bluuebunny/crossref_metadata_2025_split huggingface dataset:
Accelerated Turnover of Hepatic Glucokinase in Starved and Streptozotocin‐Diabetic Rat
Authors: SIBROWSKI Walter , STAEGEMANN Ulrich , SEITZ Hans J. 
Published: July 2010

Rat liver glucokinase synthesis and degradation was estimated in fasted/fed and diabetic/diabetic‐insulin‐treated rats by the radioimmunological technique. Starvation and Streptozotocin‐diabetes led to basal rates of synthesis and, consequently, to low levels in enzyme activity. In addition, a decrease in the apparent half‐life from about 19 h in the fed or diabetic‐insulin substituted to about 11 h in the starved or diabetic rat, respectively, was observed. Injection of Bt2cAMP into glucose‐fed animals reduced glucokinase synthesis to basal levels within 90 min, without affecting enzyme activity.It is concluded that in metabolic states associated with elevated levels in tissue cAMP glucokinase synthesis is reduced to basal values and, in addition, its rate of degradation is significantly enhanced.
Structure of Ribosomal Protein L6 from Escherichia coli
Authors: STEINHÄUSER Klaus G. , WOOLLEY Paul , EPE Bernd , DIJK Jan 
Published: July 2010

Protein L6 from the 50‐S ribosomal subunit has been investigated using fluorimetric techniques. The intrinsic fluorophore Trp‐61 and fluorescent labels (acetylaminoethyl‐dansyl and acetylaminofluorescein) attached to the residue Cys‐124 were used. It proved possible to incorporate fluorescence‐labelled L6 into the 50‐S ribosome.Trp‐61 is exposed to solvent, as shown by its emission wavelength and by quenching experiments; the latter also show that it lies in a pocket with a high positive charge due to the basic residues in the N‐terminal fragment. Cys‐124 lies in a less strongly positive region. Upon incorporation into the 50‐S subunit, the label on Cys‐124 becomes less accessible for quenching but its positive potential rises, showing the absence of direct contact with 23‐S RNA.Analysis of anisotropy data indicates a considerable degree of asphericity of free L6. Energy transfer between Trp‐61 and the dansyl label on Cys‐124, measured by donor quenching and acceptor enhancement, reveals a separation of 3.5 ± 0.4 nm (35 ± 4Å) between fluorophores.
A Hypothesis for the Role of Dithiol‐Disulfide Interchange in Solute Transport and Energy‐Transducing Processes
Authors: ROBILLARD George T. , KONINGS Wil N. 
Published: July 2010

We have recently shown that the physical mechanism for Δ+ ‐driven changes in the Km for three different transport systems is an oxidation‐reduction reaction involving a dithiol‐disulfide interconversion [Robillard, G. T. and Konings, W. N. (1981) Biochemistry, 20, 5025–5032; Konings, W. N. and Robillard, G. T. (1982) Proc. Natl Acad. Sci. USA, in the press]. Based on the similarities between the data from these three systems and published data from other systems, we now propose that dithiol‐disulfide interchange may play a general role in membrane‐related processes such as transport, energy transduction and hormone‐receptor interactions.We propose that the affinities of the substrate‐binding sites are regulated by a dithiol and a disulfide situated at different depths in the membrane. In addition we propose that the oxidation states of these two redox centers are coupled by dithiol‐disulfide interchange such that, when one is oxidized, the other is reduced. Since a transmembrane electrical potential, Δ;ψ, or a pH gradient, ΔpH, can alter the redox state, it can change the affinity of the substrate‐binding sites. The Δ;H+ ‐induced changes in affinity are sufficient to drive active transport (symport or antiport) and energy‐transducing processes. A similar mechanism can be applied to transport systems driven by phosphorylated enzyme intermediates instead of Δ;H+.Changes of the redox potential in a given compartment during metabolism could also control the affinity of ligand binding even in the absence of a Δ;H+. The ligand‐binding affinities of facilitated diffusion transport systems and receptor proteins may be regulated in this manner.
A Pulse‐Radiolysis Study of Cytochrome c3
Authors: VAN LEEUWEN Johan W. , VAN DIJK Cees , GRANDE Hans J. , VEEGER Cees 
Published: July 2010

Pulse‐radiolysis experiments were performed in the presence of methyl viologen and cytochrome c3. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c3 are studied. The reaction between cytochrome c3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH‐dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c3 molecule was Z =+ 4.7 ± 0.7.
After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome c3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, α. With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0=− 0.250–0.088α (in V).
An estimation is made of the energy of interaction between the haems due to electrostatic interactions (Δɛ < 32 mV) and due to ionic strength effects (−12 mV < Δɛ < 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c3 molecule are dependent on the degree of reduction of the other haems in the molecule.
The reaction of cytochrome c3 with MV+ or with ethanol radicals (EtOH) has been compared with the reactions of horse‐heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV+ or EtOH with horse‐heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.
Short‐Term Regulation of the Nitrogenase Activity in Rhodopseudomonas sphaeroides
Authors: HAAKER Huub , LAANE Colja , HELLINGWERF Klaas , HOUWER Berend , KONINGS Wil N. , VEEGER Cees 
Published: July 2010

The nitrogenase activity in whole cells of Rhodopseudomonas sphaeroides could be inhibited by lowering the electrical potential across the cytoplasmic membrane. The membrane potential was partly dissipated either by lowering the light intensity or by the addition of a lipophilic cation, tetraphenylphosphonium. Under these circumstances, it was shown that the intracellular ATP/ADP ratio was not affected and that the inhibition of the whole cell nitrogenase activity was not due to an inactivation of the nitrogenase enzyme. From these results it is concluded that electron transport to nitrogenase in Rps. sphaeroides is dependent on a high membrane potential.The nitrogenase enzyme in whole cells could be inactivated by lowering the membrane potential across the cytoplasmic membrane by incubating the cells in the dark or in the light in the presence of uncouplers. Nitrogenase could be reactivated in the light in the absence of uncouplers.Some possible mechanisms of action of NH+4 inhibition of whole cell nitrogenase activity could be excluded. Inhibition by NH4Cl of whole cell nitrogenase activity in Rps. sphaeroides could neither be explained by a rapid inactivation of the nitrogenase enzyme, nor by an effect on the intracellular ATP/ADP ratio or the membrane potential. NH+4 inhibits whole cell nitrogenase activity not directly but probably after being assimilated by glutamine synthetase. The role of glutamine, glutamate and 2‐oxoglutarate on the regulation of electron transport to nitrogenase will be discussed.
Synthesis of the Proteins of Complex III of the Mitochondrial Respiratory Chain in Heme‐Deficient Cells
Authors: LIN Ching‐I P. , GOLLUB Edith G. , BEATTIE Diana S. 
Published: July 2010

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5‐aminolevulinic acid synthase and, hence, devoid of heme. The two ‘core’ proteins, apocytochrome b and the iron‐sulfur protein, were observed in equal amounts in the heme‐deficient and heme‐sufficient cells with antiserum against complex III and the sensitive immunotransfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1.By contrast, when mitochondria from the heme‐deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.
Isoleucyl‐tRNA Synthetase from Escherichia coli MRE 600
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich 
Published: July 2010

The substrate specificity of isoleucyl‐tRNA synthetase from Escherichia coli MRE 600 with regard to ATP analogs has been compared with the results obtained with isoleucyl‐tRNA synthetase from yeast. The enzyme from E. coli is less specific, the two enzymes exhibit different topographies of their active centres.The order of substrate addition to isoleucyl‐tRNA synthetase from E. coli MRE 600 has been investigated by bisubstrate kinetics, product inhibition and inhibition by substrate analogs. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. As found for isoleucyl‐tRNA synthetase from yeast, the results of the pyrophosphate exchange studies indicate the possibility of formation of E · Ile‐AMP · ATP complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, release of pyrophosphate and subsequent addition of a second molecule of ATP. For the aminoacylation in the absence of pyrophosphatase, a rapid‐equilibrium random ter addition of the substrates is found whereas the enzyme from yeast exhibits a steady‐state ordered ter‐ter mechanism; in the presence of pyrophosphatase the mechanism is bi‐uni uni‐bi ping‐pong similarly as observed for the yeast enzyme.A comparison of inhibition patterns obtained with N(6)‐benzyladenosine 5′‐triphosphate under different assay conditions (spermine or magnesium ions, addition of pyrophosphatase) indicates that even more than two pathways of the aminoacylation may exist.The catalytic cycles of the two mechanisms derived from the observed orders of substrate addition and product release include the same enzyme substrate complex (E · tRNA · Ile‐AMP) for the aminoacyl transfer reaction. The kcat values, however, are considerably different: kcat of the sequential pathway is about 40% lower than kcat of the ping‐pong mechanism.
Porcine Pancreatic Lipase
Authors: BENKOUKA Fatima , GUIDONI Andrea A. , DE CARO Josiane D. , BONICEL Jacques J. , DESNUELLE Pierre A. , ROVERY Mireille 
Published: July 2010

Following complete sequence analysis of the 449 amino acids in porcine pancreatic lipase [J. De Caro et al. (1981) Biochim. Biophys. Acta, 671, 129–138], the position of the six disulfide bridges and of the two free thiols of the protein was investigated using a variety of techniques. Three bridges (Cys‐4–Cys‐10, Cys‐237–Cys‐261 and Cys‐433–Cys‐449) were easily identified in the peptic digest of lipase at pH 2.0. In the latter digest, two other bridges (Cys‐285–Cys‐296 and Cys‐299–Cys‐304) were also identified by means of the cystine peptide constituted by two peptide segments: Ala281‐Gly‐Phe‐Pro‐Cys‐Asp‐Ser287 and Thr292‐Ala‐Asn‐Lys‐Cys‐Phe‐Pro‐Cys‐Pro‐Ser‐Glu‐Gly‐Cys‐Pro‐Gln‐Met307. A disulfide bridge, formed by Cys‐285 and one of the three half‐cystines of the other segment, connected the two peptide moieties. A second disulfide bridge linked the two remaining half‐cystines. It was not possible to split any peptide bond between Cys‐296 and Cys‐304 with proteolytic enzymes. The determination of the pairing of the four half‐cystines was resolved as follows. Bond Lys‐295–Cys‐296 was cleaved with trypsin. A single cycle of Edman degradation was then performed on the peptide compound, thus freeing, in particular, Cys‐296 of the peptide bond (296–297). Cys‐296 only retained its S‐S connection with the half‐cystine partner. At the completion of the above operations, the two peptide segments (282–287) and (297–307) could be separated. Consequently it was concluded that Cys‐285 is linked to Cys‐296. Therefore Cys‐299 and Cys‐304 are paired.The most reactive SHI group of the enzyme was localized on Cys‐181 by condensation of the native protein with radioactive N‐ethylmaleimide. The alkylation of the SHII group required previous denaturation of the molecule at alkaline pH. The results obtained for the characterization of the SHII group suggested the existence of two isomeric forms, the SHII being either on Cys‐101 or Cys‐103 and the bridge alternately between Cys‐90–Cys‐103 or Cys‐90–Cys‐101.It is not yet known whether these two forms pre‐exist in native lipase or result from an exchange reaction. The bridge Cys‐90–Cys‐101 was characterized in a thermolytic digest of a cyanogen bromide fragment (CN II) of the protein. However, another bridge involving Cys‐181 was also found in the digest. This bridge is considered as being an artefact. It is possible that considering the treatments undergone by the large peptide (CN II), the SH groups of lipase were oxidized and transformed in an S‐S bridge.The disulfide bridges of lipase form relatively small loops along the main chain. This arrangement is consistent with a high flexibility of the molecule. As reported earlier [R. Verger et al. (1971) Biochim. Biophys. Acta, 242, 580–592], the SHI group is not essential for lipase activity. The role of the SHII group should be more precisely investigated.
A Coupled Diffusion and Barrier Model for the Recombination Kinetics of Myoglobin with Carbon Monoxide
Authors: MARDEN Michael C. 
Published: July 2010

The rebinding kinetics of myoglobin (Mb) with carbon monoxide (CO) after photodissociation have been analyzed using a single energy barrier coupled by a two‐dimensional channel to the solvent. The total binding rate is written as the product of the barrier rate coefficient and the probability of the ligand being at the barrier. Assuming that the photodissociated ligands are initially localized near the barrier, the probability then decays as 1/tas the ligands migrate away toward the solvent. The time‐dependent rate coefficient yields power‐law kinetics as observed in the MbCO recombination kinetics.At a given temperature two parameters are sufficient to simulate power‐law kinetics of arbitrary slope. Using a model of diffusion from a region of lower to higher diffusivity, four parameters can simulate an initial power law followed by a final slow exponential process. Estimates of the diffusion coefficients inside the protein are two to three orders of magnitude slower than the ligand diffusivity in the external solvent. A single Arrhenius rate coefficient for the barrier can be used for the entire range from 60 K to 320 K. The barrier/diffusion model implies a certain shape of the kinetics which is in good agreement with the observed recombination kinetics of many heme protein/ligand systems.
NMN Adenylyltransferase: Its Association with Chromatin and with Poly(ADP‐ribose) Polymerase
Authors: UHR Marie Louise , SMULSON Mark 
Published: July 2010

The nuclear location of NMN adenylyltransferase, which catalyses the formation of NAD and pyrophosphate from ATP and NMN, has been examined to ascertain if the enzyme is bound to the domains of chromatin which undergo poly(ADP‐ribos)ylation. This latter reaction utilizes much of the cellular NAD.A radioisotope assay using [α‐32P]ATP was developed to enable precise measurement of picomole amounts of NAD. With this assay, it appeared that the reaction catalysed by NMN adenylyltransferase proceeded with a rapid, early ‘burst’ of NAD before steady‐state velocities were established. From this it was calculated that there could be 107 active sites of NMN adenylyltransferase per HeLa nucleus in asynchronously growing cells; that is, approximately one per 10–20 nucleosomes.Very little enzyme activity was liberated by digesting HeLa nuclei with micrococcal nuclease in 80 mM NaCl, and the enzyme which was solubilized was not bound to oligonucleosomes separated by electrophoresis on polyacrylamide gels. In contrast, poly(ADP‐ribose) polymerase activity was clearly demonstrated on these particles. The enzyme was readily liberated by DNase I digestion, especially when the digestion was carried out in low‐ionic‐strength buffer.The results demonstrated that the enzyme was neither bound to oligonucleosomes nor part of the nuclear envelope or matrix. Preliminary results suggested that there could be some direct channelling of NAD between the two enzymes in intact nuclei. It appears that NMN adenylyltransferase is bound within rather than to chromatin.
Role of Gangliosides in Reception of Influenza Virus
Authors: BERGELSON Lev D. , BUKRINSKAYA Alla G. , PROKAZOVA Nina V. , SHAPOSHNIKOVA Galina I. , KOCHAROV Sergey L. , SHEVCHENKO Valeriy P. , KORNILAEVA Galina V. , FOMINA‐AGEEVA Elena V. 
Published: July 2010

The ganglioside composition of Ehrlich ascites carcinoma (EAC) cells and the role of the individual gangliosides in binding and penetration into the cell of influenza virus were determined. EAC gangliosides identical with or close to GM3, GM2, GM1, GT1a and GT1b were characterized by thin‐layer chromarography, compositional analyses, methylation analysis and mass‐spectrometry. The ganglioside uptake capacity of native and neuraminidase‐treated EAC cells was studied with tritium‐labeled gangliosides of definite structure and the binding of influenza virus to cells was determinated by using [3H]uridine‐labeled virus and by hemagglutination studies. Treatment of the cells with Vibrio cholerae neuraminidase largely decreased binding of the virus. Exogenous gangliosides with a terminal galactose unit or a penultimate galactose masked by neuraminic acid were able to restore the virus‐binding capacity of neuraminidase‐treated cells, however, the main ganglioside of EAC cells, GM2, which carbohydrate chain is terminated by N‐acetylgalactosamine, was completely ineffective. The common carbohydrate sequence of the ganghosides showing binding activity

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is proposed to be the main recognition structure of the influenza virus receptor on the surface of EAC cells.Penetration of labeled influenza virus into the nuclei of EAC cells was evaluated by measuring the radioactivity of the nuclei of neuraminidase‐treated ganglioside‐loaded cells after exposition to the labeled virus. Of all gangliosides tested only trisialogangliosides of the GT1b type were able to induce increased entry of the virus into the cells and accumulation of its radioactive component into the nuclei. It is suggested that GT1b gangliosides react specifically with the virus protein responsible for membrane fusion (apparently the hemagglutinin HA2 subunit) and thus are involved in virus penetration and delivery of the virus genome to the nuclei.
Detection and Localization of a Class of Proteins Immunologically Related to a 100‐kDa Nucleolar Protein
Authors: BUGLER Beatrix , CAIZERGUES‐FERRER Michèle , BOUCHE Gérard , BOURBON Henri , AMALRIC François 
Published: July 2010

A high‐molecular‐mass nucleolar protein (100‐kDa protein) is associated with nascent pre‐rRNA and preribosomes in Chinese hamster ovary cells. We have prepared antiserum against the 100‐kDa protein and we have used it to study the intracellular localization and the possible processing of this protein.Serologically related proteins were detected in the nucleolus and in ribosomes. Proteins of various subcellular fractions were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, reacted with serum and the protein‐immunoglobulin complexes were revealed by 125I‐labeled protein A. In the nucleolus, four proteins with molecular masses of 100 kDa, 95 kDa, 76 kDa and 70 kDa were thus visualized. In the ribosomes, two proteins (of 100 kDa and 76 kDa) gave a strong reaction while six others (of 70 kDa, 60 kDa, 50 kDa, 30 kDa, 21 kDa, 18 kDa) reacted slightly. By immunological precipitation of total cell extracts, we have shown that the 100‐kDa protein antiserum cross‐reacts with five proteins with molecular masses of 120 kDa, 100 kDa, 95 kDa, 70 kDa and 60 kDa.Specific degradation of the 100‐kDa protein into similar peptides with molecular masses of 95 kDa, 76 kDa, 70 kDa, 60 kDa and 50 kDa can be achieved by incubation of isolated nucleoli or of purified 100‐kDa protein in vitro. Cleavage of the protein is due to a thiol endoprotease which is tightly bound to the 100‐kDa protein. Possible relations between the maturation of this preribosomal protein into ribosomal proteins and the processing of preribosomal RNA into mature ribosomal RNA are discussed.
Transient Kinetics of the One‐Electron Transfer Reaction between Reduced Flavocytochrome b2 and Oxidized Cytochrome c
Authors: CAPEILLERE‐BLANDIN Chantal 
Published: July 2010

The one‐electron transfer reaction from reduced flavocytochrome b2 (fully reduced by three electron equivalents) to ferricytochrome c, both purified from the yeast Hunsenula anomala, has been studied using stopped‐flow spectrophotometry in the course of a single turnover, for reactants initially mixed in a heme molar ratio equal to one. The cytochrome c reduction proceeded to completion through an apparently first‐order process. Depending on the experimental conditions (concentrations and/or ionic strength), the reduction is of second‐order or first‐order character.To interpret these kinetic results computer simulation studies have been performed based on a kinetic scheme involving, besides the formation of a complex before the electron transfer step, intramolecular electron transfer steps within flavocytochrome b2 to maintain the concentration of the specific electron donor center, the reduced cytochrome b2. As far as the cytochrome c reduction rate constant, ka, and its variations were concerned the simulated data showed that this complicated scheme could approximate a mechanism which is by far the simplest, involving only the two former steps. Such a scheme accounts firstly for the hyperbolic dependence of the rate of reduction of cytochrome c, ka, upon reductant concentrations which had provided clear evidence for the kinetic existence of a complex in the reaction pathway. At 5°C the rate constant for the electron transfer is 380 s−1 with an activation energy of 13.8 kJ mol,−1 (3.3 kcal mol−1). Secondly it predicts the observed variations of ka with ionic strength and provides estimates of the rate constants of the binding step.