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PubMed1976

Dihydrodipicolinic acid synthase of Bacillus licheniformis. Quaternary structure, kinetics, and stability in the presence of sodium chloride and substrates.

Dihydrodipicolinic acid synthase (L-aspartate-beta-semialdehyde hydro-lyase (adding pyruvate and cyclising), EC 4.2.1.52) obtained from Bacillus licheniformis was purified to homogeneity. Its molecular weight was 108 000 to 117 500, depending on the concentration of NaCl and substrates present, and it contained four subunits of identical molecular weight (28000). The Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 Km values for pyruvate and L-aspartic semialdehyde were approximately 5.3 and 2.6 mM, respectively. It was previously shown that pyruvate and a high sodium chloride concentration contributed to the stability of the enzyme. The effect of these substances and the other substrate, L-aspartic semialdehyde, on molecular weight was determined. None of these three substances significantly affected the apparent molecular weight. The effect of sodium chloride, pyruvate, and L-aspartic semialdehyde on enzyme structure was studied by determining the effect of their presence on inactivation of the enzyme by several chemical denaturants and heat. Pyruvate dramatically protected against inactivation by all of the denaturants. Sodium chloride protected against inactivation by sodium dodecyl sulfate, guanidine-HCl, urea, and heat, but somewhat facilitated inactivation by ethanol. L-Aspartic semialdehyde had no significant effect on inactivation by sodium dodecyl sulfate and ethanol; it rendered the enzyme slightly more sensitive to inactivation by guanidine-HCl and urea. The thermal melting curve obtained for the enzyme in the presence of L-aspartic semialdehyde was biphasic. The activity was reduced approximately 50% by heating for 30 min at temperatures between 50 and 80 degrees C. Only by heating at temperatures above 80 degrees C did the inactivation become complete. The partially inactivated enzyme could be reactivated by heating after removal of the L-aspartic semialdehyde. Pyruvate prevented the partial inactivation and facilitated reactivation. The only difference detected between the native enzyme and the partially inactivated form of the enzyme was that the latter had a reduced V. It is known that in other spore-formers, dihydrodipicolinate synthase increases in activity late in sporulation. This increase may be important for normal sporulation to occur. The possibility is discussed that the intracellular pool sizes of pyruvate and L-aspartic semialdehyde might have an influence on the level of dihydrodipicolinate synthase activity, by controlling the amount of partial inactivation of the enzyme that occurs in vivo.

Aldehydes↗

PubMed1989

Studies of glycogen synthesis and the Krebs cycle by mass isotopomer analysis with [U-13C]glucose in rats.

Starved rats were infused intragastrically via indwelling duodenal cannulae with glucose at a rate of 30 mg/min/kg. The infusate contained [U-13C]glucose at an enrichment of 32 or 17%. At the end of the infusion, after 160 min, glucose and lactate were isolated from arterial and portal blood and from liver, and liver glycogen was isolated and hydrolyzed to glucose. The enrichment in glucose and lactate and the isotopomer distribution in glucose of masses from 180 to 186 were determined by gas chromatography-mass spectrometry (GC-MS). From analysis of these data we determined (a) gluconeogenesis proceeds at half the basal rate in the presence of a large infused glucose load, (b) one-quarter of the hepatic pyruvate pool is derived from nonglucose carbon, (c) half of the labeled molecules in liver glycogen are of mass 186 from the infused glucose and half are of masses 181-183, (d) the contribution of the indirect path from pyruvate when corrected for synthesis from unlabeled pyruvate ranges from 55 to 65%, (e) the rate of pyruvate carboxylase averages 90% that of citrate synthase, and (f) the rate of exchange of oxaloacetate with fumarate is about three times the rate of flux in the Krebs cycle (four times in the "forward" direction), and the enrichment in carbon 1 of oxaloacetate was 2.3 times that in carbon 4. In the Appendix a method to obtain the isotopomer distribution of newly formed glucose and glycogen glucose is described. An algorithm to correct for the contribution of natural abundance of 13C and the presence of 12C in commercial [U-13C]glucose is presented. A novel mathematical analysis to obtain the parameters of the Krebs cycle from the isotopomer distribution is developed in the Appendix. Equations to calculate the relative rates of pyruvate carboxylase (y), and the equilibration of oxaloacetate with fumarate from the isotopomer distribution are derived. Mass isotopomer analysis provides a novel and powerful tool for the study of carbohydrate metabolism and the operation of the Krebs cycle.

Animals↗

PubMed1991

Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H-NMR and 13C-NMR spectroscopy.

We propose an experimental approach combining 1H-NMR and 13C-NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters. 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyl-CoA and glutamate via the Krebs cycle in glucose-grown repressed Saccharomyces cerevisiae cells fed with [1-13C]glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2-13C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acetyl-CoA as expected, these results indicate that (a) the probability for citrate and 2-oxoglutarate to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent. The flux of ethanol formation from pyruvate is about three-times and nine-times greater than that of ethanol consumption and acetyl-CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2 + C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1.1. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data obtained from similar experiments using unlabeled ethanol and non-NMR techniques.

Acetyl Coenzyme A↗

PubMed1984

Inhibition by alloxan of mitochondrial aconitase and other enzymes associated with the citric acid cycle.

Considerable variations were found in the in vitro effect of alloxan on mouse liver enzymes associated with the citric acid cycle. The following approximative alloxan concentrations induced 50% inhibition of enzyme activity: 10(-6)M for aconitase, 10(-4)M for NAD-linked isocitrate dehydrogenase, glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase and fumarase, and 10(-3)M for citrate synthase and NADP-linked isocitrate dehydrogenase. Pyruvate dehydrogenase, succinate dehydrogenase and malate dehydrogenase were not inhibited by 10(-3)M alloxan. The inhibition of aconitase was competitive both when using mouse liver and purified porcine heart enzyme. The Ki values for the purified enzyme in the presence of 5 microM alloxan were 0.22 microM with citrate, 4.0 microM with cis-aconitate and 0.62 microM with isocitrate as substrate. The high sensitivity of aconitase for inhibition by alloxan probably plays a prominent role for the toxic effects of alloxan.

Aconitate Hydratase↗

PubMed1998

Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase.

NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via alpha-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ ratios in the cells.

Acetoin↗

PubMed1988

Enzymic capacities of amyloplasts from wheat (Triticum aestivum) endosperm.

Lysates of protoplasts from the endosperm of developing grains of wheat (Triticum aestivum) were fractionated on density gradients of Nycodenz to give amyloplasts. Enzyme distribution on the gradients suggested that: (i) starch synthase and ADP-glucose pyrophosphorylase are confined to the amyloplasts; (ii) pyrophosphate: fructose-6-phosphate 1-phosphotransferase and UDP-glucose pyrophosphorylase are confined to the cytosol; (iii) a significant proportion (23-45%) of each glycolytic enzyme, from phosphoglucomutase to pyruvate kinase inclusive, is in the amyloplast. Starch synthase, ADP-glucose pyrophosphorylase and each of the glycolytic enzymes showed appreciable latency when assayed in unfractionated lysates of protoplasts. No activity of fructose-1,6-bisphosphatase was found in amyloplasts or in homogenates of endosperm. Antibody to plastidic fructose-1,6-bisphosphatase did not react positively, in an immunoblot analysis, with any protein in extracts of wheat endosperm. It is argued that wheat endosperm lacks significant plastidic fructose-1,6-bisphosphatase and that carbon for starch synthesis does not enter the amyloplast as a C-3 compound but probably as hexose phosphate.

Antibodies↗

PubMed2005

Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation.

Dietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element-binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes.

AMP-Activated Protein Kinases↗

PubMed1983

Effects of repetitive exercise on neonatal rat skeletal muscle oxidative capacity.

Since little is known about the training response to exercise in neonatal animals, this study was undertaken to elucidate the potential of oxidative system adaptations in developing skeletal muscle of rats during 50 days of daily treadmill running. The training regimen involved male and female rats (10 days old) initially running 0.1 mph, 0% grade, for 15 min. The program progressed to 1 mph, 25% grade, for 60 min by 50 days of age. At 25 days of age, pyruvate and palmitate oxidative capacity, and citrate synthase activity in red vastus muscle homogenates were elevated in the trained group (T) compared with age- and sex-matched controls (C). These increases were also observed for each subsequent time point tested and occurred in spite of the fact that the peak oxidative capacity of neonatal red vastus muscle was 46% greater than adult values. Further, trained animals tested at 45 days of age responded with a 12% increase in maximal oxygen consumption (Vo2max) compared with controls (P less than 0.05). Assays of muscle phosphofructokinase and of creatine phosphokinase activity conducted at this time point revealed no difference between T and C groups. Collectively, these data suggest that neonatal rats can be successfully trained and that they respond to an endurance-type program qualitatively similarly to adult rats.

Animals↗

PubMed1978

Pyruvate and citrate metabolism in the muscle tissue of Ascaris lumbricoides.

Aconitase and NAD linked isocitrate dehydrogenase were present in Ascaris lumbricoides muscle at only very low activities, whilst there were significant levels of citrate synthase, NADP linked isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase and succinic thiokinase. Pyruvate dehydrogenase was present in A. lumbricoides muscle at levels comparable with mammalian tissues and results suggest that it is modulated via a phosphotransferase/phosphatase system. The tricarboxylic acid cycle intermediates, citrate, isocitrate and 2-oxoglutarate were all detected in freeze clamped muscle, but their steady state levels were considerably lower than those found in mammalian tissues.

Aconitate Hydratase↗

PubMed2003

Effect of reduced maternal inspired oxygen on hepatic glucose metabolism in the rat fetus.

Perturbations in glucose metabolism in the fetus and in the neonate are a consistent finding in several different animal models of intrauterine growth retardation (IUGR) as well as in humans. Studies in rats who have undergone IUGR have shown decreased hepatic glycogen stores in the fetus and delayed induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) at birth. Hepatic transcription factors CCAAT enhancer binding protein (C/EBP)alpha and C/EBPbeta and the increase in cyclic AMP at birth have been implicated in the initial appearance of PEPCK-C. We have examined the effect of IUGR induced by reduced maternal inspired oxygen (fractional inspired oxygen concentration 0.14) on a) the expression of genes for hepatic C/EBPalpha, C/EBPbeta, PEPCK-C and glycogen synthase; and b) transcription of the genes for C/EBPbeta and PEPCK-C by dibutyryl cyclic AMP in the fetus. Three days (d 18-21) of decrease in maternal inspired oxygen resulted in lower maternal arterial PO(2) and a lower birth weight of the pups (p < 0.01). Fetuses that underwent IUGR had significantly lower concentrations of plasma glucose, hepatic glycogen, and glycogen synthase mRNA and a higher hepatic lactate:pyruvate ratio. They also had lower levels of hepatic PEPCK-C mRNA at birth. The concentration of hepatic mRNA for C/EBPalpha and C/EBPbeta as well as the transcription factors themselves were not affected by the decreased maternal inspired oxygen. Fetal injection of dibutyryl cyclic AMP after 24 h of decreased maternal inspired oxygen (d 18-19) had no effect on the expression of C/EBPbeta. However, it resulted in an attenuated induction of PEPCK-C in the fetuses with IUGR. We speculate that a decrease in maternal inspired oxygen induced certain mediators, either in the mother or in the placenta, that caused lower fetal glucose concentration and affected the transcription of genes involved in fetal hepatic glucose metabolism. IUGR, as a result of decreased fractional inspired oxygen concentration may also be the consequence of pH-mediated changes in uterine blood flow. However, these remain to be examined in this experimental model.

Adenine Nucleotides↗

PubMed2000

Dietary fat-induced suppression of lipogenic enzymes in B/B rats during the development of diabetes.

This study was designed to determine the level of inhibition of gene transcription by the reduction in insulin levels upon the onset of diabetes in spontaneously diabetic B/B rats and if reducing the level of polyunsaturated fatty acids (PUFA) in the diet will increase lipogenic enzyme activity. Control (eight animals per group) and spontaneously diabetic B/B male weanling rats (25 animals per group) were fed semipurified diets containing 20% (w/w) fat of either low (0.25) or high (1.0) polyunsaturated to saturated (P/S) fatty acid ratio. Rats were killed at the onset of diabetes [blood glucose level of approximately/= 100 mg/dL (5.55 mM)] and as they became highly diabetic [blood glucose level of approximately/= 400 mg/dL (22.22 mM)]. Total RNA was extracted from liver, and the relative amount of mRNA coding for fatty acid synthase (FAS), acetyl-CoA carboxylase, malic enzyme, pyruvate kinase, and phosphoenolpyruvate carboxykinase was determined. Liver enzyme activities were also measured. The mRNA levels for FAS, acetyl-CoA carboxylase, and malic enzyme decreased compared to control animals. The mRNA level for pyruvate kinase decreased at the onset of diabetes as compared to control animals. Feeding animals the low P/S diet treatment elevated the level of mRNA and lipogenic enzyme activity compared to animals fed the high P/S diet treatment, suggesting that the effect of PUFA on lipogenic enzymes is through a direct effect on gene expression.

Acetyl-CoA Carboxylase↗

PubMed1971

Citrate cycle and related metabolism of Listeria monocytogenes.

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.

Carbohydrate Metabolism↗

PubMed2005

p-Hydroxybenzoic acid synthesis in Mycobacterium tuberculosis.

Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipid-producing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The recombinant enzyme catalyzed the removal of the pyruvyl moiety of chorismate to form p-hydroxybenzoate with an apparent K(m) value for chorismate of 19.7 microm and a k(cat) value of 0.102 s(-1). Strong inhibition of the reaction by p-hydroxybenzoate but not by pyruvate was observed. These results establish Rv2949c as a chorismate pyruvate-lyase responsible for the direct conversion of chorismate to p-hydroxybenzoate and identify Rv2949c as the sole enzymatic source of p-hydroxybenzoic acid in M. tuberculosis.

Chorismic Acid↗

PubMed1999

Engineering of a novel biochemical pathway for the biosynthesis of L-2-aminobutyric acid in Escherichia coli K12.

L-2-Aminobutyric acid was synthesised in a transamination reaction from L-threonine and L-aspartic acid as substrates in a whole cell biotransformation using recombinant Escherichia coli K12. The cells contained the cloned genes tyrB, ilvA and alsS which respectively encode tyrosine aminotransferase of E. coli, threonine deaminase of E. coli and alpha-acetolactate synthase of B. subtilis 168. The 2-aminobutyric acid was produced by the action of the aminotransferase on 2-ketobutyrate and L-aspartate. The 2-ketobutyrate is generated in situ from L-threonine by the action of the deaminase, and the pyruvate by-product is eliminated by the acetolactate synthase. The concerted action of the three enzymes offers significant yield and purity advantages over the process using the transaminase alone with an eight to tenfold increase in the ratio of product to the major impurity.

Acetolactate Synthase↗

PubMed1998

Action of L-acetylcarnitine on different cerebral mitochondrial populations from cerebral cortex.

The maximum rate (Vmax) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg.kg-1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic "light" and "heavy" ones. In fact, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and alpha-ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.

Acetylcarnitine↗

PubMed1993

Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli. Evidence that the genes for phosphofructokinase and pyruvate kinase constitute an operon.

Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5-phosphate but not by fructose 1,6-bisphosphate. The gene for the enzyme was cloned in Escherichia coli and its entire nucleotide sequence was determined. The deduced amino acid sequence consisted of 587 residues and the molecular mass was calculated to be 62 317 Da. The sequence was highly similar to other pyruvate kinases, indicating that they have the same evolutional origin. Similarly to the E. coli enzymes, the enzyme does not contain an N-terminal domain, in contrast to the eukaryotic pyruvate kinases. However, the Bacillus stearothermophilus enzyme had an extra C-terminal sequence consisting of about 110 amino acid residues. A phosphoenolpyruvate-binding motif, which is observed in pyruvate phosphate dikinase, phosphoenolpyruvate: sugar phosphotransferase system enzyme I and phosphoenolpyruvate synthase, was present in the extra C-terminal sequence. There was an open reading frame upstream of the pyruvate kinase gene. The homology of the sequence showed that the gene encodes phosphofructokinase. Both phosphofructokinase and pyruvate kinase were expressed in E. coli cells, and the evidence suggesting that both genes constitute an operon is presented.

Amino Acid Sequence↗

PubMed1975

The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria.

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.

Acetyl Coenzyme A↗

PubMed1992

Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder.

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose. The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed. In these studies we determined: 1) the rate of 14C-pyruvate metabolism to 14CO2 in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase. The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the Kd for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction.

Animals↗