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Results for “Substrate Cycling”

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PubMed1996

EPR detection and characterization of lignin peroxidase porphyrin pi-cation radical.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes the H2O2 dependent one- and two-electron oxidations of substrates. The catalytic cycle involves the oxidation of ferric-LiP by H2O2 by two electrons to compound I, which is an oxoferryl heme and a free radical. It has been speculated that the unpaired electron is in a pi delocalized porphyrin radical. However, no direct evidence for the presence of the free radical has been reported. We present electron paramagnetic resonance (EPR) detection and characterization of compound I of LiP. The LiP compound I EPR signal is different than those reported previously for compound I of horseradish peroxidase and chloroperoxidase. However, the EPR signal of compound I of LiP (axial g tensor extending from gperpendicular = 3.42 to gparallel approximately 2) is very similar to the EPR signals of compound I of ascorbate peroxidase and catalase from Micrococcus lysodeikticus, in which the radical has been identified as a porphyrin pi-cation radical. On the basis of the analysis of our data and comparison with the earlier published results for compounds I of other peroxidases, we interpret the LiP compound I signal by a model for exchange coupling between an S = 1 oxyferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S = 1/2) [Schulz, C.E., et al. (1979) FEBS Lett. 103, 102-105]. The exchange coupling is characterized by ferromagnetic rather than an antiferromagnetic interaction between the two species. The ferric-Lip EPR signal suggests that the iron in the heme is in near perfect orthogonal symmetry and provides additional evidence of the ferromagnetic interaction between the oxoferryl iron center and the porphyrin pi-cation radical.

Basidiomycota↗

PubMed2005

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae.

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.

Adenosine Triphosphatases↗

PubMed2001

Cleavage of E2F-1-regulating proteins and activation of E2F-1 during CD95-induced death of thymocytes.

The CD95 death receptor activates caspases that cleave a variety of intracellular substrates, including cell cycle control proteins. However, the significance of this cleavage for the induction of apoptosis is unclear. In this study, CD95-induced cleavage of the G1/S checkpoint regulator proteins, retinoblastoma protein (pRb) and murine-double-minute-2 (mdm-2), was associated with an increased protein concentration of a key transcription factor, E2F-1, which is regulated by both of them. Furthermore, DNA-binding activity to E2F sites is increased. In thymocytes, CD95-induced apoptosis was associated with increased E2F-1 DNA-binding activity, while thymocytes that lacked E2F-1 were less susceptible to CD95-induced apoptosis. We conclude that the G1/S checkpoint is an important target of CD95 signalling. CD95-activated caspases cleave regulator proteins to increase E2F-1 activity, and inappropriate activation of E2F-1 is part of the mechanism of CD95-induced apoptosis.

Animals↗

PubMed1984

Base (O.-2, e-, or OH-)-induced autoxygenation of organic substrates: a model chemical system for cytochrome P-450-catalyzed monoxygenation and dehydrogenation by dioxygen.

When catalytic quantities of superoxide ion (O.-2; or of electrons from electrolysis or of OH-) are introduced into a dry acetonitrile solution that contains excess substrate (RH), ambient air (O2), 1,2-diphenylhydrazine (PhNHNHPh), and iron(II), the substrate is rapidly and efficiently monoxygenated (e.g., triphenylphosphine----triphenylphosphine oxide, benzyl alcohol----benzaldehyde, diphenylsulfoxide----diphenylsulfone) or dehydrogenated (1,4-cyclohexadiene----benzene). The model consists of (i) an O2-activation segment that produces H2O2 from an O.-2-initiated autoxidation of disubstituted hydrazine (a model for reduced flavin), (formula; see text) and (ii) a H2O2-activation segment via the iron(II)-induced formation of ferryl ion (FeO2+), Fe(II) + H2O2----FeO2 + H2O, an effective monoxygenating agent: FeO2+ + RH----Fe(II) + ROH. The combination of i and ii provides a catalytic system for the autoxidation of organic substrates with reaction cycles that are similar to those for cytochrome P-450 monoxygenases.

Cytochrome P-450 Enzyme System↗

PubMed1998

A novel method for selection of chymotrypsin inhibitors from a phage peptide library.

A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library. In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates. After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing. These peptides share a consensus sequence motif as (S/T)RVPR(R/H). Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin. The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Bacteriophages↗

PubMed1990

The O-methylation of 4-hydroxyestradiol is inhibited by 2-hydroxyestradiol: implications for estrogen-induced carcinogenesis.

O-Methylation of catecholestrogens catalyzed by catechol-O-methyltransferase provides a major route for the rapid metabolic clearance of these steroids. However, the metabolic clearance rate of 4-hydroxyestradiol (4-OH-E2) is considerably lower than that of 2-hydroxyestradiol, although 2- and 4-hydroxycatecholestrogens (2- and 4-OH-CE) have similar apparent affinities for the enzyme. To determine the reason for this apparent paradox we have examined whether the efficiency of O-methylation of 4-OH-E2 could be affected by other catecholestrogens or their O-methyl ethers. The ratio of 4-methoxyestradiol:4-hydroxyestradiol 3-methyl ether was 2.6 at pH 8.5, the pH optimum for the reaction. The O-methylation of 4-OH-E2 (apparent Km 10 microM) was inhibited by 2-hydroxyestradiol (2-OH-E2) but not by 2- or 4-methyoxyestrogens. The values for Km, Vmax as well as the slope for the methylation of 4-OH-E2 were altered by 2-OH-E2 indicating a mixed inhibition. The inhibition constant for the intercept 1/V'max versus 2-OH-E2 concentrations and the inhibition constant for the slope versus 2-OH-E2 concentrations were 35 and 5.7 microM, respectively. The inhibition of O-methylation of 4-OH-E2 by 2-OH-E2 increased with the pH. In target tissues of the carcinogenic action of estrogens such as the rat pituitary, hamster kidney, or mouse uterus in which 2- and 4-OH-CE are both generated in almost equal amounts, the inactivation of 4-OH-CE by O-methylation may be impeded. Consequently, 4-OH-E2 would remain available as substrate for redox cycling, generation of active radicals and DNA damage.

Animals↗

PubMed2003

SMG-5, required for C.elegans nonsense-mediated mRNA decay, associates with SMG-2 and protein phosphatase 2A.

mRNAs that contain premature stop codons are degraded selectively and rapidly in eukaryotes, a phenomenon termed 'nonsense-mediated mRNA decay' (NMD). We report here molecular analysis of smg-5, which encodes a novel protein required for NMD in Caenorhabditis elegans. Using a combination of immunoprecipitation and yeast two-hybrid assays, we identified a series of protein-protein interactions involving SMG-5. SMG-5 interacts with at least four proteins: (i) SMG-7, a previously identified protein required for NMD; (ii) SMG-2, a phosphorylated protein required for NMD in worms, yeasts and mammals; (iii) PR65, the structural subunit of protein phosphatase 2A (PP2A); and (iv) PP2A(C), the catalytic subunit of PP2A. Previous work demonstrated that both SMG-5 and SMG-7 are required for efficient dephosphorylation of SMG-2. Our results suggest that PP2A is the SMG-2 phosphatase, and the role of SMG-5 is to direct PP2A to its SMG-2 substrate. We discuss cycles of SMG-2 phosphorylation and their roles in NMD.

Animals↗

PubMed2005

The CB1 receptor antagonist rimonabant reverses the diet-induced obesity phenotype through the regulation of lipolysis and energy balance.

We investigated the molecular events involved in the long-lasting reduction of adipose mass by the selective CB1 antagonist, SR141716. Its effects were assessed at the transcriptional level both in white (WAT) and brown (BAT) adipose tissues in a diet-induced obesity model in mice. Our data clearly indicated that SR141716 reversed the phenotype of obese adipocytes at both macroscopic and genomic levels. First, oral treatment with SR141716 at 10 mg/kg/d for 40 days induced a robust reduction of obesity, as shown by the 50% decrease in adipose mass together with a major restoration of white adipocyte morphology similar to lean animals. Second, we found that the major alterations in gene expression levels induced by obesity in WAT and BAT were mostly reversed in SR141716-treated obese mice. Importantly, the transcriptional patterns of treated obese mice were similar to those obtained in the CB1 receptor knockout mice fed a high-fat regimen and which are resistant to obesity, supporting a CB1 receptor-mediated process. Functional analysis of these modulations indicated that the reduction of adipose mass by the molecule resulted from an enhanced lipolysis through the induction of enzymes of the beta-oxidation and TCA cycle, increased energy expenditure, mainly through futile cycling (calcium and substrate), and a tight regulation of glucose homeostasis. These changes accompanied a significant cellular remodeling and contributed to a reduction of the obesity-related inflammatory status. In addition to a transient reduction of food consumption, increases of both fatty acid oxidation and energy expenditure induced by the molecule summate leading to a sustained weight loss. Altogether, these data strongly indicate that the endocannabinoid system has a major role in the regulation of energy metabolism.

Adipocytes↗

PubMed2005

Reduction in muscle glycogen and protein utilization with glucose feeding during exercise.

Effects of feeding glucose on substrate metabolism during cycling were studied. Trained (60.0 +/- 1.9 mL x kg(-1) x min(-1)) males (N = 5) completed two 75 min, 80% VO(2max) trials: 125 g 13(C)-glucose CHO); 13(C)-glucose tracer, 10 g (C). During warm-up (30 min 30% VO2max) 2 . 2 g 13(C)-glucose was given as bicarbonate pool primer. Breath samples and blood glucose were analyzed for 13(C/12)C with IRMS. Protein oxidation was estimated from urine and sweat urea. Indirect calorimetry (protein corrected) and 13(C/12)C enrichment in expired CO(2)and blood glucose allowed exogenous (Gexo), endogenous (Gendo), muscle (Gmuscle), and liver glucose oxidation calculations. During exercise (75 min) in CHO versus C (respectively): protein oxidation was lower (6.8 +/- 2.7, 18.8 +/- 5.9 g; P = 0.01); Gendo was reduced (71.2 +/- 3.8, 80.7 +/- 5.7% P = 0.01); Gmuscle was reduced (55.3 +/- 6.1, 65.9 +/- 6.0%; P = 0.01) compensated by increased Gexo(58.3 +/- 2.1, 3.87 +/- 0.85 g; P = 0.000002). Glucose ingestion during exercise can spare endogenous protein and carbohydrate, in fed cyclists, without glycogen depletion.

Adolescent↗

PubMed2003

Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation.

Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to alpha-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native beta-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to alpha-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.

Animals↗

PubMed2006

The cellular phenotype of AZ703, a novel selective imidazo[1,2-a]pyridine cyclin-dependent kinase inhibitor.

Because the majority of cancers exhibit direct or indirect deregulation of cyclin-dependent kinase (CDK) function, members of the CDK family are attractive targets for the development of anticancer agents. As part of an ongoing program, novel imidazopyridines were identified and developed as potent and selective CDK inhibitors. Here, we describe data on the in vitro biological activities of one of these compounds, AZ703. The selectivity profile of AZ703 was investigated in kinase assays against a range of CDK enzymes as well as a panel of protein kinases in vitro. IC50s were assessed against different tumor cell lines in vitro. The mechanism of action of AZ703 was determined by observing changes in phosphorylation of CDK substrates and cell cycle effects on tumor and normal cells. In vitro studies revealed that AZ703 is a selective inhibitor of CDK1 and CDK2 and displays a mode of action consistent with the induction of G1-, S-, and G2-M-phase arrest. AZ703 also showed potent antiproliferative activity across a wide range of tumor cell lines in vitro. Moreover, AZ703 induced reversible blockade of normal cells while causing tumor cells to undergo apoptosis. We have identified AZ703 as a novel selective imidazo[1,2-a]pyridine CDK inhibitor that shows promising antitumor properties in vitro.

Apoptosis↗

PubMed2004

Protecting muscle ATP: positive roles for peripheral defense mechanisms-introduction.

Skeletal muscle has evolved an impressive array of mechanisms for peripherally mediated control of ATP homeostasis. Some of these mechanisms are intracellular, and others are extracellular and include influences on the cross-bridge cycle itself and substrate supply. This paper introduces three distinctly different topics that nevertheless all have ATP defense in common. The role of ADP in fatigue is controversial but has recently been more clearly delineated so that an effect on alleviating force declines during extreme fatigue is plausible. AMP plays its role by activating the protein-kinase, AMPK, which is a key sensor of cellular energy stress. AMPK has different isoforms, is not uniformly distributed in the cell, and its activation is carefully controlled. It has multiple effects including improvements in substrate supply for the metabolic pathways producing ATP and inhibition of anabolic processes to further spare ATP. Red blood cells have the capacity to sense hypoxia and to release vasodilators where there is a locally increased demand for blood supply. The papers in this series emphasize the important positive roles of metabolites and sensors of fatigue in the balance between ATP supply and demand.

Adenosine Triphosphate↗

PubMed1991

Preventive effect of isocitrate on glucocorticoid-induced cataract formation of developing chick embryo.

Glucocorticoid-induced cataract formation appears to proceed via oxidation or peroxidation steps possibly caused by multiple activities of glucocorticoid in the living system. Attempts were made to modify GC-induced metabolic changes and prevent cataract formation using intermediates of the citric acid cycle. The compounds were applied to the embryos at 3, 10 and 20 hr after the administration of hydrocortisone succinate sodium (HC:0.25 mumol/egg) to 15-day-old eggs. At 48 hr after HC treatment the lenses were classified and analyzed. Almost all lenses were classified as stage IV-V (greater than 94%). However, the application of sodium isocitrate (IC:15 mumol/egg) which was the most potent among several intermediates tested showed a significant preventive effect against cataract formation. The administration of IC prevented the decline of GSH, the elevations of LPO and reduced the marked elevation of glucose in the lens caused by HC. The IC treatment also diminished the elevation of LPO in blood and liver. The above effects by IC on HC-induced events may be due to the action of IC in preventing the early decline of hepatic GSH caused by HC. Possibly IC was utilized as an intermediate of the citric acid cycle and a substrate for isocitrate dehydrogenase in cytosol to modify GC-induced metabolic changes.

Animals↗

PubMed1975

Metabolic changes in the cerebral cortex of the rat induced by intravenous pentothalsodium.

The effects of thiopental on cerebral metabolism were evaluated by means of measurements of cerebral metabolic rate for oxygen (CMRO2) and of tissue levels of organic phosphates, glycolytic substrates, citric acid cycle intermediated and selected amino acids. Shortly after the beginning of thiopental administration, CMRO2 was reduced to half the normal value, and there were signs of retardation of glycolytic flux at the phosphofructokinase step. Inhibition at this regulatory enzymatic step could be related to an increase in phosphocreatine and a fall in inorganic phosphate concentration. The rise in phosphocreatine, and the unchanged levels of ATP, ADP and AMP demonstrate that induction of anaesthesia with thiopental is unrelated to energy failure. Changes in citric acid intermediated included a fall in malate, and probably also in citrate, alpha-ketoglutarate and fumarate, with a tendency towards reduction in the pool of citric acid cycle intermediates; and the amino acid changes were dominated by a progressive rise in aspartate. It is suggested that these changes are secondary to a reduced rate of pyruvate delivery, and to a decrease in malate/oxaloacetate ratio.

Adenosine Diphosphate↗

PubMed1992

Role of the membrane surface in the activation of human coagulation factor X.

Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.

Animals↗

PubMedDate not supplied

[Photoinduced reduction of NAD(P) in the cells of green sulfur bacteria].

The spectrum of a photoinduced increase in luminescence of the cells of the gree sulphur bacterium Chlorobium limicola f. thiosulfatophilum, within the range of 400 to 520 nm, was found to correspond to the spectrum of luminescence of NADH in the protein-bound form. Photoinduced reduction of NAD(P) in green bacteria, contrary to purple bacteria, is not susceptible to the action of p-chlorocarbonylcyanide phenlhydrazone which uncouples photophosphorylation. Therefore, in Chlorobium limicola f. thiosulfatophilum, NAD(P) is reduced by direct non-cyclic transport of electrons via the photosynthetic chain. NAD(P)H is utilized mainly in the system of CO2 fixation; the process is inhibited by fluoroacetate, and the inhibition is eliminated by substrates of the cycle of carboxylic acids.

Carbon Dioxide↗

PubMed1984

Persistent hyperinsulinemia due to induced euglycemia in awake endotoxic minipigs.

Transient hyperinsulinemia has been incriminated as a contributing factor to endotoxin-induced hypoglycemia. However, a recent study using endotoxic minipigs noted an increase in the rate of glucose utilization prior to when hyperinsulinemia was seen. Based on this, the "misinformed beta-cell" hypothesis was proposed. If endotoxic pancreatic beta-cells experience an early increase in glucose uptake, they could over-estimate the true glycemic state and release insulin, further contributing to the hypoglycemia. Hypoglycemia would offset increased glucose uptake by the beta-cell and insulin secretion would return to basal rates, explaining the transient nature of the insulin response. According to this hypothesis, maintenance of euglycemia should elicit a sustained rather than transient insulin release. The hypothesis was tested with a euglycemic clamp technique in a group of five endotoxic (Difco 055:B5; 15 micrograms/kg/hr) minipigs (EC) compared to three nonclamped endotoxic minipigs (E). Persistent, profound hyperinsulinemia (15 ng/ml) occurred in the EC group, further supporting the hypothesis. The clamp appeared to be beneficial, since arterial pressure and pH were significantly (P less than or equal to 0.05) higher and arterial lactate levels were lower in EC pigs compared to E pigs. A significant discrepancy was noted between the radioisotope ( [6-3H] glucose)-derived rate of glucose appearance and the rate of exogenous glucose infusion (clamp), which may have been due to unaccounted for changes in glucose pool size and/or futile cycling of glucogenic substrates or glucose through glycogen and lipid.

Acid-Base Equilibrium↗

PubMed1983

Action of testosterone on some biochemical parameters related to the energy metabolism of the skeletal muscle.

The effect of intramuscular administration of testosterone propionate was studied on rat gastrocnemius muscle. Muscular glycolytic substrates and Krebs' cycle metabolites (glycogen, glucose, glucose-6-phosphate, pyruvate, lactate, citrate, alpha-ketoglutarate, succinate, malate), related aminoacids (glutamate, alanine, ammonia), energy store and mediators (creatine phosphate, ATP, ADP, AMP) and the energy charge potential were evaluated. The influence of the factors: testosterone dose, time course of treatment and sex of animals was investigated, no relevant changes being noticed.

Amino Acids↗