bluuebunny/crossref_metadata_2025_split huggingface dataset:
Properties of vasoactive‐intestinal‐peptide receptors and β‐adrenoceptors in the murine radiation leukemia‐virus‐induced lymphoma cell line BL/VL3
Authors: ABELLO Jacques , DAMIEN Catherine , DE NEEF Philippe , TASTENOY Michèle , HOOGHE Robert , ROBBERECHT Patrick , CHRISTOPHE Jean 
Published: March 2005

Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with β2‐adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T‐cell lymphoma induced by a radiation leukemia virus.
The relative potency of VIP‐related peptides to stimulate adenylate cyclase activity was: helodermin > VIP > peptide histidine isoleucinamide. Five VIP analogs inhibited 125I‐iodo‐VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP > [D‐Asp3]VIP > [D‐Ser2]VIP > [D‐Ala4]VIP = [D‐His1]VIP = [D‐Phe2]VIP. [D‐Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin‐ and VIP‐stimulated adenylate cyclase activity with a similar Ki (0.07–0.10 μM). These data suggest the existence, in this murine T‐cell lymphoma, of VIP receptors of the ‘helodermin‐preferring’ subtype that are coupled to adenylate cyclase.
Homologous and heterologous regulation of the helodermin/vasoactive‐intestinal‐peptide response in the murine radiation leukemia‐virus‐induced lymphoma cell line BL/VL3
Authors: ABELLO Jacques , DAMIEN Catherine , ROBBERECHT Patrick , HOOGHE Robert , VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , CHRISTOPHE Jean 
Published: March 2005

Functional vasoactive intestinal peptide (VIP)/helodermin receptors and β2‐adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T‐cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal).
Short‐term (5–30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase.
Long‐term (16‐h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D‐Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.
Direct and energy‐transfer photolabelling of brain muscarinic acetylcholine receptors
Authors: ILIEN Brigitte , HIRTH Christian 
Published: March 2005

Efficient photolabelling of muscarinic acetylcholine receptors was obtained using either two aryldiazonium salts or an azido derivative. These probes did not discriminate between muscarinic binding subtypes or affinity states and became irreversibly bound to the receptor sites, in an entirely atropine‐protectable manner, upon ultraviolet irradiation. The extent of labelling was dependent both on probe concentration and on time of irradiation and reached up to 80% of the receptor population, under optimal alkylating conditions.In contrast to the azido derivative, both diazonium salts behave as potent irreversible labels of muscarinic receptors, provided energy‐transfer photolabelling conditions were followed. Such an indirect activation of diazonium ligands, through an energy transfer from photoexcited tryptophan residues, has been previously found to increase the site‐specificity and the rate of labelling of other acetylcholine binding proteins. Analogies in the photolabelling process of acetylcholinesterase or of nicotinic and muscarinic receptors by the two diazonium salts are discussed.Altogether, these findings suggest that these new probes may be promising tools to investigate the location and the topography of the agonist‐antagonist binding domain on purified muscarinic receptors, through amino acid and/or sequence analyses of radioactive, photolabelled residues.
Kinetics of bilirubin oxidation catalysed by bilirubin oxidase in a water‐in‐oil microemulsion system
Authors: OLDFIELD Christopher , FREEDMAN Robert B. 
Published: March 2005

Bilirubin oxidase can catalyse the oxidation of its primary substrate, bilirubin, in a water‐in‐oil microemulsion, which consists of discrete nanometer‐diameter water droplets dispersed in a continuous water‐immiscible oil medium. The droplets are stabilized by a monolayer of the surfactant, cetyltrimethylammonium bromide present at the oil/water interface.
Spectroscopic evidence is presented to show that bilirubin solubilized in this system is located mainly in the surfactant layer, in a form accessible to the enzyme molecule.
Studies are presented on the enzyme‐catalysed rate of bilirubin oxidation in this system, as a function of temperature, pH, water content, and substrate and enzyme concentrations.
The main conclusions are that the enzyme can efficiently oxidise bilirubin in microemulsions of low water content. The reaction obeys Michaelis‐Menten kinetics. The optimal pH for the catalysis is 8.0. The efficiency of catalysis decreases sharply as the water content increases.
The visualization by affinity electrophoresis of a specific association between the consecutive citric acid cycle enzymes fumarase and malate dehydrogenase
Authors: BEECKMANS Sonia , VAN DRIESSCHE Edilbert , KANAREK Louis 
Published: March 2005

Evidence is growing that the citric acid cycle, like many other metabolic pathways, might exist in vivo as a more or less tightly organized multi‐enzyme cluster. The term ‘metabolon’ [Robinson, J. B. & Srere, P. A. (1985) J. Biol. Chem. 260, 10800–10805] was recently introduced to describe such a complex of sequential metabolic enzymes.We adopted the technique of affinity electrophoresis for the study of interactions between the cycle enzymes fumarase and malate dehydrogenase. This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J. Biochem. 117, 527–535], one of which is the fact that the interaction can be directly visualized. The observed association is specific since both metabolically unrelated proteins and the cytoplasmic isoenzyme of malate dehydrogenase do not interact with fumarase.Several metabolites (citrate, isocitrate, 2‐oxoglutarate, succinate, fumarate, malate, oxaloacetate, Pi, AMP, ADP, NAD+, NADH) were found not to affect the association between fumarase and mitochondrial malate dehydrogenase. Both ATP, Mg2+‐ATP and GTP disrupt the association when they are present at 1 mM concentrations. Lower non‐physiological ATP concentrations do not, however, disturb the interaction. The presence of 1 mM ADP was found to abolish the disrupting effect of 1 mM ATP. The latter findings are suggestive of an interruption of the citric acid cycle at the level of fumarase under conditions of high energy load (i.e. high ATP/ADP ratios)
Reactivity of the P‐site‐bound donor in ribosomal peptide‐bond formation
Authors: SYNETOS Dennis , COUTSOGEORGOPOULOS Charalambos 
Published: March 2005

The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)‐site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat).If we assume that the puromycin‐reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P‐site binding of the donor substrates AcPhe‐tRNA or fMet‐tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3= 0.8 min−1 versus 8.3 min−1). On the other hand there are conditions, under which the proportion of P‐site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min−1. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell‐free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis.
Methyl‐coenzyme‐M reductase from Methanobacterium thermoautotrophicum (strain Marburg)
Authors: ELLERMANN Joachim , ROSPERT Sabine , THAUER Rudolf K. , BOKRANZ Martin , KLEIN Albrecht , VOGES Markus , BERKESSEL Albrecht 
Published: March 2005

Methyl‐coenzyme‐M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the α, β and γ subunits, no additional polypeptides were detectable in the preparation. Under appropriate conditions the enzyme was found to catalyze the reduction of methyl‐CoM with 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP) to CH4 at a specific rate of 2.5 μmol · min−1· mg protein−1. This finding contradicts a recent report that methyl‐CoM reductase is only active when some contaminating proteins are present.The two polypeptides encoded by the open reading frames ORF1 and ORF2 of the methyl‐CoM reductase transcription unit did not co‐purify with the α, β and γ subunits. They were neither required nor did they stimulate the activity under the assay conditions.3‐Bromopropanesulfonate (apparent Ki= 0.05 μM) and 2‐azidoethanesulfonate (apparent Ki= 1 μM) were found to be two new competitive inhibitors of methyl‐CoM reductase. Both inhibitors were considerably more effective than the “classical” 2‐bromoethanesulfonate (apparent Ki= 4 μM).
Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca2+‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP
Authors: GIRARDET Jean‐Luc , DUPONT Yves , LACAPERE Jean‐Jacques 
Published: March 2005

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+‐ATPase. Three classes of Tb3+‐binding sites have been found: a first class of low‐affinity (Kd= 10 μM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (<0.1 μM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+‐ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP.Substitution of H2O by D2O shows that Tb‐FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+‐ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site.Addition of calcium quenches the fluorescence signal of the terbium‐FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2→ E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1↔ E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high‐affinity calcium sites are located very close to the Tb‐FTP complex bound to the nucleotide site.
13C‐NMR study of autotrophic CO2 fixation in Thermoproteus neutrophilus
Authors: SCHÄFER Sigrid , GÖTZ Martina , EISENREICH Wolfgang , BACHER Adelbert , FUCHS Georg 
Published: March 2005

The pathway of autotrophic CO2 fixation has been investigated in the extremely thermophilic sulfur‐respiring anaerobic archaebacterium Thermoproteus neutrophilus. [1,4‐13C2]Succinate was used as a tracer since this compound was incorporated in small amounts virtually into all cell compounds without affecting the organism's ability to synthesize all cell constituents from CO2. Three representative amino acids, glutamate, aspartate and alanine were isolated from cells after growth for several generations in the presence of [1,4‐13C2]succinate and their labelling patterns were determined by 13C NMR spectroscopy.The data is consistent with CO2 fixation by a reductive citric acid cycle, as proposed earlier for the green sulfur bacterium Chlorobium limicola, the sulfate‐reducing Desulfobacter hydrogenophilus and the microaerophilic Knallgasbacterium Hydrogenobacter thermophilus. The presence of a reductive citric acid cycle in archaebacteria indicates that this CO2 fixation mechanism which is an alternative to the Calvin cycle is present in many anaerobic or facultative anaerobic microorganisms.
Structural changes in positively and negatively supercoiled DNA
Authors: BRAHMS Sabine , NAKASU Syo , KIKUCHI Aki , BRAHMS J. Georges 
Published: March 2005

The effect of superhelical constraint on the structure of covalently closed circular DNA (cccDNA; pBR322) with positive and negative writhe (superturn) has been investigated as a function of decreasing and increasing specific linking difference (mean superhelical density σ¯). At low and moderate negative superhelical densities σ¯, the overall average structure is maintained in an unwound B‐form slightly modified.The overwound cccDNAs with positive writhe differ from those with negative writhe by an absence of cruciform structure. At high negative densities of supercoiling different changes involving the reversal of twist handedness are shown to lead to the formation of DNA segments in a conformation identical to the left‐handed component of form V DNA.
Serological analysis and characterization of calf thymus ribonuclease H IIb
Authors: VONWIRTH Heiner , FRANK Peter , BÜSEN Werner 
Published: March 2005

Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52‐kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52‐kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52‐kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric.The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N‐ethylmaleimide, and exhibits a pH optimum of 9.0–9.5. The enzyme releases oligoribonucleotides with 3′‐OH and 5′‐phosphate ends, probably in an exonucleolytical manner.The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431–433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.
Purification and molecular properties of rabbit liver esterase ES‐1A
Authors: van LITH Hein A. , BIEMAN Maria den , van ZUTPHEN Bert F. M. 
Published: March 2005

The isolation of esterase ES‐1A from rabbit liver microsomes/lysosomes is reported. The purification, as measured by methylbutyrate‐hydrolysing activity, was about 27‐fold with a recovery of 2.4%.
The resulting product is apparently homogeneous by polyacrylamide (gradient) gel electrophoresis and sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis after protein staining. The enzyme exhibits heterogeneity after staining for esterase activity and in isoelectric focusing.
The molecular mass of the native protein was found to be about 183 kDa (determined by gel filtration and polyacrylamide gel electrophoresis) with a subunit mass of about 63 kDa, indicating a trimeric structure of the enzyme, with subunits of equal size.
ES‐1A is a glycoprotein and is classified as a carboxylesterase (EC 3.1.1.1).
The high degree of similarity of the properties of rabbit ES‐1A with those of mouse ES‐6A and rat ES‐10 suggests that these three esterases may have a common evolutionary origin.
Molecular and cellular studies of tryptophanyl‐tRNA synthetase using monoclonal antibodies
Authors: BERESTEN Sergey F. , ZARGAROVA Tatyana A. , FAVOROVA Ol'ga O. , RUBIKAITE Birute I. , RYAZANOV Alexey G. , KISSELEV Lev L. 
Published: March 2005

Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl‐tRNA synthetase were prepared.Am2 antibodies inhibit the Trp‐tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40‐kDa or 51‐kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp‐tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp‐tRNA synthetase molecule. Am1 cross‐reacts with Trp‐tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis.A rapid two‐step technique was developed for isolating electrophoretically homogeneous Trp‐tRNA synthetase from Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaroytic Trp‐tRNA synthetase, Am1 did not influence the activity of Trp‐tRNA synthetase from E. coli.From the aforementioned results it follows that: (a) the conservation of part of the Trp‐tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp‐tRNA synthetases suggests that the dispensalbe part of the molecule might be involved in some additional biological function(s) of Trp‐tRNA synthetase besides tRNATrp charging; (b) the common antigenic determinant in Trp‐tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.
Molecular and cellular studies of tryptophanyl‐tRNA synthetases using monoclonal antibodies
Authors: FAVOROVA Olga O. , ZARGAROVA Tatyana A. , RUKOSUYEV Vadim S. , BERESTEN Sergey F. , KISSELEV Lev L. 
Published: March 2005

The content of Trp‐tRNA synthetase in pancreas and liver of cattle, sheep, swine, rat, rabbit and man was assayed by direct radioimmunoblotting with a 125I‐labelled monoclonal antibody Am1, specifically interacting with any cukaryotic Trp‐tRNA synthetase. Its content in the organs studied, with the exception of bovine and sheep pancreas, was found to be 0.002–0.012% of total proteins. The enzyme content in bovine pancreas was about 0.2% of total proteins, i.e. 70 times higher than in bovine liver; similar correlations were found for sheep. The Trp‐tRNA synthetase levels in each organ varied from animal to animal of the same species by not more than a factor of four; these individual variations cannot affect the conclusion about the profound differences in the levels of the enzyme in pancreases of Ruminantia and of the other mammalians.As shown by indirect immunofluorescence technique, bovine Trp‐tRNA synthetase is mainly located in the exocrine part of the pancreas. Moreover, the immunoreactive material is detectable also in bovine (not human) pancreatic juice. The abnormally high Trp‐tRNA synthetase content in the ruminant pancreas may be connected with unknown function(s) of this protein somehow related to the peculiarities of digestion of these mammals.
The transitory complex between photoexcited rhodopsin and transducin
Authors: BORNANCIN Frédéric , PFISTER Claude , CHABRE Marc 
Published: March 2005

In the first step of the visual transduction cascade a photoexcited rhodopsin molecule, Rret*, binds to a GDP‐carrying transducin molecule, TGDP. The R*‐T interaction causes the opening of the nucleotide site in T and catalyzes the GDP/GTP exchange by allowing the release of the GDP. We have studied the influences on this R*‐T transitory complex of the occupancies of the nucleotide site in T and the retinal site in rhodopsin. After elimination of the GDP released from the bound transducin, the complex, named Rret*‐Te (ret for retinal present, e for nucleotide site empty) remains stabilized almost indefinitely in a medium whose ionic composition is close to physiological. In this complex the bound Te retains a lasting ability to interact with GDP or GTP, and Rret* remains spectroscopically in the meta‐II state, by contrast with free Rret* which decays to opsin and free retinal. Hence the R*‐T interaction which opens the nucleotide site in T conversely blocks the retinal site in Rret*. Upon prolonged incubation in a low‐ionic‐strength medium the Rret*‐Te complex dissociates partially, but the liberated Te is then unable to rebind GDP or GTP, even in the presence of Rret*; it is probably denaturated. Upon treatment of the Rret*‐Te complex by a high concentration of hydroxylamine, the retinal can be removed from the rhodopsin. The Re‐Te complex remains stable and the complexed transducin keeps its capacity to bind GTP. TGTP then dissociates from Re. The liberated Re loses its capacity to interact with a new transducin.These data are integrated into a discussion of the development of the cascade. We stress that affinities, i.e. dissociation equilibrium constants, are insufficient to describe the flow of reactions triggered by one Rret* molecule. It depends on a few critical rapid binding and dissociation processes, and is practically insensitive to other slow ones, hence to the values of affinities that express only the ratio of kinetics constants.The effect of the R*‐T interaction on the retinal site in rhodopsin is analogous to the effect of the binding of a G‐protein on the apparent affinity of a receptor for its agonist.