acetyl-CoA Concentration Research Articles

Interactive effect of sulphur and nitrogen on lipid accumulation, acetyl-CoA concentration and acetyl-CoA carboxylase activity in developing seeds of oilseed crops ( Brassicacampestris L. and Erucasativa Mill.)

Field experiments were conducted to determine the interactive effect of sulphur (S) and nitrogen (N) on lipid accumulation, acetyl-CoA concentration and acetyl-CoA carboxylase activity in the developing seeds of oilseed crops viz. rapeseed ( Brassica campestris L. cv. Pusa Gold) and taramira ( Eruca sativa Mill.) differing in their lipid content. Two combinations of S and N (in kg/ha): 0S + 100N (−S+N) and 40S + 100N (+S+N) were used. The period between 10 to 40 days after flowering was observed as the active period of lipid accumulation in the seeds of both the genotypes. The accumulation of the lipid was preceded by a marked rise in acetyl-CoA carboxylase activity and acetyl-CoA concentration, which declined rapidly when lipid accumulation reached a plateau. Total sugar content decreased, while protein content increased during the active period of lipid accumulation in both the genotypes. The application of sulphur and nitrogen together significantly ( P < 0.05) enhanced the lipid accumulation in both the genotypes at all the growth stages. The increase in the lipid content was 4–214% in rapeseed and 12–227% in taramira with +S+N treatment over −S+N treatment. The application of sulphur and nitrogen together (+S+N) also increased acetyl-CoA concentration, acetyl-CoA carboxylase activity, soluble protein, and sugar content in the developing seeds of both the genotypes. It is suggested that the increase in the lipid content with combined application of S and N may be associated with increased acetyl-CoA carboxylase activity through the enhancement of acetyl-CoA concentration. Further, the increased sugar content because of combined application of S and N provided enough carbon source and energy for lipid biosynthesis.

Expression of a yeast acetyl CoA hydrolase in the mitochondrion

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Effect of ppc gene knockout on the metabolism of Escherichia coli in view of gene expressions, enzyme activities and intracellular metabolite concentrations.

Physiological characteristics and regulatory mechanism were investigated based on comparative analyses of gene expressions, enzyme activities and intracellular metabolite concentrations between the ppc knockout Escherichia coli strain JWK3928 and its parent strain BW25113. RT-PCR was used to detect gene expressions in both strains. It was observed that the ppc knockout strain excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate together with a lower glucose consumption rate, as compared with the parent strain. Accordingly, the biomass yield on glucose was improved in the ppc(-) mutant. It was found that genes such as gltA, icd, aceA and mdh involved in the glyoxylate shunt and part of the tricarboxylic acid cycle were significantly upregulated, whereas genes involved in glycolysis and the pentose phosphate pathway, such as pgi, fba, gapA, zwf and gnd, were significantly downregulated in response to ppc knockout. It was also found that the intracellular metabolites in glycolysis and the pentose phosphate pathway, such as phosphoenolpyruvate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-phosphate and 6-phosphogluconate, increased, whereas oxaloacetate and acetyl-CoA concentrations decreased in ppc(-) mutant, as compared with those in the parent strain. Comparison between the gene expressions and enzyme activities implies that most of them were well correlated, except a few genes such as icd. It can be said from the present investigation that the combination of the information from gene expressions, enzyme activities and intracellular metabolite concentrations provides a comprehensive understanding of the physiological consequences and regulatory mechanisms for the specific gene-knockout E. coli.

Equilibrium concentrations for pyruvate dehydrogenase and the citric acid cycle at specified concentrations of certain coenzymes

It is of interest to calculate equilibrium compositions of systems of biochemical reactions at specified concentrations of coenzymes because these reactants tend to be in steady states. Thermodynamic calculations under these conditions require the definition of a further transformed Gibbs energy G″ by use of a Legendre transform. These calculations are applied to the pyruvate dehydrogenase reaction plus the citric acid cycle, but steady-state concentrations of CoA, acetyl-CoA and succinyl-CoA cannot be specified because they are involved in the conservation of carbon atoms. These calculations require the use of linear algebra to obtain further transformed Gibbs energies of formation of reactants and computer programs to calculate equilibrium compositions. At specified temperature, pH, ionic strength and specified concentrations of several coenzymes, the equilibrium composition depends on the specified concentrations of the coenzymes and the initial amounts of reactants.

Equilibrium concentrations for pyruvate dehydrogenase and the citric acid cycle at specified concentrations of certain coenzymes

It is of interest to calculate equilibrium compositions of systems of biochemical reactions at specified concentrations of coenzymes because these reactants tend to be in steady states. Thermodynamic calculations under these conditions require the definition of a further transformed Gibbs energy G ″ by use of a Legendre transform. These calculations are applied to the pyruvate dehydrogenase reaction plus the citric acid cycle, but steady-state concentrations of CoA, acetyl-CoA and succinyl-CoA cannot be specified because they are involved in the conservation of carbon atoms. These calculations require the use of linear algebra to obtain further transformed Gibbs energies of formation of reactants and computer programs to calculate equilibrium compositions. At specified temperature, pH, ionic strength and specified concentrations of several coenzymes, the equilibrium composition depends on the specified concentrations of the coenzymes and the initial amounts of reactants.

Acetyl coenzyme A concentrations in plant tissues

Despite the importance of acetyl coenzyme A in many facets of metabolism and the availability of methods for estimation of its concentration, data for acetyl-CoA concentrations in plant tissues have been very scarce. A method using reversed phase HPLC for the quantitative estimation of acetyl-CoA was applied to a variety of plant tissues. In three different developing oilseeds the bulk acetyl-CoA concentration ranged from 5 to 25 nmoles/g fresh weight. In Arabidopsis thaliana leaves it was 5 nmoles/g fresh weight, and in Spinacia oleracea leaves 6.8 nmoles/g fresh weight. Immediate quenching of the harvested tissue in liquid nitrogen is needed to obtain high recoveries of acetyl-CoA.

The role of adenosine triphosphate citrate lyase in the metabolism of acetyl coenzyme a and function of blood platelets in diabetes mellitus

Diabetes is known to increase blood platelet activity. Activities of pyruvate dehydrogenase (PDH), adenosine triphosphate (ATP)-citrate lyase (ATPCL), acetyl-coenzyme A (acetyl-CoA) content, malonyl dialdehyde (MDA), synthesis, and platelet aggregation in resting conditions and after activation with thrombin were measured in diabetic subjects and in age- and sex-matched healthy subjects. Activities of ATPCL and PDH, acetyl-CoA content, and thrombin-evoked MDA synthesis as well as platelet aggregation in diabetes were 31%, 51%, 62%, 35%, and 21%, respectively, higher than in healthy subjects. In addition, activation of diabetic platelets caused 2 times greater release of acetyl-CoA from their mitochondria than in controls. Both 1.0 mmol/L (−)hydroxycitrate and 0.1 mmol/L SB-204490 decreased acetyl-CoA content in platelet cytoplasm along with suppression of MDA synthesis and platelet aggregation. These inhibitory effects were about 2 times greater in diabetic than in control platelets. The data presented indicate that the ATPCL pathway is operative in human platelets and may be responsible for provision of about 50% of acetyl units from their mitochondrial to cytoplasmic compartment. Increased acetyl-CoA synthesis in diabetic platelets may be the cause of their excessive activity in the course of the disease. ATPCL may be a target for its specific inhibitors as factors decreasing platelet activity.

SKELETAL MUSCLE FAT AND CARBOHYDRATE METABOLISM DURING RECOVERY FROM GLYCOGEN DEPLETING EXERCISE IN HUMANS

The primary aim of the present study was to determine whether intramuscular triacylglycerol (IMTG) utilization contributed significantly to the increase in lipid oxidation during recovery from exercise, as determined from the muscle biopsy technique. In addition, we also examined the regulation of pyruvate dehydrogenase (PDHa) and changes in muscle acetyl units during an 18 h recovery period after glycogen-depleting exercise. Eight endurance-trained males completed an exhaustive bout of exercise (~90 min) on a cycle ergometer followed by ingestion of carbohydrate (CHO)-rich meals (64–70 % of energy from carbohydrate) at 1, 4 and 7 h of recovery. Duplicate muscle biopsies were obtained at exhaustion, and 3, 6 and 18 h of recovery. Despite the large intake of CHO during recovery (491 ± 28 g or 6.8 ± 0.3 g kg _1 ), respiratory exchange ratio values of 0.77 to 0.84 indicated a greater reliance on lipid as an oxidative fuel. However, there was no net IMTG utilization during recovery. IMTG content at exhaustion was 23.5 ± 3.5 mmol (kg dry wt) _1 , and remained constant at 24.6 ± 2.6, 25.7 ± 2.8 and 28.4 ± 3.0 mmol (kg dry wt) _1 after 3, 6 and 18 h of recovery. Muscle glycogen increased significantly from 37 ± 11 mmol (kg dry wt) _1 at exhaustion, to 165 ± 13, 250 ± 18, and 424 ± 22 mmol (kg dry wt) _1 at 3, 6 and 18 h of recovery, respectively. PDHa was reduced at 6 and 18 h when compared to exhaustion, but did not change during the recovery period. Acetyl-CoA, acetylcarnitine and pyruvate contents declined significantly after 3 h of recovery compared to exhaustion, and thereafter remained unchanged. We conclude that IMTG has a negligible role in contributing to the enhanced fat oxidation during recovery from exhaustive exercise. Despite the elevation of glucose and insulin following high-CHO meals during recovery, CHO oxidation and PDH activation were decreased, supporting the hypothesis that glycogen resynthesis is of high metabolic priority. Plasma fatty acids, very low density lipoprotein triacylglycerols, as well as intramuscular acetylcarnitine stores are likely to be important fuel sources for aerobic energy, particularly during the first few hours of recovery.

Functional Characterization and Localization of Acetyl-CoA Hydrolase, Ach1p, in Saccharomyces cerevisiae

Acetyl-CoA hydrolase (Ach1p), catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating intracellular acetyl-CoA or CoASH pools; however, its intracellular functions and distribution remain to be established. Using site-directed mutagenesis analysis, we demonstrated that the enzymatic activity of Ach1p is dependent upon its putative acetyl-CoA binding sites. The ach1 mutant causes a growth defect in acetate but not in other non-fermentable carbon sources, suggesting that Ach1p is not involved in mitochondrial biogenesis. Overexpression of Ach1p, but not constructs containing acetyl-CoA binding site mutations, in ach1-1 complemented the defect of acetate utilization. By subcellular fractionation, most of the Ach1p in yeast was distributed with mitochondria and little Ach1p in the cytoplasm. By immunofluorescence microscopy, we show that Ach1p and acetyl-CoA binding site-mutated constructs, but not its N-terminal deleted construct, are localized in mitochondria. Moreover, the onset of pseudohyphal development in homozygote ach1-1 diploids was abolished. We infer that Ach1p may be involved in a novel acetyl-CoA biogenesis and/or acetate utilization in mitochondria and thereby indirectly affect pseudohyphal development in yeast.

Effects of benfluorex on fatty acid and glucose metabolism in isolated rat hepatocytes: from metabolic fluxes to gene expression.

The effects of benfluorex and two of its metabolites (S 422-1 and S 1475-1) on fatty acid and glucose metabolic fluxes and specific gene expression were studied in hepatocytes isolated from 24-h fasted rats. Both benfluorex and S 422-1 (0.1 or 1 mmol/l) reduced beta-oxidation rates and ketogenesis, whereas S 1475-1 had no effect. At the same concentration, benfluorex and S 422-1 were more efficient in reducing gluconeogenesis from lactate/pyruvate than S 1475-1. Benfluorex inhibited gluconeogenesis at the level of pyruvate carboxylase (45% fall in acetyl-CoA concentration) and of glyceraldehyde-3-phosphate dehydrogenase (decrease in ATP/ADP and NAD(+)/NADH ratios). Accordingly, neither benfluorex nor S 422-1 inhibited gluconeogenesis from dihydroxyacetone, but both stimulated gluconeogenesis from glycerol. In hepatocytes cultured in the presence of benfluorex or S 422-1 (10 or 100 micromol/l), the expression of genes encoding enzymes of fatty acid oxidation (carnitine palmitoyltransferase [CPT] I), ketogenesis (hydroxymethylglutaryl-CoA synthase), and gluconeogenesis (glucose-6-phosphatase, PEPCK) was decreased, whereas mRNAs encoding glucokinase and pyruvate kinase were increased. By contrast, Glut-2, acyl-CoA synthetase, and CPT II gene expression was not affected by benfluorex or S 422-1. In conclusion, this work suggests that benfluorex mainly via S 422-1 reduces gluconeogenesis by affecting gene expression and metabolic status of hepatocytes.

Changes in intracellular metabolite pools, and acetate formation in Escherichia coli are associated with a cell-density-dependent metabolic switch

A cell density-dependent metabolic switch in amino acid metabolism occurs in E. coli W3110 batch cultures at 1.15 g dry wt l−1 (Han L, Doverskog M, Enfors S-O, Haggstrom L, 2002, J. Biotechnol.92: 237–249). A two- to three-fold decrease of the concentration of most glycolytic and citric acid cycle metabolites, and an increase in acetyl-CoA concentration after the switch, indicates that the central metabolism also is affected. The specific acetate production rate decreases throughout the culture, except for a temporary increase at the switch point. The intracellular acetate concentration remains relatively constant during the culture.

Ranolazine, a partial fatty acid oxidation inhibitor, reduces myocardial infarct size and cardiac troponin T release in the rat

Ranolazine reduces cellular acetyl-CoA content via inhibition of fatty acid β-oxidation and activates pyruvate dehydrogenase. This metabolic switch increases ATP production per mole of oxygen consumed, reduces the rise in lactic acid and acidosis, and maintains myocardial function under conditions of reduced myocardial oxygen delivery. It is still unclear whether ranolazine causes a reduction of (i) infarct size and (ii) cardiac troponin T release, in a male Wistar rat model of left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h). Rats were subjected to saline infusion ( n=12) or ranolazine (bolus injection: 10 mg/kg plus infusion: 9.6 mg/kg/h, n=12), 30 min prior to left anterior descending coronary artery occlusion–reperfusion, respectively. Ranolazine caused a significant reduction in myocardial infarct size of approximately 33% compared to saline control ( P<0.05). In addition, infusion of ranolazine significantly attenuated the release of cardiac troponin T into the plasma from 65±14 (controls) to 12±2 ng/ml. This study demonstrates for the first time that ranolazine significantly reduces (i) infarct size and (ii) cardiac troponin T release in rats subjected to left anterior descending coronary artery occlusion–reperfusion.

Caffeine ingestion does not alter carbohydrate or fat metabolism in human skeletal muscle during exercise.

This study examined the effect of ingesting caffeine (6 mg kg-1) on muscle carbohydrate and fat metabolism during steady-state exercise in humans. Young male subjects (n = 10) performed 1 h of exercise (70% maximal oxygen consumption (VO2,max)) on two occasions (after ingestion of placebo and caffeine) and leg metabolism was quantified by the combination of direct Fick measures and muscle biopsies. Following caffeine ingestion serum fatty acid and glycerol concentration increased (P< or =0.05) at rest, suggesting enhanced adipose tissue lipolysis. In addition circulating adrenaline concentration was increased (P< or =0.05) at rest following caffeine ingestion and this, as well as leg noradrenaline spillover, was elevated (P< or =0.05) above placebo values during exercise. Caffeine resulted in a modest increase (P< or =0.05) in leg vascular resistance, but no difference was found in leg blood flow. Arterial lactate and glucose concentrations were increased (P< or =0.05) by caffeine, while the rise in plasma potassium was dampened (P< or =0.05). There were no differences in respiratory exchange ratio or in leg glucose uptake, net muscle glycogenolysis, leg lactate release or muscle lactate, or glucose 6-phosphate concentration. Similarly there were no differences between treatments in leg fatty acid uptake, glycerol release or muscle acetyl CoA concentration. These findings indicate that caffeine ingestion stimulated the sympathetic nervous system but did not alter the carbohydrate or fat metabolism in the monitored leg. Other tissues must have been involved in the changes in circulating potassium, fatty acids, glucose and lactate.

The Role of Pyruvate Ferredoxin Oxidoreductase in Pyruvate Synthesis during Autotrophic Growth by the Wood-Ljungdahl Pathway

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO(2). The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO(2) fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase. The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V(max) = 1.6 unit/mg (k(cat) = 3.2 s(-1)), K(m)(Acetyl-CoA) = 9 micrometer, and K(m)(CO(2)) = 2 mm. The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of its k(cat)/K(m) values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis.

Acetyl-CoA metabolism in cholinergic neurons and their susceptibility to neurotoxic inputs.

Cholinergic neurons, unlike other brain cells utilize acetyl-CoA not only for energy production but also for acetylcholine (ACh) synthesis. Therefore, suppression of acetyl-CoA metabolism by different neurotoxic inputs may be particularly harmful for this group of cells. Differentiation of SN56 cholinergic hybrid cells increased their choline acetyltransferase (ChAT) activity and ACh content but depressed pyruvate dehydrogenase activity and acetyl-CoA content. Differentiated cells were more susceptible to acute and chronic influences of aluminum, NO and amyloid-beta. Al decreased acetyl-CoA content, ACh release and increased Ca accumulation in differentiated cells (DC) to much higher degree than in non-differentiated ones (NC). NO strongly depressed acetyl-CoA level and increased ACh release in DC but did not affect NC. Additive effects of Al and NO were seen in DC but not in NC. Also long term suppressory effects of amyloid-beta, Al and NO on cholinergic phenotype and morphologic maturation were more evident in DC than in NC. Thus, relative shortage of acetyl-CoA in highly differentiated cholinergic neurons could make them particularly susceptible to degenerative insults in the course of different cholinergic encephalopathies.

Effect of sulfur fertilisation on oil accumulation, acetyl-CoA concentration, and acetyl-CoA carboxylase activity in the developing seeds of rapeseed (Brassica campestris L.)

The effect of sulfur (S) fertilisation on oil accumulation, acetyl-CoA concentration, and activity of acetyl-CoA carboxylase (EC 6.4.1.2) was determined in the developing seeds of rapeseed (Brassica campestris L. cv. Pusa Gold) grown in the field with and without S. The period between 14 and 35 days after flowering (DAF) was identified as the active period of oil accumulation in the developing seeds of rapeseed. The accumulation of oil was preceded by a marked rise in acetyl-CoA carboxylase activity and acetyl-CoA concentration, which declined rapidly when oil accumulation reached a plateau. Starch and soluble sugar content decreased, while protein content increased during the period of active oil accumulation in the developing seeds (i.e. 14–35 DAF). Sulfur fertilisation significantly (P &lt; 0.05) enhanced the oil accumulation in the developing seeds at all the growth stages except at 7 DAF. The increase in the oil content was 13.0–52.0% with S fertilisation over the control treatment. Sulfur fertilisation also increased acetyl-CoA concentration, acetyl-CoA carboxylase activity, and soluble protein, sugar, and starch content in the developing seeds. It is suggested that the increase in the oil content with S fertilisation may be associated with increases in acetyl-CoA carboxylase activity through the enhancement of acetyl-CoA concentration. Further, the increased sugar content due to S fertilisation provided enough carbon sources for oil biosynthesis.

Acute and chronic effects of aluminum on acetyl-CoA and acetylcholine metabolism in differentiated and nondifferentiated SN56 cholinergic cells.

Mechanisms of preferential loss of cholinergic neurons in the course of neurodegenerative diseases are unknown. Therefore, we investigated whether differentiation-evoked changes in acetyl-CoA and acetylcholine metabolism contribute to the susceptibility of cholinergic neuroblastoma to cytotoxic effects of Al. In SN56 cells differentiated with retinoic acid and dibutyryl cAMP (DC), pyruvate utilization and acetyl-CoA content were lower and acetylcholine level higher than in nondifferentiated cells (NC), respectively. In DC Al and Ca accumulations were 50% and 100%, respectively higher than in NC. Acute Al addition caused inhibition, whereas its chronic application had no effect on pyruvate utilization both in NC and in DC. On the other hand, in both experiments, Al evoked a greater decrease of acetyl-CoA level in DC than in NC. Acute addition of Al depressed acetylcholine release from DC to two times lower values than in NC. On the other hand, chronic addition of Al increased ACh release from DC over twofold, being without effect on its release from NC. These findings indicate that higher accumulation of Ca, along with low levels of acetyl-CoA, could make DC more susceptible to neurotoxic inputs than NC. Excessive acetylcholine release, evoked by Al, is likely to increase acetyl-CoA utilization for resynthesis of the neurotransmitter pool and cause deficit of this metabolite in DC. On the other hand, NC, owing to lower Ca accumulation, slower ACh metabolism, and higher level of acetyl-CoA, would be less prone to these harmful conditions.

Biochemical controls of citrate synthase in chickpea bacteroids.

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 +/- 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The Km value for acetyl-CoA was 174 microM in the absence of added KCl, and 88 microM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 microM without added KCl and 14 microM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 microM NADH and more than 90% by 200 microM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (Kis = 23.1 +/- 3 microM) and linear noncompetitive with respect to oxaloacetate (Kis = 56 +/- 3.8 microM and Kii = 115 +/- 15.4 microM). NADH inhibition was relieved by NAD+ and by micromolar concentrations of 5'-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD+ and 5'-AMP, allowing carbon to enter the tricarboxylic acid cycle.

Regulating the molar fraction of 4-hydroxybutyrate in poly(3-hydroxybutyrate-4-hydroxybutyrate) biosynthesis by Ralstonia eutropha using propionate as a stimulator

The regulation of the molar fraction of 4-hydroxybutyrate (4-HB) in the poly(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] biosynthesis by Ralstonia eutropha (formerly Alcaligenes eutrophus) was attempted by the supplemental addition of propionate. The molar fraction of 4-HB in P(3HB-4HB) was increased significantly from 12.3 to 51.8 mol% by the addition of a small amount of propionate along with γ-butyrolactone commonly used as a precursor for the biosynthesis of P(3HB-4HB). The mechanism of regulation by propionate was investigated by measuring the variation of enzyme activities related to the biosynthesis of P(3HB-4HB) and the level of intermediate metabolite acetyl-CoA. PHB synthase activity was induced significantly by propionate, and the acetyl-CoA concentration also increased significantly due to the additional supply of propionate. The overflowing acetyl-CoA seems to cause an inhibitory effect on the ketolysis reaction catalysing the lysis of 4-hydroxybutyryl-CoA to two molecules of acetyl-CoA; consequently, the 4-HB fraction available for polymerization increased. Accordingly, the molar fraction of 4-HB in P(3HB-4HB) biosynthesis seems to be regulated by both an increased 4-HB fraction and an activated PHB synthase due to the supplemental addition of propionate as a stimulator.

Effect of induced metabolic acidosis on human skeletal muscle metabolism during exercise

The roles of pyruvate dehydrogenase (PDH), glycogen phosphorylase (Phos), and their regulators in lactate (Lac(-)) metabolism were examined during incremental exercise after ingestion of 0.3 g/kg of either NH(4)Cl [metabolic acidosis (ACID)] or CaCO(3) [control (CON)]. Subjects were studied at rest, at rest postingestion, and during continuous steady-state cycling at three stages (15 min each): 30, 60, and 75% of maximal oxygen uptake. Radial artery and femoral venous blood samples, leg blood flow, and biopsies of the vastus lateralis were obtained during each power output. ACID resulted in significantly lower intramuscular concentration of [Lac(-)] (ACID 40.8 vs. CON 56.9 mmol/kg dry wt), arterial whole blood [Lac(-)] (ACID 4.7 vs. CON 6.5 mmol/l), and leg Lac(-) efflux (ACID 3.05 vs. CON 6.98 mmol. l(-1). min(-1)). The reduced intramuscular [Lac(-)] resulted from decreases in pyruvate production due to inhibition of glycogenolysis, at the level of Phos a, and phosphofructokinase, together with an increase in the amount of pyruvate oxidized relative to the total produced. The reduction in Phos a activity was mediated through decreases in transformation, decreases in free inorganic phosphate concentration, and decreases in the posttransformational allosteric regulator free AMP. Reduced PDH activity occurred with ACID and may have resulted from alterations in the concentrations of acetyl-CoA, free ADP, pyruvate, NADH, and H(+), leading to greater relative activity of the kinase. The results demonstrate that imposed metabolic acidosis in skeletal muscle results in decreased Lac(-) production due to inhibition of glycogenolysis at the level of Phos and increased pyruvate oxidation at PDH.