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PubMed2003

Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout.

The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C(6)]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed.

Acetyl Coenzyme A↗
PubMed2006

Accessing the dynamics of end-grafted flexible polymer chains by atomic force-electrochemical microscopy. Theoretical modeling of the approach curves by the elastic bounded diffusion model and Monte Carlo simulations. Evidence for compression-induced lateral chain escape.

The dynamics of a molecular layer of linear poly(ethylene glycol) (PEG) chains of molecular weight 3400, bearing at one end a ferrocene (Fc) label and thiol end-grafted at a low surface coverage onto a gold substrate, is probed using combined atomic force-electrochemical microscopy (AFM-SECM), at the scale of approximately 100 molecules. Force and current approach curves are simultaneously recorded as a force-sensing microelectrode (tip) is inserted within the approximately 10 nm thick, redox labeled, PEG chain layer. Whereas the force approach curve gives access to the structure of the compressed PEG layer, the tip-current, resulting from tip-to-substrate redox cycling of the Fc head of the chain, is controlled by chain dynamics. The elastic bounded diffusion model, which considers the motion of the Fc head as diffusion in a conformational field, complemented by Monte Carlo (MC) simulations, from which the chain conformation can be derived for any degree of confinement, allows the theoretical tip-current approach curve to be calculated. The experimental current approach curve can then be very satisfyingly reproduced by theory, down to a tip-substrate separation of approximately 2 nm, using only one adjustable parameter characterizing the chain dynamics: the effective diffusion coefficient of the chain head. At closer tip-substrate separations, an unpredicted peak is observed in the experimental current approach curve, which is shown to find its origin in a compression-induced escape of the chain from within the narrowing tip-substrate gap. MC simulations provide quantitative support for lateral chain elongation as the escape mechanism.

Aluminum Oxide↗
PubMed1990

Redox cycling of radical anion metabolites of toxic chemicals and drugs and the Marcus theory of electron transfer.

A wide variety of aromatic compounds are enzymatically reduced to form anion free radicals that generally contain one more electron than their parent compounds. In general, the electron donor is any of a wide variety of flavoenzymes. Once formed, these anion free radicals reduce molecular oxygen to superoxide and regenerate the parent compound unchanged. The net reaction is the oxidation of the flavoenzyme's coenzymes and the reduction of molecular oxygen. This catalytic behavior has been described as futile metabolism or redox cycling. Electron transfer theory is being applied to these reactions and, in some cases, has successfully correlated Vmax and Km with the reduction potentials of the aromatic compounds.

Anions↗
PubMedDate not supplied

Stimulation of platelet serotonin transport by substituted 1,4-naphthoquinone-induced oxidant stress.

The effect of oxidant stress produced by redox cycling of substituted 1,4-naphthoquinones on the activity of platelet (Na(+)-K+)ATPase and the active transport of serotonin (5-HT) was studied. 2-Methyl-1,4-naphthoquinone (menadione) produced a concentration-dependent (0-100 microM) and time-dependent (2-20 min) stimulation of platelet 5-HT transport. Exogenous superoxide dismutase (250 units) and/or catalase (500 units) failed to block the stimulation. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked menadione-induced stimulation of 5-HT uptake as did ouabain, an inhibitor of platelet (Na(+)-K+)ATPase. The structure-activity relationship of select 1,4-naphthoquinones suggested that stimulation was due to redox cycling and not arylation. The kinetics of 5-HT transport revealed that menadione markedly increased the maximal rate of 5-HT transport (Vmax control = 20.6 +/- 2.0 pmol/10(8) platelets/4 min vs Vmax menadione = 46.4 +/- 3.9 pmol/10(8) platelets/4 min) but did not significantly alter the Km values. The activity of (Na(+)-K+)ATPase was determined by measuring the uptake of 86Rb+ into intact platelets. Menadione produced a concentration-dependent and time-dependent stimulation of platelet 86Rb+ uptake. These changes in platelet (Na(+)-K+)ATPase activity paralleled the changes observed in 5-HT transport and were inhibited in a concentration-dependent manner by ouabain. The data have shown that the redox cycling of 1,4-naphthoquinones caused an increase in (Na(+)-K+)ATPase activity that resulted in the stimulation of the rate of platelet 5-HT transport.

Animals↗
PubMed1999

Cold stress increases lipolysis, FFA Ra and TG/FFA cycling in humans.

BACKGROUND: To characterize the important changes in the selection and mobilization of metabolic fuel during cold stress, six males rested for 3 h at 29 degrees C and at 5 degrees C dressed only in shorts while 2H5 glycerol, 1-13C palmitate and 6,6 2H2 glucose were continuously infused for 3 h in each condition to determine their rate of turnover (Ra). METHODS: Metabolic rate (M) as well as rates of carbohydrate (CHOox) and lipid oxidation (FATox) were assessed by indirect calorimetry whereas all isotopic enrichments were determined by mass spectrometry. RESULTS: Cold exposure decreased rectal and mean skin temperatures and increased M, FATox and CHOox compared with the same test at thermal neutrality (p<0.05). As expected, cold increased plasma glucose Ra and plasma FFA Ra (from 4.58+/-0.19 to 14.69+/-1.07 micromol kg(-1) x min(-1); p < 0.05). However, in absolute terms, plasma FFA Ra in the cold remained more than twice greater than FATox (FATox only increased up to 6.9 +/-0.85 micromol kg(-1) x min(-1)), suggesting an enhanced non-oxidative disposal of fatty acids (i.e., TG/FFA cycling) to account for all FFA Ra. Indeed, cold increased extracellular TG/FFA recycling rate (2.23+/-0.40 vs 7.77+/-1.19 micromol kg(-1) x min(-1); p<0.05) whereas intracellular cycling was unaffected. CONCLUSION: Even though lipolysis and FFA Ra are greatly increased by cold stress in humans, the present results demonstrate that only about half the rate of FFA Ra is ultimately oxidized, suggesting that under the present cold conditions: 1) non-oxidative FFA disposal or TG/FFA cycling is significantly enhanced; 2) white adipose tissue-derived fatty acids could easily account for most of FATox. The results further emphasize the importance of the TG/FFA cycle in amplifying the ability of stored TG to react quickly to major changes in energy expenditure induced by a sustained cold stress.

Adipose Tissue↗
PubMed2003

A novel colorimetric assay for the determination of lysophosphatidic acid in plasma using an enzymatic cycling method.

BACKGROUND: Several methods for measuring concentrations of lysophosphatidic acid (LPA), a lipid mediator, have been reported to date. However, these methods are not routinely used because most of them require specialized instrument and a complicated protocol. METHODS: We developed a novel LPA assay using enzymatic cycling. LPA in a sample is hydrolyzed with lysophospholipase to glycerol-3-phosphate, followed by enzymatic cycling using glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase. Amplified concentrations of hydrogen peroxides, a product of the enzymatic cycling, are then colorimetrically measured. RESULTS: This method was specific for LPA, being insensitive to the presence of phosphatidic acid or lysophosphatidylcholine. The within-run and between-run CVs were 1.31-1.32% and 0.73-1.03%, respectively. The recoveries of exogenous LPA added to plasma were 100.3-101.6%. In males, LPA concentrations (mean+/-S.D.) of human serum and EDTA-plasma were 0.41+/-0.14 and 0.08+/-0.02 micromol/l, respectively. In females, they were 0.41+/-0.12 and 0.09+/-0.02 micromol/l, respectively. CONCLUSIONS: This novel colorimetric assay for determination of LPA using enzymatic cycling is simple and highly sensitive. It can be used with an automatic analyzer. It may also be useful for further studies of the biological functions of LPA as well as clinical applications in various disorders.

Adult↗
PubMed2005

Catalysis of protein disulfide bond isomerization in a homogeneous substrate.

Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a beta hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N and C terminus contain a fluorescence donor (tryptophan) and acceptor (N(epsilon)-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E(o') = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys-Gly-His-Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/K(M) = 1.7 x 10(5) M(-1) s(-1), which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.

Amino Acid Sequence↗
PubMed1991

Intracellular xylitol-phosphate hydrolysis and efflux of xylitol in Streptococcus sobrinus.

The parental strain Streptococcus sobrinus (Streptococcus mutans ATCC 27352), which is known to transport, phosphorylate and accumulate xylitol intracellularly as nonmetabolizable xylitol-phosphate (xylitol-sensitive (XS) strain) and its xylitol-resistant (XR) spontaneous mutant were used to further investigate the inhibitory action of xylitol on oral streptococci. Fructose-grown XR cells did not accumulate xylitol-phosphate, indicating that the inducible fructose PTS is incapable of transporting the pentitol. The intracellularly accumulated pentitol-phosphate by the XS cells did not prevent the subsequent uptake and degradation of glucose or fructose, despite a drop in the PEP pool and a 50% inhibition of the glucose but not the fructose catabolism. Intracellular dephosphorylation of the pentitol-phosphate and release of xylitol in the extracellular medium resulted in a rapid decrease of the intracellular level of this nonmetabolizable product. A Mg(++)- or Mn(++)-independent sugar-phosphate hydrolysing activity capable of splitting xylitol-phosphate was demonstrated in both XS and XR strains. Preincubation in the presence of N1-ethylmaleimide (NEM) and xylitol or NEM and fructose resulted in the subsequent inhibition of both xylitol uptake and efflux. The efflux kinetic at various temperatures is compatible with a facilitated diffusion by the phosphotransferase system EIIfru without, however, excluding the existence of an additional exit route, but it excludes a simple diffusion exit process. The results are consistent with the existence of a xylitol futile cycle contributing to the growth inhibition of S. sobrinus by the pentitol without excluding a toxic effect of xylitol-phosphate. Discrepancies in the literature on the action of xylitol on S. mutans could be explained in the light of the evidence presented.

Dental Plaque↗
PubMed1991

Signal transmission through a metabolic cycle follows the compression hypothesis or a weak Weber's law.

Biological signal transduction often involves a metabolic cycle in which the flux at one point is driven by the input signal and the concentration of one of the metabolites of the cycle serves as the output signal. A kinetic analysis of such a metabolic cycle is made under an assumption that the law of mass action applies. The resultant kinetic model can produce a response that overshoots, quickens, and eventually saturates as the input intensity is increased. The possible model behavior ranges parametrically from non-adaptive (compression hypothesis) to weakly adaptive (limited Weber's law).

Adaptation, Physiological↗
PubMed2001

Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by (13)C NMR spectroscopy with multiple (13)C labels.

The intracellular compartmentation of pyruvate in primary cultures of cortical neurons was investigated by high resolution (13)C NMR using mixtures of different pyruvate precursors conveniently labeled with (13)C or unlabeled. Cells were incubated with 1-5 mM (1-(13)C, 1,2-(13)C(2) or U-(13)C(6)) glucose only or with mixtures containing 1.5 mM (1-(13)C or U-(13)C(6)) glucose, 0.25-2.5 mM (2-(13)C or 3-(13)C) pyruvate and 1 mM malate. Extracts from cells and incubation media were analyzed by (13)C NMR to determine the relative contributions of the different precursors to the intracellular pyruvate pool. When ((13)C) glucose was used as the sole substrate fractional (13)C enrichments and (13)C isotopomer populations in lactate and glutamate carbons were compatible with a unique intracellular pool of pyruvate. When mixtures of ((13)C) glucose, ((13)C) pyruvate and malate were used, however, the fractional (13)C enrichments of the C2 and C3 carbons of lactate were higher than those of the C2 and C3 carbons of alanine and depicted a different (13)C isotopomer distribution. Moreover, neurons incubated with 1 mM (1,2-(13)C(2)) glucose and 0.25-5 mM (3-(13)C) pyruvate produced exclusively (3-(13)C) lactate, revealing that extracellular pyruvate is the unique precursor of lactate under these conditions. These results reveal the presence of two different pools of intracellular pyruvate; one derived from extracellular pyruvate, used mainly for lactate and alanine production and one derived from glucose used primarily for oxidation. A red-ox switch using the cytosolic NAD(+)/NADH ratio is proposed to modulate glycolytic flux, controlling which one of the two pyruvate pools is metabolized in the tricarboxylic acid cycle when substrates more oxidized or reduced than glucose are used.

Animals↗
PubMed1993

Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine.

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

DNA single-strand breakage in mammalian cells induced by redox cycling quinones in the absence of oxidative stress.

Quinone-induced cell death is often attributed to oxidative stress during which the formation of DNA strand breaks is thought to play an important role. In this study, extensive DNA damage was observed in human chronic myelogenous leukemic cells (K562) exposed for 15 minutes to low concentrations (15-100 microM) of the redox cycling quinones 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) and menadione. However, DNA strand breakage and cell death could not be attributed to oxidative stress as the intracellular level and redox status of the reducing equivalents NADP(H) and GSH were unaffected. The intracellular level of NAD+ was found to correlate well with the extent of DNA repair (r = 0.93, P < 0.02) and cell proliferation (r = 0.96, P < 0.01) in cells exposed to the quinones. In contrast, a significant decrease in the level of intracellular ATP was only observed in cells exposed to menadione (50-100 microM). These results suggest that redox cycling quinones are capable of inducing DNA damage in mammalian cells by a mechanism that does not involve oxidative stress. Following DNA damage, cell death is dependent on the availability of NAD+, which may be key to the rapid repair of strand breaks.

Adenosine Triphosphate↗
PubMed2001

Sulfation is rate limiting in the futile cycling between estrone and estrone sulfate in enriched periportal and perivenous rat hepatocytes.

The metabolic activities and tissue binding of estrone (E1) and estrone sulfate (E1S) on futile cycling were examined. Desulfation of E1S in the 9000g supernatant fraction (S9) of periportal (PP) and perivenous (PV) rat hepatocytes were of similar V (2.9 +/- 1.0 and 2.4 +/- 0.9 nmol/min/mg of S9 protein), K (30.4 +/- 8.3 and 34.8 +/- 6.6 microM), and desulfation intrinsic clearances (V/K of 77 and 55 microl/min/10(6) cells). The intrinsic clearance towards E1 sulfation (1 microM) in cytosolic preparations of PV hepatocytes was 4 times that of PP hepatocytes (V/K of 26.4 +/- 9.5 versus 6.1 +/- 2.2 microl/min/mg of cytosolic protein or 13 +/- 5 versus 3.1 +/- 1.1 microl/min/10(6) cells). The observation was consistent with the immunolocalization of estrogen sulfotransferase (PV/PP ratio of 3.4 +/- 1.1) but not hydroxysteroid sulfotransferase (PV/PP ratio of 0.29 +/- 0.21) nor phenol sulfotransferase (PV/PP ratio of 1.13 +/- 0.23). Upon incubation of E1S (1-125 microM) with hepatocytes (30 min), higher concentrations of E1S and E1 were observed within PP than in PV cells, and saturation was evident at the higher concentrations. Based on the in vitro metabolic and tissue binding parameters for E1S and E1 and the published zonal uptake clearances of E1S (116 microl/min/10(6) cells), fitting revealed that uptake of E1 (1484 and 1463 microl/min/10(6) cells) by PP and PV cells was rapid and similar, and E1 sulfation was the slowest step in futile cycling. The greater metabolism of E1 in PV region led to higher levels of E1 and E1S in PP hepatocytes, and the nonlinear uptake, binding, and vesicular accumulation of E1S resulted in different t(1/2) values for E1S and E1.

Animals↗
PubMed2000

Energy-wasteful total Ca(2+) handling underlies increased O(2) cost of contractility in canine stunned heart.

Postischemic myocardial stunning halved left ventricular contractility [end-systolic maximum elastance (E(max))] and doubled the O(2) cost of E(max) in excised cross-circulated canine heart. We hypothesized that this increased O(2) cost derived from energy-wasteful myocardial Ca(2+) handling consisting of a decreased internal Ca(2+) recirculation, some futile Ca(2+) cycling, and a depressed Ca(2+) reactivity of E(max). We first calculated the internal Ca(2+) recirculation fraction (RF) from the exponential decay component of postextrasystolic potentiation. Stunning significantly accelerated the decay and decreased RF from 0.63 to 0. 43 on average. We then combined the decreased RF with the halved E(max) and its doubled O(2) cost and analyzed total Ca(2+) handling using our recently developed integrative method. We found a decreased total Ca(2+) transport and a considerable shift of the relation between futile Ca(2+) cycling and Ca(2+) reactivity in an energy-wasteful direction in the stunned heart. These changes in total Ca(2+) handling reasonably account for the doubled O(2) cost of E(max) in stunning, supporting the hypothesis.

Animals↗
PubMed2002

A futile metabolic cycle activated in adipocytes by antidiabetic agents.

Thiazolidinediones (TZDs) are effective therapies for type 2 diabetes, which has reached epidemic proportions in industrialized societies. TZD treatment reduces circulating free fatty acids (FFAs), which oppose insulin actions in skeletal muscle and other insulin target tissues. Here we report that TZDs, acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma, markedly induce adipocyte glycerol kinase (GyK) gene expression. This is surprising, as standard textbooks indicate that adipocytes lack GyK and thereby avoid futile cycles of triglyceride breakdown and resynthesis from glycerol and FFAs. By inducing GyK, TZDs markedly stimulate glycerol incorporation into triglyceride and reduce FFA secretion from adipocytes. The 'futile' fuel cycle resulting from expression of GyK in adipocytes is thus a novel mechanism contributing to reduced FFA levels and perhaps insulin sensitization by antidiabetic therapies.

Adipocytes↗
PubMed1995

Energetics of bacterial growth: balance of anabolic and catabolic reactions.

Biomass formation represents one of the most basic aspects of bacterial metabolism. While there is an abundance of information concerning individual reactions that result in cell duplication, there has been surprisingly little information on the bioenergetics of growth. For many years, it was assumed that biomass production (anabolism) was proportional to the amount of ATP which could be derived from energy-yielding pathways (catabolism), but later work showed that the ATP yield (YATP) was not necessarily a constant. Continuous-culture experiments indicated that bacteria utilized ATP for metabolic reactions that were not directly related to growth (maintenance functions). Mathematical derivations showed that maintenance energy appeared to be a growth rate-independent function of the cell mass and time. Later work, however, showed that maintenance energy alone could not account for all the variations in yield. Because only some of the discrepancy could be explained by the secretion of metabolites (overflow metabolism) or the diversion of catabolism to metabolic pathways which produced less ATP, it appeared that energy-excess cultures had mechanisms of spilling energy. Bacteria have the potential to spill excess ATP in futile enzyme cycles, but there has been little proof that such cycles are significant. Recent work indicated that bacteria can also use futile cycles of potassium, ammonia, and protons through the cell membrane to dissipate ATP either directly or indirectly. The utility of energy spilling in bacteria has been a curiosity. The deprivation of energy from potential competitors is at best a teleological explanation that cannot be easily supported by standard theories of natural selection. The priming of intracellular intermediates for future growth or protection of cells from potentially toxic end products (e.g., methylglyoxal) seems a more plausible explanation.

Adenosine Triphosphate↗
PubMed2001

Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data.

Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za.

Agriculture↗
PubMed1997

A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.

Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.

Carbon Dioxide↗