Analytical solutions for (13)C isotopomer analysis of complex metabolic conditions: substrate oxidation, multiple pyruvate cycles, and gluconeogenesis.
Explore the source record for details and available documents.
Carbon Isotopes↗
SEARCH · Search PubMed
Results for “Substrate Cycling”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Cs(2) fixation by carbonic anhydrase model systems-a new substrate in the catalytic cycle.
The conversion of CS(2) with common carbonic anhydrase model systems has been studied using Hartree-Fock and density-functional theory methods employing the 6-311+G basis set. The calculated geometries and energetical parameters for [L(3)ZnOH](+)/CS(2) model systems (L = NH(3), imidazole) are compared with those obtained previously for the CO(2) hydration. While the same reaction mechanism applies for both heterocumulenes, the hypothetical conversion of CS(2) to give [L(3)ZnSC(O)SH](+) is characterized by a higher barrier and is much more exothermic than the corresponding CO(2) reaction cascade. Due to the increased number of heteroatoms, additional intermediates and product structures (compared with those involved in the CO(2) conversion) must be taken into account and have been analyzed in detail. The smaller electrophilicity of CS(2) is the reason for the higher activation energies, while the significantly increased exothermicity is due to the strong zinc(II)/sulfur interaction. The reversibility and therefore the existence of a catalytic cycle which could allow comparable CS(2) transformations must be questioned. Nevertheless, an interesting field of stoichiometric zinc-mediated CS(2) transformations is conceivable.
Carbon Dioxide↗
Effect of medium-chain triacylglycerol and carbohydrate ingestion during exercise on substrate utilization and subsequent cycling performance.
The aim of this study was to investigate the effect of medium-chain triacylglycerol (MCT) ingestion during exercise on subsequent time-trial cycling performance. Seven well-trained cyclists performed four exercise trials consisting of 2 h at 60% of maximal oxygen uptake followed by a simulated time trial (ie, completion of a preset amount of work as fast as possible) of approximately 15 min duration. During the trials, subjects ingested 1) a 10% carbohydrate solution (CHO; 170 +/- 6 g glucose), 2) a 10% carbohydrate electrolyte with 5% MCT solution (CHO + MCT; 85 +/- 3 g MCT), 3) a 5% MCT solution, or 4) artificially colored and flavored water (placebo). Neither CHO nor CHO + MCT ingestion had any effect on performance compared with placebo ingestion, whereas ingestion of MCT had a negative effect on performance. Average work rates during the time trial were 314 +/- 19, 314 +/- 13, and 312 +/- 18 with CHO, CHO + MCT, and placebo, respectively, and was 17-18% lower in the MCT trial (263 +/- 22 W). In addition, compared with placebo ingestion, MCT ingestion had no effect on total rates of fat or carbohydrate oxidation, nor did it affect exogenous or endogenous carbohydrate utilization. The negative effect of MCT ingestion was associated with increased gastrointestinal complaints (ie, intestinal cramping). These data suggest that large amounts of MCTs (85 g) ingested during prolonged submaximal exercise may provoke gastrointestinal problems leading to decreased exercise performance.
3-Hydroxybutyric Acid↗
Aerobic conditioning effects on substrate responses during graded cycling in pregnancy.
This study was conducted to test the hypothesis that aerobic conditioning prevents exercise-induced hypoglycemia and preserves the capacity to utilize carbohydrates and to produce lactate during heavy exercise in late gestation. The effects of closely monitored cycle ergometer conditioning (heart rate = 143 +/- 2 beats/min, 25 min/day, 3 days/week) during the second and third trimesters were studied in 18 previously sedentary women (exercised group, EG). A nonexercising pregnant control group (CG, n = 9) was also studied. Data collection times for both groups were as follows: start of the second trimester (Entry), ends of the second (TM2) and third (TM3) trimesters (post-training), and 4-6 months postpartum (nonpregnant control). Respiratory gas exchange was studied and venous blood samples were obtained before, during, and after a graded cycle ergometer test that was terminated at a peak heart rate of 170 beats/min. Measurements included plasma glucose, insulin, free fatty acids, the respiratory exchange ratio at peak exercise, and peak postexercise lactate concentration. A significant aerobic conditioning effect in the EG was confirmed by a 17% increase in O2 pulse at peak exercise between Entry and TM3. As expected, values for free fatty acids in the CG rose with advancing gestational age. The CG showed a clear trend for a rise in plasma insulin with advancing gestational age, under all experimental conditions. Also, peak exercise respiratory exchange ratio and peak postexercise lactate concentration were significantly reduced in late gestation, and plasma glucose decreased significantly during and following the end of TM3 testing. Effects of pregnancy to reduce peak postexercise lactate and to reduce plasma glucose during and after exercise at the end of the third trimester were significantly attenuated in the EG. These effects were attributed to attenuation of pregnancy-induced insulin resistance (as reflected by insulin/glucose ratio) by physical conditioning. These findings support our original experimental hypothesis that aerobic conditioning prevents exercise-induced hypoglycemia and preserves the ability to utilize carbohydrate and produce lactate during heavy exercise in late gestation.
Adult↗
[Effect of hydrocortisone on the oxidation of substrates in the Krebs cycle].
Explore the source record for details and available documents.
Animals↗
Letter: The presence of the homoanalogues of substrates of the urea cycle in the presence of argininosuccinate synthetase deficiency.
Explore the source record for details and available documents.
Amino Acid Metabolism, Inborn Errors↗
Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalysed by a human placental 17beta-hydroxysteroid dehydrogenase.
The human placental 17beta-hydroxysteroid dehydrogenase reduces a number of polycyclic aromatic hydrocarbon (PAH) o-quinones; some of the quinones undergo redox cycling at rates that approach or exceed the rate of reduction of estrone by the enzyme. The non-K-region o-quinone, 7,8-benzo[a]pyrenequinone, is the best o-quinone substrate tested. Cycling of all the quinone substrates is inhibited by superoxide dismutase; cycling is also inhibited by 17beta-estradiol and other estrogens. Since 19 alpha-estradiol is a competitive inhibitor of 9,10-phenanthrenequinone by the 17beta-hydroxysteroid dehydrogenase, it is likely that both reactions occur at the same active site on the enzyme. In the presence of the 17beta-hydroxysteroid dehydrogenase, the equilibrium between 17beta-estradiol, estrone, NADP, and NADPH is shifted by 7,8-benzo[a]pyrenequinone because the rapid redox cycling of this quinone results in the oxidation of NADPH. Unlike a number of hydroxysteroid dehydrogenases, the placental 17beta-hydroxysteroid dehydrogenase does not oxidize any of the six PAH trans-dihydrodiols tested.
17-Hydroxysteroid Dehydrogenases↗
Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone.
Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release. ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70. This key step is strongly stimulated by DnaJ cochaperones. We show for the Escherichia coli Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK. Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK. It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK. Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins.
Adenosine Triphosphatases↗
Protein targeting by the bacterial twin-arginine translocation (Tat) pathway.
The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.
Bacteria↗
Crystal structure of a thwarted mismatch glycosylase DNA repair complex.
The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase. The structure of this complex explains the preference for G:U over G:T mispairs, and reveals an essentially non-specific pyrimidine-binding pocket that allows MUG/TDG enzymes to excise the alkylated base, 3, N(4)-ethenocytosine. Together with structures for the free enzyme and for an abasic-DNA product complex, the MUG-substrate analogue complex reveals the conformational changes accompanying the catalytic cycle of substrate binding, base excision and product release.
Base Pair Mismatch↗
Glucose kinetics and exercise performance during phases of the menstrual cycle: effect of glucose ingestion.
To study the effect of menstrual cycle phase and carbohydrate ingestion on glucose kinetics and exercise performance, eight healthy, moderately trained, eumenorrheic women cycled at 70% of peak O(2) consumption for 2 h and then performed a 4 kJ/kg body wt time trial. A control (C) and a glucose ingestion (G) trial were completed during the follicular (F) and luteal (L) phases of the menstrual cycle. Plasma substrate concentrations were similar before the commencement of exercise. Glucose rates of appearance and disappearance were higher (P < 0.05) during the 2nd h of exercise in FC than in LC. The percent contribution of carbohydrate to total energy expenditure was greater in FC than in LC, and subjects performed better (13%, P < 0.05) in FC. Performance improved (19% and 26% in FG and LG compared with FC and LC, respectively, P < 0.05) with the ingestion of glucose throughout exercise. These data demonstrate that substrate metabolism and exercise performance are influenced by the menstrual cycle phase, but ingestion of glucose minimizes these effects.
Adult↗
Depression of neuron responses to acetylcholine by combined application of norepinephrine and substrates of the tricarboxylic acid cycle.
The possible relationship between the function of nicotinic acetylcholine receptors in Lymnaea stagnalis neurons and energy metabolism was studied. Oxidative phosphorylation was activated by treatment of neurons with substrates of the tricarboxylic acid cycle and norepinephrine. Transmembrane currents induced by acetylcholine in isolated neurons were measured by voltage clamp. Succinate dehydrogenase activity was determined histochemically in the same neurons. Cyclic adenosine monophosphate concentration in ganglia were assayed by the protein saturation method of Gilman (1970). When used alone, succinate depressed the responses of about 50% of neurons to acetylcholine. Norepinephrine did not affect the acetylcholine-induced currents but almost doubled the inhibitory action of succinate. The mixture of norepinephrine and isocitrate also diminished the responses to acetylcholine but to a lesser extent than norepinephrine with succinate. A short-term exposure of the ganglia to succinate with norepinephrine led to the activation of succinate dehydrogenase in neurons and a threefold increase in cyclic adenosine monophosphate concentrations in ganglia. When used alone, norepinephrine doubled the cyclic adenosine monophosphate concentration. The results obtained suggest energy-dependent regulation of acetylcholine receptors.
Acetylcholine↗
Effect of total hepatectomy on selected cerebral substrates and enzymes of the glycolytic pathways and Krebs cycle.
Posthepatectomy coma was produced in 13 dogs and the cerebrums were biopsied for analysis of concentrations of glucose, glucose-6-phosphate, dihydroxyacetone-phosphate, phosphoenolpyruvate, pyruvate, lactate, citrate, alpha-ketogulutarate, fumarate, malate, oxaloacetate, adenosinetriphosphate, ammonia, and glutamine as well as for activities of glucokinase, phosphofructokinase, pyruvate kinase, isocitrate dehydrogenase, glutamate dehydrogenase, malate dehydrogenase, and malic enzyme. There were no differences from normal in the brain glycolytic substrate concentrations. Four of the Krebs cycle substrates were significantly reduced, but not differently than in dogs sedated for 24 hours. The glycolytic pathway, Krebs cycle, and related enzyme activities were not significantly altered. Cerebral adenosine triphosphate concentration was unchanged but the concentrations of ammonia and glutamine increased threefold.
Adenosine Triphosphatases↗
The chaperonin cycle and protein folding.
The process of protein folding in the cell is now known to depend on the action of other proteins. These proteins include molecular chaperones, which interact non-covalently with proteins as they fold and improve the final yields of active protein in the cell. The precise mechanism by which molecular chaperones act is obscure. Experiments reported recently show that for one molecular chaperone (Cpn60, typified by the E. coli protein GroEL), the folding reaction is driven by cycles of binding and release of the co-chaperone Cpn10 (known as GroES in E. coli). These alternate with binding and release of the unfolded protein substrate. These cycles come about because of the opposite effects of Cpn10 and unfolded protein on the Cpn60 complex: the former stabilises the ADP-bound state of Cpn60, whereas the latter stimulates ADP-ATP exchange. This model proposes that the substrate protein goes through multiple cycles of binding and release, and is released into the cavity of the Cpn60 complex where it can undergo folding without interacting with other nearby folding intermediates. This is consistent with the ability of Cpn60 proteins to enhance folding by blocking pathways to aggregation.
Adenosine Triphosphate↗
Cell-cycle regulated expression and serine phosphorylation of the myristylated protein kinase C substrate, SSeCKS: correlation with culture confluency, cell cycle phase and serum response.
We recently identified a novel myristylated protein kinase C (PKC) substrate, named SSeCKS (pronounced essex), whose transcription is suppressed > 15 fold in src- or ras-transformed rodent fibroblasts, but not in raf-transformed cells [1, 2]. SSeCKS associates with and controls the elaboration of a cortical cytoskeletal matrix in response to phorbol esters [2], and overexpression of SSeCKS causes growth arrest of untransformed NIH3T3 cells [3]. Our preliminary data suggested that SSeCKS functions as a negative mitogenic regulator by controlling cytoskeletal architecture and that serine phosphorylation of SSeCKS by kinases such as PKC alters its interaction with cytoskeletal matrices and its ability to control mitogenesis. Here, we determine the effects of culture confluency, growth arrest and serum response on the steady-state abundance of SSeCKS RNA and protein and on the relative level of phosphoserine-free SSeCKS. SSeCKS transcription is initially induced by serum factors and by contact-inhibited growth rather than by cell-cycle arrest induced by serum starvation, hydroxyurea or nocodazole, and following serum-induced G1/S progression, SSeCKS transcription is suppressed. SSeCKS protein is hyperphosphorylated on serine residues during G1/S progression but not during the G2/M phase. Finally, we show that the induction of SSeCKS protein expression by contact inhibition is independent of SSeCKS' serum responsiveness. These data suggest that SSeCKS expression and function can be controlled at either the transcriptional or post-translational level in response to serum factors and culture confluency. The data strengthen the notion that SSeCKS plays an important, yet transient, role in cell cycle progression from G0 to G1 that differs from its role in controlling contact-inhibited growth.
A Kinase Anchor Proteins↗
Substrate responses to submaximal exercise in the midfollicular and midluteal phases of the menstrual cycle.
This study examined substrate metabolism responses of eumenorrheic women to different intensities of submaximal exercise at the midfollicular (MF) and the midluteal (ML) phases of the menstrual cycle. Nine women performed a 30-min treadmill run in which the exercise intensity was made more difficult every 10 min (35%, 60%, and 75%). Carbohydrate (CHO) utilization and oxidation rates for the 35% and 60% intensities during the ML session were significantly lower than during the comparable intensities in the MF. Conversely, lipid utilization and oxidation were significantly greater during the 35% and 60% ML session than in the MF session. At 75%, however, the ML and MF CHO-lipid utilization and oxidation rates were not significantly different from one another. Thus, the phase of the menstrual cycle in eumenorrheic women does influence metabolic substrate usage during low- to moderate-intensity submaximal exercise, probably due to changes in the endogenous levels of the female sex hormones.
Adult↗
Contribution of various substrates to total citric acid cycle flux and anaplerosis as determined by 13C isotopomer analysis and O2 consumption in the heart.
A simple relationship between parameters derived from a 13C NMR isotopomer analysis and O2 consumption is presented that allows measurement of the absolute rate of acetyl-CoA oxidation and anaplerotic flux in tissues oxidizing a mixture of four substrates. The method was first applied in a study of the effects of work state and beta-adrenergic stimulation on net acetate oxidation and anaplerosis in the isolated working rat heart. The results demonstrate that the anticipated ratio of 2 between O2 consumption and TCA cycle flux for hearts oxidizing only acetate holds at low workload when anaplerosis is low, but deviates toward a factor of 3 under high workload conditions when anaplerosis is increased. This analysis was also extended to hearts that oxidize a more physiological mixture of substrates including long-chain fatty acids, acetoacetate, lactate, pyruvate, and glucose. We show that the contribution each substrate makes to total TCA cycle flux can be determined by combined 13C NMR and O2 consumption measurements. The present study also demonstrates that stimulation of anaplerosis (by addition of propionate) can significantly alter the relative contribution each substrate makes to total TCA cycle flux. We conclude if 13C labeling patterns are selected appropriately, a comprehensive picture of flux through all major metabolic pathways feeding the cycle can be determined in a single experiment even when complex physiological mixtures of substrates are provided.
Acetates↗
Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase.
Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.
Adenosylhomocysteinase↗