Presence Of Other Substrates Research Articles

Increased dietary protein rather than fiber supports key metabolic and intestinal tissue functions in pigs, without increasing post-weaning diarrhea.

The post-weaning period in pigs is a critical window where nutritional interventions are implemented to prevent post-weaning diarrhea (PWD) and antibiotic use. One common strategy is feeding of low protein diets immediately following weaning. This intervention may reduce protein fermentation and pathogen proliferation, therefore decreasing the incidence of post-weaning diarrhea. These effects may also be mitigated by providing dietary fiber. However, studies examining the role of protein and fiber on gastrointestinal microbiota and metabolism are complicated by the presence of other substrates, including polyphenols and antinutritional factors in complex ingredients. In this study, semi-purified diets formulated to meet nutrient requirements were fed to 40 weaned pigs (n = 10/diet) to examine the effects of high protein (HP), high fiber (HF), or both (HFHP) compared to a control (CON) diet with industry standard crude protein and fiber content. Critical alterations in host metabolism and cecal transcriptome were identified in response to the CON diet. Diets with lower protein levels (CON and HF) induced alteration in transcripts from the serine synthesis pathways and integrated stress response in cecal tissue alongside systemic increases in metabolic pathways related to lysine degradation. High protein diets did not induce increases in gastrointestinal pathogen abundance. These results challenge the practice of feeding low protein diets post-weaning, by demonstrating a detrimental effect on intestinal cell function and muscle accretion. This suggests that with careful ingredient selection, increased dietary protein post-weaning could better pig health and growth compared to a standard diet.

Inducible and reversible SOD2 knockdown in mouse skeletal muscle drives impaired pyruvate oxidation and reduced metabolic flexibility.

Skeletal muscle mitochondrial dysfunction is a key characteristic of aging muscle and contributes to age related diseases such as sarcopenia, frailty, and type 2 diabetes. Mitochondrial oxidative distress has been implicated as a driving factor in these age-related diseases, however whether it is a cause, or a consequence of mitochondrial dysfunction remains to be determined. The development of more flexible genetic models is an important tool to test the mechanistic role of mitochondrial oxidative stress on skeletal muscle metabolic dysfunction. We characterize a new model of inducible and reversible mitochondrial redox stress using a tetracycline controlled skeletal muscle specific short hairpin RNA targeted to superoxide dismutase 2 (iSOD2). iSOD2 KD and control (CON) animals were administered doxycycline for 3- or 12- weeks and followed for up to 24 weeks and mitochondrial respiration and muscle contraction were measured to define the time course of SOD2 KD and muscle functional changes and recovery. Maximum knockdown of SOD2 protein occurred by 6 weeks and recovered by 24 weeks after DOX treatment. Mitochondrial aconitase activity and maximum mitochondrial respiration declined in KD muscle by 12 weeks and recovered by 24 weeks. There were minimal changes in gene expression between KD and CON muscle. Twelve-week KD showed a small, but significant decrease in muscle fatigue resistance. The primary phenotype was reduced metabolic flexibility characterized by impaired pyruvate driven respiration when other substrates are present. The pyruvate dehydrogenase kinase inhibitor dichloroacetate partially restored pyruvate driven respiration, while the thiol reductant DTT did not. We use a model of inducible and reversible skeletal muscle SOD2 knockdown to demonstrate that elevated matrix superoxide reversibly impairs mitochondrial substrate flexibility characterized by impaired pyruvate oxidation. Despite the bioenergetic effect, the limited change in gene expression suggests that the elevated redox stress in this model is confined to the mitochondrial matrix.

236 Increased dietary protein supports key metabolic and intestinal tissue functions in weaned pigs, without increasing gastrointestinal pathogen abundance

Abstract A common dietary strategy to prevent diarrhea in weaned pigs is feeding low protein diets in the period immediately following weaning. The goal of this intervention is to decrease dietary protein sufficiently to reduce pathogen proliferation and protein fermentation without negatively impacting growth performance. One potential alternative to low protein diets is provision of dietary fiber. However, many studies examining the role of protein and fiber on gastrointestinal microbiota and post-weaning diarrhea are complicated by the presence of other substrates, including polyphenols and antinutritional factors in complex ingredients. To assess the role of increased protein and fiber on gut microbiota and host metabolism, semi-purified diets differing in crude protein (CP) and crude fiber (CF) were fed to weaned pigs (n = 40; 28 d of age). Diets with high protein (HP; CP 24.3%, CF 6.4%), high fiber (HF; CP 18.9%, CF 9.4%), or both (HFHP; CP 24.9%, CF 9.5%) were compared with a control (CON; CP 18.1%, CF 6.3%) diet with industry standard crude protein and fiber levels (n = 10/group). Growth performance and diarrhea prevalence were measured alongside gut microbiota composition and metabolites. Pigs fed the HP and HFHP diets had significantly improved feed efficiency compared with both CON and HF diets. Increased protein fermentation, measured through production of biogenic amines, was observed in HP fed animals (P < 0.001). These effects were mitigated by addition of fiber in the HFHP fed pigs. Despite increased markers of protein fermentation, post-weaning diarrhea incidence was not greater in HP fed animals and no increases in gastrointestinal pathogen abundance were detected. Metabolomic and transcriptomic analyses identified critical alterations in host metabolism and cecal transcriptome in the CON compared with the HP fed animals. While all diets were formulated to meet the dietary requirements of weaned pigs, diets with lower protein levels (CON and HF) induced alteration in transcripts from the serine synthesis pathways (P < 0.001) and integrated stress response (P < 0.001) in cecal tissue alongside increases in metabolic pathways related to lysine degradation (P = 0.005). These results challenge the current practice of low protein feeding following weaning by demonstrating a detrimental effect of low protein diets on intestinal cell function and muscle accretion. This suggests that with careful ingredient selection, increased dietary protein post-weaning could better support pig health and performance compared with current industry standard diets.

Plasticity of microbial substrate carbon use efficiency in response to changes in plant carbon input and soil organic matter status

The ability of the soil's biological community to immobilise carbon (C) from substrates, often referred to as carbon use efficiency (CUE), has been shown to be dependent on the prevailing soil conditions and management regime, potentially leading to changes in C storage and functioning. However, there remains a lack of understanding about how soil CUE is affected by different common labile substrates (and combinations thereof) and native soil organic matter (SOM) status. Here we studied the CUE of three ubiquitous soil C substrates central to microbial metabolism, namely glucose, glutamic acid and citric acid, in soils with and without long-term C deprivation and associated differences in microbial biomass and community structure. We hypothesised that C deprivation-induced stress would reduce substrate CUE due to the investment of more C into stress-alleviation metabolic pathways, while conversely a balanced mixture of C substrates (i.e., sugars, amino acids and organic acids) would promote more efficient growth and substrate CUE. Our results showed that CUE was substrate-specific following the series glucose > glutamic acid > citric acid. CUE values for glucose demonstrated plasticity, being significantly lower in the soils experiencing C deprivation for both 6 and 16 years. Further, the CUE of glucose was increased when supplied alongside citrate and glutamate suggesting that substrate mixture may promote more efficient microbial growth. In contrast, the CUE for glutamic acid and citric acid showed no plasticity, being unaffected by both SOM status and the presence of other substrates. In conclusion, we found evidence to support both our hypotheses indicating that the type of C entering soil alongside native SOM levels may have a strong influence on overall CUE and thus C storage potential.

Modulation of the Activity of the Insulin-Degrading Enzyme by Aβ Peptides.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved protease implicated in the degradation of insulin and amyloidogenic peptides. Most of the biochemical and biophysical characterization of IDE's catalytic activity has been conducted using solutions containing a single substrate, i.e., insulin or Aβ(1-40). IDE's activity toward a particular substrate, however, is likely to be influenced by the presence of other substrates. Here, we show by a kinetic assay based on insulin's helical circular dichroic signal and MALDI TOF mass spectrometry that Aβ peptides modulate IDE's activity toward insulin in opposing ways. Aβ(1-40) enhances IDE-dependent degradation of insulin, whereas Aβ(pyroE3-42), the most pathogenic pyroglutamate-modified Aβ peptide in AD, inhibits IDE's activity. Intriguingly, Aβ(pyroE3-42) also inhibits IDE's ability to degrade Aβ(1-40). Together, our results implicate Aβ peptides in the abnormal catabolism of IDE's key substrates.

Laccase-Catalyzed Oxidation of Mixed Aqueous Phenolic Substrates at Low Concentrations

It has been proposed that Trametes versicolor laccase can be used to detoxify wastewaters that are contaminated with phenolic pollutants. However, the oxidation of phenols at low concentrations may be impacted if other substrates tend to interfere with or enhance the oxidation of the target substrate. To test this, experiments were conducted to evaluate effects arising from the simultaneous presence of mixed substrates including phenol (P), estradiol (E2), cumylphenol (CP), and triclosan (TCL), each of which are characterized by different rates of oxidation and tendencies to inactivate laccase. Slower and faster substrates were found to have only minor negative impacts upon the rate of conversion of targeted substrates, except where they tended to cause inactivation. No enhancements in substrate oxidation were observed. A multi-substrate kinetic model was shown to be able to accurately predict the time course of reactions of mixed substrates over extended periods at micromolar and sub-micromolar concentrations, except when estradiol and triclosan were simultaneously present. In this case, more enzyme inactivation was observed than would be expected from the oxidation of individual substrates alone. The utility of the model for providing insights into the reaction phenomenon and for evaluating the feasibility of oxidizing targeted substrates in the presence of other substrates is demonstrated.

Ca2+-Dependent Mitochondrial Permeability Transition Pore Opening in Kidney is Substrate Dependent

Oxidative stress within the kidney is associated with disease states including hypertension, renal diseases. The metabolic functions of the mammalian kidneys result in consumption of nearly 20% of total oxygen utilization. There is limited information regarding the effects of oxygen free radicals upon kidney mitochondrial functions. Not only mitochondria oxidize numerous metabolic substrates but also play a key role in maintaining cellular Ca2+ homeostasis. Failure of cellular Ca2+ homeostasis and excessive mitochondrial Ca2+ (mCa2+) lead to opening of a mitochondrial permeability transition pore (mPTP), which is associated with loss of IMM potential and uncoupling of oxidative phosphorylation. We investigated whether mitochondrial metabolic substrates have a differential impact on the Ca2+-induced mPTP opening in the kidney, which plays a critical role in hypertension. For this purpose, mitochondria isolated from Sprague Dawley (SD) rat kidney outer medulla (OM) were energized using mechanistically distinct substrate combinations, Pyruvate+Malate, Glutamate+Malate(GM), Succinate+Rotenone, Succinate, α-KetoGlutarate+Malate, or PalmitoylCarnitine+Malate. Fluorescence-based analysis using Ca2+ indicator Fura-4F assessed the functional consequences of Ca2+-induced mPTP opening. Results show that in the presence of GM, mitochondria can tolerate significantly greater mCa2+ loads as compared to that in the presence of other substrates. Succinate+Rotenone addition significantly delayed mPTP opening over controls (succinate alone). A pore inhibitor CyclosporinA(CsA) and Na+/Ca2+exchanger inhibitor(CGP) were used with all substrates for comparisons. The present study reports variations in mitochondrial substrates sensitivity to Ca2+-induced mPTP opening and that the metabolic fluxes determine the mitochondrial functional responses to mCa2+ overload. The results underscore the effects of various mitochondrial substrates and their electron flux in regulating mPTP opening, which is a determinant of cell death in many diseases. These data are important in order to understand the different pathways/proteins involved in mPTP opening to identify effective interventions to prevent/limit it.

Stoichiometry and kinetics of single and mixed substrate uptake in Aspergillus niger

In its natural environment, the filamentous fungus Aspergillus niger grows on decaying fruits and plant material, thereby enzymatically degrading the lignocellulosic constituents (lignin, cellulose, hemicellulose, and pectin) into a mixture of mono- and oligosaccharides. To investigate the kinetics and stoichiometry of growth of this fungus on lignocellulosic sugars, we carried out batch cultivations on six representative monosaccharides (glucose, xylose, mannose, rhamnose, arabinose, and galacturonic acid) and a mixture of these. Growth on these substrates was characterized in terms of biomass yields, oxygen/biomass ratios, and specific conversion rates. Interestingly, in combination, some of the carbon sources were consumed simultaneously and some sequentially. With a previously developed protocol, a sequential chemostat cultivation experiment was performed on a feed mixture of the six substrates. We found that the uptake of glucose, xylose, and mannose could be described with a Michaelis–Menten-type kinetics; however, these carbon sources seem to be competing for the same transport systems, while the uptake of arabinose, galacturonic acid, and rhamnose appeared to be repressed by the presence of other substrates.

Glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

Chronic alcohol intoxication is associated with increased oxidative stress. However, the mechanisms by which ethanol triggers an increase in the production of reactive oxygen species (ROS) and the role of mitochondria in the development of oxidative stress has been insufficiently studied. The biochemical and proteomic data obtained in the present work suggest that one of the main causes of an increase in ROS generation is enhanced oxidation of glutamate in response to long-term alcohol exposure. In the course of glutamate oxidation, liver mitochondria from alcoholic rats generated more superoxide anion and H2O2 than in the presence of other substrates and more than control organelles. In mitochondria from alcoholic rats, rates of H2O2 production and NAD reduction in the presence of glutamate were almost twice higher than in the control. The proteomic study revealed a higher content of glutamate dehydrogenase in liver mitochondria of rats subjected to chronic alcohol exposure. Simultaneously, the content of mitochondrial catalase decreased compared to control. Each of these factors stimulates the production of ROS in addition to ROS generated by the respiratory chain complex I. The results are consistent with the conclusion that glutamate contributes to alcohol hepatotoxicity by enhancing oxidative stress in mitochondria.

Substrate Competition Studies Demonstrate Oxidative Metabolism of Glucose, Glutamate, Glutamine, Lactate and 3-Hydroxybutyrate in Cortical Astrocytes from Rat Brain

It is well established that astrocytes can utilize many substrates to support oxidative energy metabolism; however, use of energy substrates in the presence of other substrates, as would occur in vivo, has not been systematically evaluated. Substrate competition studies were used to determine changes in the rates of (14)CO(2) production since little is known about the interaction of energy substrates in astrocytes. The rates of (14)CO(2) production from 1 mM D-[6-(14)C]glucose, L-[U-(14)C]glutamate, L-[U-(14)C]glutamine, D-3-hydroxy[3-(14)C]butyrate, L-[U-(14)C]lactate and L-[U-(14)C]malate by primary cultures of astrocytes from rat brain were determined to be 1.17 ± 0.19, 85.30 ± 12.25, 28.04 ± 2.84, 13.55 ± 4.56, 14.84 ± 2.40 and 5.20 ± 1.20 nmol/h/mg protein (mean ± SEM), respectively. The rate of (14)CO(2) production from glutamate oxidation was higher than that of the other substrates Addition of unlabeled glutamate significantly decreased the rates of (14)CO(2) production from all other substrates studied; however, glutamate oxidation was not altered by the addition of any of the other substrates. The rate of (14)CO(2) production of glutamine was decreased by glutamate, but not altered by other substrates. The rate of (14)CO(2) production from glucose was significantly decreased by the addition of unlabeled glutamate, glutamine or lactate, but not by 3-hydroxybutyrate or malate. Addition of unlabeled glucose did not significantly alter the (14)CO(2) production from any other substrate. (14)CO(2) production from lactate was decreased by the addition of unlabeled glutamine or glutamate and increased by addition of malate. The (14)CO(2) production from malate was decreased by the addition of unlabeled glutamate or lactate, but was not altered by the other substrates. The substrate utilization for oxidative energy metabolism in astrocytes is very different than the profile previously reported for synaptic terminals. These studies demonstrate the potential use of multiple substrates including glucose, glutamate, glutamine, lactate and 3-hydroxybutyrate as energy substrates for astrocytes. The data also provide evidence of interactions of substrates and multiple compartments of TCA cycle activity in cultured astrocytes.

Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Aerobic degradation of 3-chlorobenzoic acid by an indigenous strain isolated from a polluted river.

An indigenous strain of Pseudomonas putida capable of degrading 3-chlorobenzoic acid as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. Aerobic biodegradation assays were performed using a 2-l microfermentor. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Lactuca sativa as test organisms. The indigenous bacterial strain degrades 100 mg l(-1) 3-chlorobenzoic acid in 14 h with a removal efficiency of 92.0 and 86.1% expressed as compound and chemical oxygen demand removal, respectively. The strain was capable of degrading up to 1,000 mg of the compound l(-1). Toxicity was not detected at the end of the biodegradation process. Besides initial concentration, the effect of different factors, such as initial pH, initial inoculum, adaptation to the compound and presence of other substrates and toxic related compounds, was studied.

Detoxification of 2,4,6-trichlorophenol by an indigenous bacterial community

An indigenous bacterial community capable of degrading 2,4,6-trichlorophenol (TCP) as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. The community consists of three gram-negative bacterial, non-fermentative strains, two of them was identified as belonging to genus Pseudomonas and the other to genus Stenotrophomonas. None of the individual strains were capable of degrading TCP as the sole carbon source. Aerobic biodegradation assays were performed using a 2-l microfermentor at 28 °C with agitation. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna as test organisms. The indigenous bacterial community degrades 100 mg l −1 of TCP in 27 h. The absence of metabolites and toxicity was proved at the end of the process. The influence of the initial concentration of compound, pH, cell inoculum, presence of other substrates and toxic related compounds in the biodegradation process was assayed. Also the application for polluted water was studied. The promising behavior of the bacterial community under the different conditions assayed allows us to suggest its possible use in remediation processes.

Butyrate metabolism in human colon carcinoma cells: Implications concerning its growth‐inhibitory effect

Butyrate and acetate are bacterial metabolites present in the large intestine lumen. Although butyrate is well known to inhibit the in vitro proliferation of human colon carcinoma cells in a process involving the hyperacetylation of specific nuclear histones, little is known about the possible link between butyrate metabolism and its growth-inhibitory effect. In a previous study (Leschelle et al., 2000, Eur J Biochem 267: 6435-6442), we showed that butyrate accumulates and is metabolized in HT-29 Glc(-/+) cells without increasing oxygen consumption. In the present study, using the same cell line incubated with (14)C-labeled butyrate, we determined that a minor part of (14)C from butyrate was recovered in nuclear histones. Unlike butyrate, acetate exerted no effect on cell growth but was a precursor for overall net histone acetylation. Although butyrate was able to increase the cellular AMP/ADP ratio, it did not affect the ATP cell content or the adenylate charge or the oxidation of endogenous L-glutamine. Butyrate oxidation was found to be markedly sensitive to the presence of other substrates with D-glucose decreasing this oxidation and L-malate stimulating it. Furthermore, in the presence of L-malate, the growth-inhibitory effect of butyrate was significantly weaker than in its absence. From these data, we conclude that the metabolism of butyrate downstream acetyl-CoA synthesis is not involved in the butyrate antiproliferative effect. The suggestion that butyrate metabolism in mitochondria is not used in these cells as a fuel but acts as a regulator of butyrate free concentrations (thus limiting its action upon cellular targets), is discussed.

Effects of environmental settings on MTBE removal for a mixed culture and its monoculture isolation

A mixed culture was utilized to evaluate methyl tert-butyl ether (MTBE) removal under various conditions and to isolate a MTBE-degrading pure culture. The results showed that high MTBE removal efficiencies can be reached even in the presence of other substrates. The biodegradation sequence of the target compounds by the mixed culture, in order of removal rate, was toluene, ethyl benzene, p-xylene, benzene, MTBE, ethyl ether, tert-amyl methyl ether, and ethyl tert-butyl ether. In addition, preincubation of the mixed cultures with benzene and toluene showed no negative effect on MTBE removal; on the contrary, it could even increase the degradation rate of MTBE. The kinetic behavior showed that the maximum specific growth rate and the saturation constant of the mixed culture degrading MTBE are 0.000778 h(-1) and 0.029 mg l(-1), respectively. However, a high MTBE concentration (60 mg l(-1)) was slightly inhibiting to the growth of the mixed culture. The pure culture isolated from the enrichments in the bubble-air bioreactor showed better efficiency in MTBE removal than the mixed culture; whereas, tert-butyl alcohol was formed as a metabolic intermediate during the breakdown of MTBE.

Storage of substrate mixtures by activated sludges under dynamic conditions in anoxic or aerobic environments

In spite of the fact that in most activated sludge plants substrate complex mixtures are removed under alternating anoxic and aerobic conditions, most studies on the dynamic response of biomass are limited to feeding a single substrate (acetate or glucose) under a single redox condition (aerobic or anoxic). In this study, the dynamic response of biomass in a sequencing batch reactor is described in terms of substrate removal and related storage as internal polymers, as functions of single or simultaneous feed of several substrates (acetate, glucose, glutamic acid and ethanol) and of anoxic vs. aerobic conditions. Under anoxic conditions, the four substrates were simultaneously removed at a significantly greater nitrate removal rate than when single substrates were present, so showing that the simultaneous removal was partially due to independent metabolic activities. On the other hand, the removal of every substrate was affected (positively or negatively) by the presence of the others, demonstrating that the substrates can be also used by the same metabolism. As an exception, acetate removal was not affected by the presence of other substrates. As for the comparison of aerobic and anoxic conditions, the acetate uptake rate almost doubled moving from anoxic to aerobic conditions, whereas other substrates were only slightly affected. This difference was probably due to the additional presence of aerobic denitrification, which was much more important for acetate. This also confirmed that acetate removal was independent from other substrates. In all cases, storage was the main mechanism of solids formation, so confirming the general importance of such phenomenon under dynamic conditions, independently from feed complexity and redox conditions.

Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy.

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn2+ complex was -8.9 kcal.mol-1, which is 3.2 kcal.mol-1 more favorable than in the complex with Mg2+. For the substrate nucleotide-metal complexes, similar binding affinities (-6.0 to -6.2 kcal.mol-1) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.

The inhibition of gluconeogenesis by chloroquine contributes to its hypoglycaemic action

The effect of chloroquine on gluconeogenesis in isolated hepatocytes and kidney-cortex tubules of rabbit has been studied. The inhibitory action of 200 μM chloroquine was the highest in hepatocytes and renal tubules incubated with glutamine and glutamate+glycerol+octanoate, respectively, while in the presence of other substrates the drug action was less pronounced. With amino acids as substrates, the inhibition of gluconeogenesis was accompanied by a decreased glutamine production, resulting from a decline of glutamate dehydrogenase activity. A decrease in the urea production by hepatocytes incubated with chloroquine in the presence of glutamine but not NH 4Cl as the source of ammonium is in agreement with this suggestion. The degree of inhibition by chloroquine of the rate of gluconeogenesis in renal tubules isolated from control rabbits was similar to that determined in diabetic animals. Chloroquine-induced changes in levels of intracellular gluconeogenic intermediates indicate a decrease in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase activities probably due to increased concentration of 2-oxoglutarate, an inhibitor of these two enzymes. In view of the data, it is likely that inhibition by chloroquine of glucose formation in liver and kidney may contribute to the hypoglycaemic action of this drug. The importance of the inhibitory effect of chloroquine on glutamate dehydrogenase activity in the antihyperglycaemic action of the drug is discussed.

Factors affecting biodegradation of 2‐chlorophenol by Alcaligenes sp. in aerobic reactors

The influence of variations in carbon source concentration, cell inocula, pH, presence of other substrates, and other organisms on the biodegradation of 2-chlorophenol (2-CP) was studied for Alcaligenes sp. isolated from natural sources. Assays of biodegradation were performed in batch and continuous-flow fluidized-bed aerobic reactors. Evaluation of biodegradation was performed by determining total phenols, chemical oxygen demand (COD), and 2-CP by ultraviolet (UV) spectrophotometry. Measurement of microbial growth was carried out by the plate count method. Bioassays of acute toxicity were performed to evaluate detoxification by using Daphnia magna. Results obtained show that under batch conditions with initial inocula of 10(6) cells/mL the strain grew exponentially with 100, 200, and 300 mg/L of 2-CP within 48 hr. A lag period was observed with low cell density inocula (10(5) cells/mL). The strain showed marked delay in the biodegradation of 2-CP at pH 5. Removal of target substrate from mixtures containing other carbon sources demonstrated the possibility of concurrent growth. Mineralization of 2-CP was assessed by gas chromatography carried out at the end of the batch assays and at the exit of the continuous-flow reactor. The presence of other organisms (bacteria, rotifers, ciliate, and algae) that developed in the fluidized-bed reactor did not affect the efficacy of the biodegradation of 2-CP. The removal of 2-CP in the two assayed systems was over 97% in all cases. Toxicity was not detected at the exit of the continuous reactor.