silver stained electropherograms, were injected into sheep. Antibody for each enzyme was purified on specific immunosorbant and its specificity was verified by immunotransfer analysis.     

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, <Emphasis Type="Italic">Streptococcus bovis</Emphasis>

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP⁺-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD⁺ ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.     

Stimulation of mutases and isomerases by vanadium.

The effects of vanadate and vanadate complexes on the rates of exchange of phosphoryl groups in the reactions catalyzed by the enzymes phosphoglucomutase and the coupled system formed by phosphoglycerate mutase and enolase, and the effects of vanadyl complexes on the interconversion of aldehyde and keto groups catalyzed by the enzymes phosphomannose isomerase, phosphoribose isomerase, and phosphoglucose isomerase, were measured using one-dimensional 31P nuclear magnetic resonance spectroscopy. Chemical exchange was investigated by observing the transfer of magnetization achieved by selective irradiation of resonances using the DANTE pulse sequence. The presence of vanadium stimulated the catalytic activity of the enzymes in vitro, with the exception of enolase whose activity was not affected. Addition of vanadate also increased the rate constants of the interconversion of glucose 6-phosphate and fructose 6-phosphate in hemolysates. 51V nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were employed to investigate the interactions between ammonium vanadate and sugar phosphates and the formation of vanadium--sugar phosphate complexes that may be involved in the stimulation of the catalytic activity of the isomerases.     

Glycolytic enzyme activity in developing red and white muscle

The activities of the constant proportion enzymes of the Embden-Meyerhof chain (triose phosphate isomerase (TIM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM) and enolase (ENOL)), and the activity of lactic dehydrogenase (LDH) were studied in developing red (trapezius) and white (longissimus) muscles of the pig from a fetal stage to 24 weeks postnatal. Both muscles were differentiated by two weeks postnatal in the sense that they had reached the adult level of enzyme activity. Enzyme activities were two- to three-fold greater in the longissimus than in the trapezius. Enzyme activity ratios based on GAPDH were not consistent in the fetal and day 1 samples but were consistent during later stages of growth. Ratios of enzyme activity based on activity at 105 days gestation revealed that TIM, PGK and PGM are grouped and follow the same pattern, but GAPDH and ENOL are quite different from each other and from the pattern shown by TIM, PGK and PGM. The constant proportion concept in developing muscle is therefore questioned.     

Molecular characterization of transketolase (EC 2.2.1.1) active in the Calvin cycle of spinach chloroplasts

A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T₇ promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.Abbreviations RPE ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - PGK phosphoglycerate kinase - FBP fructose-1,6-bisphosphatase - SBP sedoheptulose-1,7-bisphosphatase - OPPP oxidative pentose phosphate pathway - Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphosphate aldolase - IPTG isopropyl -d-thiogalactoside - TPI triosephosphate isomerase     

Distribution and phylogenies of enzymes of the Embden-Meyerhof-Parnas pathway from archaea and hyperthermophilic bacteria support a gluconeogenic origin of metabolism

Enzymes of the gluconeogenic/glycolytic pathway (the Embden-Meyerhof-Parnas (EMP) pathway), the reductive tricarboxylic acid cycle, the reductive pentose phosphate cycle and the Entner-Doudoroff pathway are widely distributed and are often considered to be central to the origins of metabolism. In particular, several enzymes of the lower portion of the EMP pathway (the so-called trunk pathway), including triosephosphate isomerase (TPI; EC 5.3.1.1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12/13), phosphoglycerate kinase (PGK; EC 2.7.2.3) and enolase (EC 4.2.1.11), are extremely well conserved and universally distributed among the three domains of life. In this paper, the distribution of enzymes of gluconeogenesis/glycolysis in hyperthermophiles--microorganisms that many believe represent the least evolved organisms on the planet--is reviewed. In addition, the phylogenies of the trunk pathway enzymes (TPIs, GAPDHs, PGKs and enolases) are examined. The enzymes catalyzing each of the six-carbon transformations in the upper portion of the EMP pathway, with the possible exception of aldolase, are all derived from multiple gene sequence families. In contrast, single sequence families can account for the archaeal and hyperthermophilic bacterial enzyme activities of the lower portion of the EMP pathway. The universal distribution of the trunk pathway enzymes, in combination with their phylogenies, supports the notion that the EMP pathway evolved in the direction of gluconeogenesis, i.e., from the bottom up.