bluuebunny/crossref_metadata_2025_split huggingface dataset:
31P Nuclear Magnetic Resonance Studies of Energy Transduction in Rhodopseudomonas sphaeroides
Authors: NICOLAY Klaas , KAPTEIN Robert , HELLINGWERF Klaas J. , KONINGS Wil N. 
Published: March 2005

31P nuclear magnetic resonance spectra of the phototrophic bacterium Rhodopseudomonas sphaeroides reveal the presence of inorganic phosphate, sugar phosphates and two non‐identified P,P1‐diesterified pyrophosphate compounds. Due to the presence of paramagnetic cations the resonances of these compounds can only be detected after repeated washing of the bacterial cells with a buffer, containing EDTA plus excess Mg2+.Washing with Mg2+‐free EDTA buffer deteriorates the structural integrity of the membranes of Rps. sphaeroides. This is indicated by the appearance of an extra resonance peak in the spectra of these cells in a region where the phosphoiipids absorb and by a fivefold increase in proton permeability of the cytoplasmic membrane of Rps. sphaeroides under these conditions. Upon illumination of the cell suspension in the NMR tube the generation of a transmembrane pH gradient can be inferred from the shift in the resonances of extracellular and intracellular inorganic phosphate. Intracellular inorganic phosphate shows one homogeneous resonance peak upon illumination. This demonstrates that the mixing system, which has been developed for this application, functions efficiently. The magnitude of the light‐dependent pH difference is 0.8 at an external pH 6.The width at half height of the internal inorganic phosphate peak is essentially independent of internal pH from pH 5–8, remains unchanged upon addition of uncoupler and is inversely proportional to the number of EDTA washings applied. These observations indicate that the inorganic phosphate NMR peak width is predominantly determined by the presence of a residual amount of paramagnetic cations, rather than by a broad distribution of internal pH values over the cells.Ionophores have an effect on the light‐dependent pH‐gradient in accordance with the chemiosmotic theory: valinomycin increases, and carbonylcyanide p‐trifluoromethoxyphenylhydrazone decreases, the magnitude of this gradient.
Conformational Changes and Possible Structure of the Oxoglutarate Translocator of Rat‐Heart Mitochondria Revealed by the Kinetic Study of Malate and Oxoglutarate Uptake
Authors: SLUSE‐GOFFART Claudine M. , SLUSE Francis E. , DUYCKAERTS Claire , RICHARD Marc , HENGESCH Paul , LIÉBECQ Claude , Bertrand Laurette , Dethier Eli , Cuvelier‐Klimek Monique , Salmon José 
Published: March 2005

Initial rates of the exchanges [14C]malateout:malatein, ([14C]oxoglutarate + malate)out: malatein and ([14C]malate + oxoglutarate)out: malatein catalysed by the oxoglutarate carrier of rat‐heart mitochondria have been studied under conditions where internal and external substrates may be varied.It is shown that contrary to external oxoglutarate which induces a conformational change of the translocator subunit to which it binds, external malate does not induce conformational changes during its binding and is a Michaelian substrate.The study of the effect of external malate on the rate of oxoglutarate uptake shows that external malate and external oxoglutarate are competitive.External oxoglutarate affects the catalytic rate constant of malate uptake in a modulated way.After substrate binding, the exchange reaction between an external dicarboxylate and an internal dicarboxylate is accompanied by conformational changes. The particular form of the rate equation strongly suggests that during a first step the external substrate bound to an external binding subunit at the external surface of the membrane, and the internal substrate bound to an internal binding subunit at the internal surface of the membrane, are transferred to a catalytic subunit (channel?) deeper in the membrane. Two models, one with a single channel, and the other with several associated channels, are proposed.It is demonstrated that a binding subunit which has transferred its substrate to a catalytic subunit is left in a conformation which does not depend on the substrate that has ‘passed through it’. It is also demonstrated that all the catalytic subunits are identical. These theoretical deductions allow a simple description of the complicated effect that external oxoglutarate has on the rate of malate uptake.The fact that all the external binding subunits are equivalent regarding external malate binding and that all the catalytic subunits are identical support the view that the mitochondrial preparation contains a single species of oxoglutarate translocator and not an isozymic mixture.
Calcium and Cadmium Binding to Troponin C
Authors: TELEMAN Olle , DRAKENBERG Torbjörn , FORSÉN Sture , THULIN Eva 
Published: March 2005

Proton NMR is used to compare the structural changes induced in bovine cardiac troponin C on binding of cadmium and calcium ions. The same spectral changes are observed for both ion species. The rate of the conformational changes associated with cadmium binding to the two high‐affinity sites is slow, that associated with cadmium ions binding to the low‐affinity site is high. 113Cd‐NMR spectra of cardiac troponin C feature two signals interpreted as due to cadmium ions bound to the strong sites.Strong arguments are given in favour of cooperativity in binding of the first two cadmium or calcium ions to cardiac and skeletal muscle troponin C.
Cis‐Platinum Induced Distortions in DNA
Authors: den HARTOG Jeroen H. J. , ALTONA Cornelis , van BOOM Jacques H. , MARCELIS Antonius T. M. , van der MAREL Gijs A. , RINKEL Lambertus J. , WILLE‐HAZELEGER Gerry , REEDIJK Jan 
Published: March 2005

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G‐G chelated deoxytrinucleosidediphosphate platinum complex cis‐Pt(NH3)2{d(GpCpG)}, on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis‐Pt(NH3)2{d(Gp)}2, cis‐Pt(NH3)2{d(pG)}2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis‐Pt(NH3)2]2+.Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring. This feature is also apparent in ribose mononucleotides and is possibly caused by an increased anomeric effect. In cis‐Pt(NH3)2{d(pG)}2 the phase angle of pseudorotation of the S‐type sugar ring is 20° higher than in ‘free’ d(pG) which might be an indication for an ionic interaction between the positive platinum and the negatively charged phosphate.It appears that d(GpCpG) reverts from a predominantly random coil to a normal right‐handed B‐DNA‐like single‐helical structure at lower temperatures, whereas the conformational features of cis‐Pt(NH3)2{d(GpCpG)} are largely temperature‐independent. In the latter compound much conformational freedom along the backbone angles is seen. The cytosine protons and deoxyribose protons exhibit almost no shielding effect as should normally be exerted by the guanine bases in stacking positions. This is interpreted in terms of a ‘turning away’ of the cytosine residue from both chelating guanines. Conformational features of cis‐Pt(NH3)2{d(GpCpG)} are compared with the ‘bulge‐out’ of the ribose‐trinucleotide m62ApUpm62A.
Characterisation of Hepatocyte Sub‐populations Generated by Centrifugal Elutriation
Authors: SUMNER Ian Gordon , FREEDMAN Robert Ben , LODOLA Alberto 
Published: March 2005

We describe protocols for the fractionation of isolated hepatocytes into eight sub‐populations using centrifugal elutriation. The distribution of fluorescein isothiocyanate and acridine orange in hepatocytes prepared from livers pre‐perfused with one of these dyes is described and used as an indicator of acinar zone derivation for each population. The cytochrome P‐450 content and response to induction by 3‐methylcholanthrene and phenobarbitone; the distribution of lactate dehydrogenase, glucose‐6‐phosphatase, pyruvate kinase and tyrosine aminotransferase activities in the sub‐populations is also reported. A marked asymmetry of distribution in all these activities was observed. On the basis of putative zone derivations (based on data of fluorescent dye distribution) of eight factors studied, the distributions of six were consistent with the sub‐populations being derived from different acinar zones. Two major discrepancies were noted however, the distribution of pyruvate kinase activity and the response of the sub‐populations to phenobarbitone.We conclude from this study that while a metabolic heterogeneity was revealed in the sub‐populations generated, further characterisation is required to determine whether acinar zone separation has occurred and if so to what extent.
Increased methyl esterification of membrane proteins in aged red‐blood cells
Authors: GALLETTI Patrizia , INGROSSO Diego , NAPPI Ausilia , GRAGNANIELLO Vincenzo , IOLASCON Achille , PINTO Luciano 
Published: March 2005

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age‐related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37°C) in the presence of l‐[methyl‐3H]methionine, the precursor in vivo of the methyl donor S‐adenosylmethionine, a fourfold increase in membrane‐protein carboxyl methylation was observed in the oldest cells compared with the youngest ones.The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age‐related increase in methyl esterification is accompanied by a significant reduction of the half‐life of methyl esters.The activity of cytoplasmic protein methylase II does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age‐related increased ability to act as methyl‐accepting substrates, when incubated in presence of purified protein methylase II and methyl‐labelled S‐adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated.Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.
Short‐term regulation of glycolysis by insulin and dexamethasone in cultured rat hepatocytes
Authors: PROBST Irmelin , JUNGERMANN Kurt , Unthan Kirsten 
Published: March 2005

Evidence for a direct metabolic effect of insulin in isolated liver preparations is scarce. The stimulation of glycolysis by insulin previously demonstrated in monolayer cultures of adult rat hepatocytes [(1982) Eur. J. Biochem. 126, 271–278] was further investigated.

The degree of stimulation varied with the age of the culture and amounted to 250%, 200%, 500% and 200% of the control value using cells at the culture age of 2 h, 24 h, 48 h, and 72 h, respectively.
Half‐maximal dose of insulin was 0.1 nM. Maximal stimulation was reached within 5 min and lasted for at least 4 h.
Dexamethasone acted both as a long‐term and short‐term modulator. Long‐term pretreatment of the cells with dexamethasone proved necessary to permit insulin action. In addition to this permissive action, pretreatment with dexamethasone reduced the insulin‐independent basal glycolytic rate.
In short‐term experiments dexamethasone decreased the basal glycolytic flux, however, it did not affect the absolute increase in glycolysis brought about by insulin. The half‐maximal dose of dexamethasone was 10 nM.
The stimulatory effects of insulin may in part be attributed to the activation of pyruvate kinase. Insulin produced a left‐shift of the substrate saturation curve, decreasing the K0.5 value for phosphoenolpyruvate.
Peroxidase catalysed aerobic degradation of 5,6,7,8‐tetrahydrobiopterin at physiological pH
Authors: ARMAREGO Wilfred L. F. , RANDLES David , TAGUCHI Hiroyasu 
Published: March 2005

The aerobic degradation of 5,6,7,8‐tetrahydrobiopterin at neutral pH is catalysed by peroxidase (EC 1.11.1.7) and provides quinonoid 7,8‐dihydro(6H)biopterin which readily loses the side chain to yield 7,8‐dihydro(3H)pterin. The latter is in equilibrium with trace amounts of 6‐hydroxy‐5,6,7,8‐tetrahydropterin (covalent hydrate) Which is irreversibly oxidised to quinonoid 6‐hydroxy‐7, 8‐dihydro(6H)pterin, and this finally rearranges to 7,8‐dihydroxanthopterin.Spectroscopic evidence (ultraviolet, 1H NMR and 13C NMR) is presented for the reversible addition of water across the 5,6‐double bond of 7,8‐dihydro(3H)pterin.The intermediate quinonoid 6‐hydroxy‐7,8‐dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a Km of 16.3 μM and kcat of 22.5s−1.The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6R)‐5,6,7,8‐tetrahydrobiopterin is several times slower than the rate for the unnatural (6S) isomer.By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (6R)‐7,8‐dihydro(6H)biopterin Km= 1.3μM and kcat= 22.8s−1; with (6S)‐7,8‐dihydro(6H)biopterin Km= 13.5μM and kcat= 51.6s−1; and with (6RS)‐7,8‐dihydro(6H)neopterin km= 19.2μM and kcat= 116s−1.
Short‐term modulation of glycogen metabolism, glycolysis and gluconeogenesis by physiological oxygen concentrations in hepatocyte cultures
Authors: WÖLFLE Detlef , SCHMIDT Helga , JUNGERMANN Kurt 
Published: March 2005

The influence of different oxygen concentrations (0% to 20%, v/v) on the main pathways of carbohydrate metabolism was studied in rat hepatocyte cultures. Cells resembling the periportal or the perivenous cell type were obtained after 48 h culture under different hormonal conditions; they are referred to as ‘periportal’ or ‘perivenous’, respectively. Using radiochemical techniques the metabolic rates of the two cell types were measured between 48 h and 50 h under different oxygen tensions. Standard physiological substrates of 5mM glucose, 2mM lactate and endogenous glycogen were used.In cells incubated under 4% O2, mimicking hepatovenous oxygen levels, compared to cells assayed under 13% O2, mimicking arterial levels, glycogen degradation to free glucose and to lactate was faster while glycogen synthesis was slower; glycolysis, from glucose to lactate, was faster and gluconeogenesis, from lactate to glucose, was only slightly slower.Under anoxic conditions glycogen breakdown was maximal and glycogen synthesis minimal; gluconeogenesis was also minimal, but glycolysis was not maximal, it reached its peak rate at 4% O2.CO2‐formation increased up to 6% and then stayed essentially constant at higher O2tensionsNet glycogen metabolism: in ‘perivenous’ cells net glycogen synthesis was observed above, net glycogen degradation below 4% O2. In ‘periportal’ cells, which had a very low glycogen content, net glycogen metabolism was very small.Net glucose metabolism: in ‘perivenous’ cells net glucose formation was only seen under anoxic conditions. Net glucose utilization was observed at about the same rate under all physiological O2tensions. In ‘periportal’ cells net glucose formation increased clearly up to 6% O2and then remained almost constant.Net lactate metabolism: in ‘perivenous’ cells net lactate formation occurred below, and net lactate utilization above, 6% O2. In ‘periportal’ cells a pronounced net lactate utilization was observed under all physiological O2tensions.Net flow between glucose‐6‐phosphate and pyruvate was observed in the glycolytic direction in ‘perivenous’ and in the gluconeogenic direction in ‘periportal’ cells except under anoxic conditions. When O2tensions were lowered, the percentage of ‘futile cycling’ was decreased in the ‘perivenous’, glycolytic hepatocytes; conversely, it was increased in the ‘periportal’, gluconeogenic hepatocytes.It is concluded that physiological oxygen concentrations modulate hepatic carbohydrate metabolism and that they contribute further to the proposed metabolic differences between periportal and perivenous cellsin vivo.
Cooperative effects in the peptidyltransferase center of Escherichia coli ribosomes
Authors: BOURD Saul B. , KUKHANOVA Marina K. , GOTTIKH Boris P. , KRAYEVSKY Alexander A. 
Published: March 2005

We have measured the binding isotherms of C‐A‐C‐C‐A(3′NH)‐[14C]Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes. Analysis of the experimental binding isotherms leads to the following conclusions. A ribosome (or subunit) binds two C‐A‐C‐C‐A(3′NH)‐Phe molecules. The binding of C‐A‐C‐C‐A(3′NH)‐Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient τ=40±5 or more.The binding of C‐A‐C‐C‐A(3′NH)‐AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5×106 M−1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10–25, the association binding constant of puromycin at the acceptor site being (1–2)×104 M−1. The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules adsorbed on a ribosome equals 100–200 M−1.
Isolation and comparison of arylsulfatase A from rat liver and Morris hepatoma 7777
Authors: HESS Grzegorz 
Published: March 2005

Rat liver and Morris hepatoma 7777 arylsulfatase A were isolated from the soluble lysosomal extract by a procedure involving blue‐Sepharose affinity chromatography, DEAE‐cellulose chromatography, hydrophobic chromatography on phenyl‐Sepharose and preparative polyacrylamide gel electrophoresis. The preparation obtained by this method was apparently homogenous in disc electrophoresis and in immunoelectrophoresis.The comparative studies revealed that the properties of arylsulfatase A from rat liver and Morris hepatoma 7777 are very similar, considering molecular weight of the native monomer and its subunits, the ability to form tetramers, isoelectric point, Michaelis constant and the anomalous kinetics of the reaction.The twofold elevation of arylsulfatase B activity found in Morris hepatoma 7777 suggests that the enzyme may have certain functions in tumor growth.
A pulse‐radiolysis study of the reduction of flavodoxin from Megasphaera elsdenii by viologen radicals. A conformational change as a possible regulating mechanism
Authors: van LEEUWEN Johan W. , van DIJK Cees , VEEGER Cees 
Published: March 2005

Pulse‐radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is –(480+30)mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change (k2∼ 105 s−1) the intermediate state decays to the stable semiquinone form of flavodoxin. The ΔG of the conformational change at pH 8 is about 29 kJ mol−1 (0.3 eV). This means that the upper limit for the pK of N‐5 in the semiquinone form will be 13. The activation energy of the conformational changes is 43 kJ mol−1 (0.45 eV).The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion‐controlled with a forward rate constant of (7 ± 1) × 108 M−1 s−1 (pH 8, I= 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be –(408 ± 5) mV.A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two‐electron process, the midpoint potential of which should be located between –440 mV < Em (Fld/FldH−) < ‐290 mV. The exact value will depend on the ΔG of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two‐electron donor.
Phenolic and tyrosyl ring deiodination in thyroxine from rat retina during postnatal development
Authors: IENTUILE Riccardo , MACAIONE Salvatore , RUSSO Pietro , PUGLIESE Gaetano , DI GIORGIO Rosa Maria 
Published: March 2005

To elucidate tetraiodothyronine (T4) metabolism in developing rat retina 5‐monodeiodinating and 5′‐monodeiodinating activities were studied. T4 was incubated with aliquots of homogenate or crude primary subcellular fraction, and the 3,3′,5′‐triiodothyronine (rT3) or 3,5,3′‐triiodothyronine (T3) produced were measured by radioimmunoassay. Reaction rates were dependent on incubation time, tissue amount, temperature and pH. The optimum pH values were 7.8 and 7.2 respectively for rT3 forming and T3‐forming systems. Conversion of T4 to either T3 or rT3 was dependent on dithiothreitol concentration, and the T4‐5′‐deiodioatrag activity was inhibited by propylthiouracil. Deiodinase activities were mainly found in the crude microsomes. The retinal 5′‐monodeiodination rate of T4 was immeasurably low by the 2nd day and the highest values were reached on 15th day of postnatal development. On the other hand deiodination of the T4 tyrosyl ring shows a progressive decline from birth, and adult values were reached on the 15th day.Data support the hypothesis that, in developing rat thyroxine, phenolic and tyrosyl‐ring deiodinase activities are present in the retina and their reciprocal changes may regulate morphological and biochemical cell maturation.