bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uptake of glutamate in Corynebacterium glutamicum
Authors: KRÄMER Reinhard , LAMBERT Camille 
Published: March 2005

The inducible glutamate uptake system in Corynebacterium glutamicum (Krämer, R., Lambert, C., Hoischen, C. & Ebbighausen, H., preceding paper in this journal) was characterized with respect to its mechanism and energy coupling. All possible secondary active uptake mechanisms can be excluded. Glutamate transport is not coupled to the translocation of H+, Na+ or K+ ions. Although changes in membrane potential and uptake activity cannot completely be separated, no correlation between these two parameters is observed.The uptake of glutamate resembles a primary active, ATP‐dependent transport mechanism in several respects. (a) The substrate affinity is very high (1.3 μM). (b) Accumulation of glutamate reaches values of greater than 2 · 105, at least as high as those reported for binding‐protein‐dependent systems in Gram‐negative bacteria. (c) The uptake is unidirectional. Even after complete deenergization, the accumulation ratio was not significantly reduced. (d) The rate of glutamate uptake is directly correlated to the cytosolic ATP content and also to the ATP/ADP ratio. This is shown by varying internal ATP by different procedures applying inhibitors (NaCN, dicyclohexyl carbodiimide), uncouplers (carbonyl m‐chlorophenylhydrazone), ionophores (valinomycin), and even by shifting the cells to anaerobiosis. Uptake is not promoted by cytosolic ATP levels below 1.5 mM, the maximum uptake rate is reached at 4–5 mM ATP.
Reaction pathways for biodehalogenation of fluorinated anilines
Authors: RIETJENS Ivonne M. C. M. , TYRAKOWSKA Bozena , VEEGER Cees , VERVOORT Jacques 
Published: March 2005

Pathways for biodehalogenation of fluorinated aniline derivatives were investigated. Microsomal NADPH‐dependent dehalogenation of fluoroanilines was shown to proceed by three different reaction pathways. The first route appeared to result in monooxygenation at a fluorinated position and release of the fluorine atom as a fluoride anion. The primary additional reaction product formed is the reactive quinoneimine, not the 4‐hydroxyaniline. In NADPH‐containing microsomal systems with 4‐fluoro‐substituted anilines, formation of the 4‐hydroxyaniline derivative is observed because NADPH chemically reduces this quinoneimine metabolite. A second pathway for dehalogenation proceeds by protein binding of a fluoro‐containing (semi)quinoneimine metabolite, the formation of which may result from the mono‐oxygenase reaction (pathway 1) and/or from (re)oxidation of a hydroxyaniline metabolite by superoxide anion radicals produced by the microsomal system. This latter reaction pathway becomes more important with increasing number of fluoro‐substituents in the fluoroaniline derivative. The higher ratio of fluoride anion formed to 4‐hydroxyaniline derivative detected in incubations with liver microsomes from dexamethasone‐treated rats, as compared to incubations with liver microsomes from control rats, can in part be explained by the higher production of superoxide anion radicals observed in the dexamethasone systems.The third mechanism was shown to proceed by formation of a hydroxylated metabolite that loses fluoride anion upon exposure to oxygen. The reactive intermediate formed upon oxygen exposure might be the semiquinoneimine which loses its fluorine atom as a fluoride anion upon dimerization or polymerization and/or protein binding. The fluorohydroxyanilines, in which the hydroxyl group is ortho or para with respect to the fluoro substituent, appear especially to be highly unstable and lose fluoride anion in the presence of oxygen.Finally, it is concluded that all three pathways for dehalogenation of fluorinated aniline derivatives are bioactivation pathways. The reactivity of the (semi)quinoneimines formed in these reactions is dependent on their substitution pattern and increases with increasing number of fluoro‐substituents. Therefore, bioactivation for a series of fluorinated aniline derivatives, can be expected to vary with the substitution pattern and to increase with increasing number of halogen substituents.
Phosphorus‐31 nuclear magnetic resonance of C6 glioma cells and rat astrocytes
Authors: PIANET Isabelle , MERLE Michel , LABOUESSE Julie , CANIONI Paul 
Published: March 2005

31P‐NMR spectroscopy has been used to study the energy metabolism and the NMR visibility of ATP and intracellular Pi of the C6 glioma cell line and rat astrocyte grown on microcarrier beads with the following results.

In vivo NMR spectra of C6 glioma cells and rat astrocytes indicate that these cells were able to maintain their level of ATP resonances during a long anoxic period (more than an hour). Both cell types were sensitive to ischemia which induced a loss of ATP resonances within 40 min. Glucose starvation induced a 40% decrease in ATP resonances correlated to a 50% increase in the intensity of the Pi signal. These changes corresponded to a new steady state which could be reversed by reperfusing the cells with a glucose‐containing medium.
In contrast to in vivo data, 31P‐NMR analyses of perchloric acid extracts of cells incubated in a glucose‐free medium showed that their ATP and Pi contents were unchanged during starvation. The changes of NMR visibility of the metabolites in living C6 cells were correlated to modifications of their macroscopic longitudinal relaxation times, evolving from 0.30 ± 0.08 s and 6.6 ± 1.5 s in the presence of glucose to 0.68 ± 0.26 s and 3.2 ± 0.9 s in the absence of glucose for ATP and Pi, respectively. The changes of the NMR detectability of ATP and Pi, indicate that changes in their microenvironment occur during glucose starvation, suggesting the existence of different pools of these metabolites within the cells.
Under various experimental conditions, i.e. anoxia, ischemia and glucose starvation, rat astrocytes in primary culture showed a very similar behavior to that of C6 cells, suggesting a similar adaptability to the nature of the energy supply for both the normal and the malignant cell.
Irreversible transitions in the 6‐phosphofructokinase/fructose 1,6‐bisphosphatase cycle
Authors: SCHELLENBERGER Wolfgang , HERVAGAULT Jean‐François 
Published: March 2005

The dynamics of the fructose 6‐phosphate/fructose‐1,6‐bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied. In addition to 6‐phosphofructokinase and fructose‐1,6‐bisphosphatase, pyruvate kinase, adenylate kinase and glucose‐6‐phosphate isomerase were involved. In that multi‐enzyme system, the main source of non‐linearity is the reciprocal effect of AMP on the activities of 6‐phosphofructokinase and fructose‐1,6‐bisphosphatase. Depending upon the experimental parameter values, stable attractors, various types of multiple states and sustained oscillations were shown to occur.In the present report we show that irreversible transitions are also likely to occur for realistic operating conditions. Two parameters of the system, that is the adenylate energy charge of the influx and the fructose‐1,6‐bisphosphatase maximal activity, are potential candidates to provoke such irreversible transitions from one steady state to the other: (a) when varying the maximal activity of fructose‐1,6‐bisphosphatase, the system can jump irreversibly from a low to a high stable steady state, and (b) when the adenylate energy charge of the influx is the changing parameter, irreversible transitions occur from a high stable steady state to a stable oscillatory state (limit cycle motion). This behavior can be predicted by constructing the loci of limit points and Hopf bifurcation points.
The amphiphilic α‐helix concept
Authors: THIAUDIÈRE Eric , SIFFERT Odile , TALBOT Jean‐Claude , BOLARD Jacques , ALOUF Joseph Elie , DUFOURCQ Jean 
Published: March 2005

Staphylococcal δ‐toxin, a synthetic analogue and a fragment were studied in order to determine their structure in solution and bound in lipids. In solution, a self‐association process is observed. Analytical ultracentrifuge and quasi‐elastic light‐scattering experiments suggest an isodesmic aggregation in the high concentration domain above 2 μM up to very large asymmetrical species. Decreasing concentrations below 2 μM of δ‐toxin and the analogue allows dissociation, probably into monomers. The self‐associated species are essentially α‐helical (70%) with buried and highly immobilized Trp either at position 15 for natural δ‐toxin or 16 for the analogue. At the lowest concentration studied, the α‐helix content severely decreases down to 35% while Trp fluorescence shows that these residues are exposed to buffer. The fragment 11–26 is always monomeric and structureless. From all the data, a structural model of aggregated species is proposed with stacked antiparallel amphipathic rods.When bound to lipids, whatever their initial structure in solution, 26‐residue long peptides mainly adopt an α‐helix conformation (80%) while fragment 11–26 exhibits about 50%α‐helix. The lipid–peptide interactions were quantitatively analysed. For fragment 11–26, a single‐step mechanism fits the spectroscopic changes and defines a single monomeric bound structure. On the other hand, for the 26‐residue‐long analogue, multiple‐step processes must occur. The data were analysed with a partition of tetramers into lipids followed by a partial dissociation. Finally, the affinity of fragment 11–26 severely decreases from micelles to fluid and gel‐state bilayers. The partition coefficient of the δ‐toxin analogue is higher than those of other more apolar peptides, such as melittin and alamethicin, correlating with Eisenberg's hydrophobic moments. It is therefore proposed that δ‐toxin probably lies parallel to the surface, only penetrating weakly in lipids, depending on their packing.
Gene for the ADP‐ribosylatable elongation factor 2 from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: SCHRÖDER Johanna , KLINK Friedrich 
Published: March 2005

The gene coding for ADP‐ribosylatable elongation factor 2 (EF‐2) from the extreme thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been cloned and its sequence is reported. Amino acid sequence comparisons showed that EF‐2 from S. acidocaldarius is more closely related to eukaryotic EF‐2 than to eubacterial EF‐G. Consensus sequences are derived from comparison of a region around the unique amino acid diphthamide, which is the target for ADP‐ribosylation by diphtheria toxin in archaebacteria and eukaryotes. The conserved positions are likely to constitute a recognition site for the toxin and the histidine‐modifying enzymes.A single transcript of approximately the size of the EF‐2 gene was observed in Northern blot experiments. Transcription initiation and termination signals were identified in the immediate vicinity of the respective translation start and stop codons of the gene. These results indicate that, in contrast to all prokaryotic EF‐2 genes studied previously, the gene of S. acidocaldarius is not located within the streptomycin operon but is transcribed separately.
Phosphate binding and ATP‐binding sites coexist in Na+/K+‐transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4
Authors: BUXBAUM Engelbert , SCHONER Wilhelm 
Published: March 2005

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K+‐ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1):

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The inactivation rate constant k2 for the formation of a stable E′2· Co(NH3)4PO4 at 37°C was 0.057 min−1; the dissociation constant, Kd= 300 μM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5′‐[β,γ‐methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks= 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks= 50 μM, as did phosphate (Ks= 16 mM) and Mg2+ (Ks= 160 μM). It is concluded that the MgPO4 analogue binds to the MgPO4‐binding subsite of the low‐affinity ATP‐binding site (of the E2 conformation). Also, Na+ (Ks= 860 μM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 μM, and for the E · Co(NH3)4PO4 complex of 720 μM, were calculated.Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K+‐ATPase and K+‐activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na+‐ATPase, the phosphorylation capacity of the high‐affinity ATP‐binding site and the ATP/ADP‐exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol/unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P‐labelled tetramminecobalt ATP {[γ‐32P]Co(NH3)4ATP} at the low‐affinity ATP‐binding site, allowed (independent of the purity of the Na+/K+‐ATPase preparation) a further incorporation of radioactivity from 32P‐labelled tetraaquachromium(III) ATP ([γ‐32P]CrATP) to the high‐affinity ATP‐binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K+‐ATPase by [γ‐32P]CrATP prevented the binding of Co(NH3)432PO4 or [γ‐32P]Co(NH3)4ATP to the enzyme. [γ‐32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (α,β)2‐diprotomer, which change their interactions during the Na+/K+‐pumpimg process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.
Control of hepatic mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase during the foetal/neonatal transition, suckling and weaning in the rat
Authors: QUANT Patti A. , ROBIN Danielle , ROBIN Pierre , FERRE Pascal , BRAND Martin D. , GIRARD Jean 
Published: March 2005

We assayed active and total (i.e. active plus succinylated) 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase in mitochondria isolated from foetal, neonatal, suckling or weaned rats.
HMG‐CoA synthase was substantially succinylated and inactivated in mitochondria isolated from termfoetal, (1‐h‐old, 6‐h‐old, 1‐day‐old) neonatal, suckling and high carbohydrate/low‐fat (hc)‐weaned rats. Succinylation of HMG‐CoA synthase was very low in mitochondria isolated from the livers of foetal, 30‐min‐old neonatal and high‐fat/carbohydrate‐free (hf)‐weaned rats.
There was a negative correlation between active HMG‐CoA synthase and succinyl‐CoA content in mitochondria isolated from term‐foetal, suckling and hc‐weaned rats.
Differences in active enzyme could not be entirely accounted for by differences in succinylation and inactivation of the synthase. Immunoassay confirmed that the absolute amounts of mitochondrial HMG‐CoA synthase increased during the foetal/neonatal transition and decreased with hc weaning. The levels remained elevated with hf weaning.
From these data we propose that mitochondrial HMG‐CoA synthase is controlled by two different mechanisms in young rats. Regulation by succinylation provides a mechanism for rapid modification of existing enzyme in response to changing metabolic states. Changes in the absolute amounts of HMG‐CoA synthase provide a more long‐term control in response to nutritional changes.
Blocking of the receptor‐mediated invasion of erythrocytes by Plasmodium knowlesi malaria with sulfated polysaccharides and glycosaminoglycans
Authors: DALTON John P. , HUDSON Diana , ADAMS John H. , MILLER Louis H. 
Published: March 2005

Invasion of human erythrocytes by Plasmodium knowlesi requires the Duffy blood group antigen. P. knowlesi merozoites synthesize a 135‐kDa polypeptide which binds to the Duffy antigen with receptor‐like specificity. In this study, we show that the sulfated polysaccharide fucoidan and the glycosaminoglycan dextran sulfate inhibit the binding of the 135‐kDa polypeptide to human Duffy‐positive and rhesus erythrocytes while the chondroitin sulfates do not. Fucoidan and dextran sulphate also blocked the in vitro invasion of human Duffy b and rhesus erythrocytes cells by P. knowlesi merozoites. These inhibitors were more effective at blocking the binding of the 135‐kDa polypeptide to human Duffy b erythrocytes than to rhesus erythrocytes, which correlated with them having a greater inhibitory effect on invasion of merozoites into human than into rhesus erythrocytes.The blocking by these sulfated sugars is not related to charge density on the polysaccharides; fucoidan with a relatively low charge density blocks binding of the 135‐kDa polypeptide at 4 μg/ml, while the highly negatively charged chondroitin sulfates do not block binding even at the concentration of 1 mg/ml. Furthermore, fucoidan‐Sepharose bound and removed the 135‐kDa polypeptide from parasite culture supernatants with a selectivity equal to that of the Duffy blood group antigen. The negatively charged sulfate groups on fucoidan and dextran sulfate and the conformation in which they are held possibly mimic similarly charged groups on the Duffy antigen which bind the 135‐kDa P. knowlesi polypeptide.
Lys631 residue in the active site of the bacteriophage T7 RNA polymerase
Authors: MAKSIMOVA Tatyana G. , MUSTAYEV Arkady A. , ZAYCHIKOV Evgeny F. , LYAKHOV Dmitry L. , TUNITSKAYA Vera L. , AKBAROV Akhror Kh. , LUCHIN Sergei V. , RECHINSKY Vladimir O. , CHERNOV Boris K. , KOCHETKOV Sergei N. 
Published: March 2005

A highly selective affinity labeling of T7 RNA polymerase with the o‐formylphenyl ester of GMP and [α‐32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N‐chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue.Site‐directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys → Gly mutant enzyme, anomalous template binding was observed.
H+‐translocating inorganic pyrophosphatase of plant vacuoles Inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases
Authors: MAESHIMA Masayoshi 
Published: March 2005

The effects of divalent cations, especially Ca2+ and Mg2+, on the proton‐translocating inorganic pyrophosphatase purified from mung bean vacuoles were investigated to compare the enzyme with other pyrophosphatases. The pyrophosphatase was irreversibly inactivated by incubation in the absence of Mg2+. The removal of Mg2+ from the enzyme increased susceptibility to proteolysis by trypsin. Vacuolar pyrophosphatase required free Mg2+ as an essential cofactor (K0.5= 42 μM). Binding of Mg2+ stabilizes and activates the enzyme. The formation of MgPPi is also an important role of magnesium ion. Apparent Km of the enzyme for MgPPi was about 130 μM.CaCl2 decreased the enzyme activity to less than 60% at 40 μM, and the inhibition was reversed by EGTA. Pyrophosphatase activity was measured under different conditions of Mg2+ and Ca2+ concentrations at pH 7.2. The rate of inhibition depended on the concentration of CaPPi, and the approximate Ki for CaPPi was 17 μM. A high concentration of free Ca2+ did not inhibit the enzyme at a low concentration of CaPPi. It appears that for Ca2+, at least, the inhibitory form is the Ca2+‐PPi complex. Cd2+, Co2+ and Cu2+ also inhibited the enzyme.The antibody against the vacuolar pyrophosphatase did not react with rat liver mitochondrial or yeast cytosolic pyrophosphatases. Also, the antibody to the yeast enzyme did not react with the vacuolar enzyme. Thus, the catalytic properties of the vacuolar pyrophosphatase, such as Mg2+ requirement and sensitivity to Ca2+, are common to the other pyrophosphatases, but the vacuolar enzyme differs from them in subunit mass and immuno‐reactivity.
Primary structure of O‐linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains
Authors: GERWIG Gerrit J. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G. , (MORGENSTERN) Ely MORAG , LAMED Raphael , BAYER Edward A. 
Published: March 2005

The cell‐free forms of the multiple cellulase‐containing protein complex (cellulosome), isolated from the celluloytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5–7% (by mass), consisting of O‐linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline‐borohydride treatment and fractionated as oligosaccharide alditols via gel‐permeation chromatography and HPLC. The fractions were investigated by 500‐MHz 1H‐NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast‐atom‐bombardment mass spectrometry (FAB‐MS). In addition to the previously described major oligosaccharide,

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[Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027 – 10351, the following partial structures of this compound could be established:

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Cell‐free and cell‐associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.
Processing of the precursor of protamine P2 in mouse Identification of intermediates by their insolubility in the presence of sodium dodecyl sulfate
Authors: ELSEVIER Susan M. , NOIRAN Joseph , CARRE‐EUSEBE Danièle 
Published: March 2005

Two basic proteins, protamines P1 and P2, are present in chromatin of mouse spermatozoa. Protamine P1, the less abundant protein in mouse, has a homolog in most mammals, and its synthesis follows a conventional route. In contrast, protamine P2 has been found only in certain other mammals, including humans, and it is synthesized as a precursor nearly twice as long as the mature protein. Processing of this precursor is not yet understood, although it necessarily takes place in elongating spermatids and is likely to play a role in the chromatin condensation occurring in these haploid cells.We have fractionated basic proteins from mouse testis chromatin and have identified six proteins on electrophoretic gels which, like protamines, are insoluble in SDS. All six were also soluble at the same trichloroacetic acid concentration as protamine P2 and were present in chromatin of elongating spermatids. Radioactive labelling patterns acquired by these SDS‐insoluble proteins during translation in vitro of testis RNA indicate that the largest represents the precursor of protamine P2, and suggest that the others represent intermediates generated by proteolytic cleavage of the precursor. Results from pulse 3H labelling in vivo were also consistent with the conclusion that a precursor/product relationship exists between these proteins and protamine P2. Conclusions concerning the kinetics of processing have, in addition, been drawn from this data. Hypotheses concerning possible functional roles played by the precursor are presented.