bluuebunny/crossref_metadata_2025_split huggingface dataset:
Galanin receptor and its ligands in the rat hippocampus
Authors: FISONE Gilberto , LANGEL ülo , CARLQUIST Mats , BERGMAN Tomas , CONSOLO Silvana , HÖKFELT Tomas , UNDÉN Anders , ANDELL Siv , BARTFAI Tamas 
Published: March 2005

Receptors for the 29‐amino‐acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine‐T‐iodinated porcine galanin as ligand. The equilibrium binding of125I‐galanin showed the presence of a high‐affinity binding site (Kd=1.91 ± 40 nM). The concentration of the high‐affinity‐binding sites was 107 ± 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 ± 0.1 M−1 min−1 at 37°C.The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The125I‐galanin‐binding site is a trypsin‐sensitive membrane protein, which is heat‐denaturated at 60°C within 5 min. The effect of GTP and its analogs and of pertussis‐toxin‐catalyzed ADP‐ribosylation on the binding of125I‐galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I‐galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I‐galanin. The 125I‐galanin‐binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1–20 and 21–29 of rat galanin and the synthetic fragments 12–29, 18–29 and 21–29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1–29 of 125I‐galanin at a concentration of 1μM.
Hepatic processing of insulin
Authors: SMITH Geoffrey D. , CHRISTENSEN Jillian R. , RIDEOUT James M. , PETERS Timothy J. 
Published: March 2005

The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied.

Methylamine, ammonium chloride and dansyl cadaverine but not chloroquine reduce the total amount of insulin endocytosed.
Ammonium chloride, dansyl cadaverine and chloroquine but not methylamine inhibit subsequent degradation and/or translocation of degradation products.
None of the weak bases changed the species of the degradation products found within the endocytic vesicles.
Kinetic analysis of intravesicular degradation indicates that dissociation from the receptor is the ratelimiting process in degradation.
Chloroquine and dansyl cadaverine but not methylamine or ammonium chloride showed specific inhibition of insulin degradation in isolated endocytic vesicles.
The effect of chloroquine and dansyl cadaverine on the kinetics of degradation suggest that they are acting by switching the receptor into a tight‐binding conformation thereby slowing dissociation.
On the role of phospholipids in the reconstituted cytochrome P‐450 system
Authors: RIETJENS Ivonne M. C. M. , ANCHER Leo J. , VEEGER Cees 
Published: March 2005

The difference in pentoxyresorufin O‐dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P‐450b (IIB1) system.The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P‐450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P‐450 for the NADPH‐cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho‐dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme.We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P‐450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P‐450 molecules in these systems.Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P‐450 molecule, but also on the efficiency of electron transfer from NADPH‐cytochrome reductase to cytochrome P‐450.
A further insight into the binding of blood clotting factors to membranes
Authors: PRIGENT‐DACHARY Jeanne , LINDHOUT Théo , BOISSEAU Michel René , DUFOURCQ Jean 
Published: March 2005

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR — factor‐Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy‐transfer experiments between the Trp residues of factor Va and pyrene‐labelled phospholipids.Prothrombin was cleaved by α‐chymotrypsin into two parts: N‐terminal residues 1–41 (peptide 1–41) containing the γ‐carboxyglutamic acid residues (Gla), and des‐(1–41)‐prothrombin; their membrane association was investigated. Peptide 1–41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C‐terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1–41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin.Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des‐(1–41)‐prothrombin. Moreover, the binding curves of des(1–41)‐prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+‐independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.
A combination of intracellular leucine with either glutamate or aspartate inhibits autophagic proteolysis in isolated rat hepatocytes
Authors: CARO L. Heleen P. , PLOMP Peter J. A. M. , LEVERVE Xavier M. , MEIJER Alfred J. 
Published: March 2005

It has been shown previously that the inhibition of autophagic proteolysis in liver by a physiological mixture of amino acids can be mimicked completely by addition of leucine in combination with alanine [Leverve, X. M., Caro, L. H. P., Plomp, P. J. A. M. and Meijer, A. J. (1987) FEBS Lett. 219, 455–458]. We have now further defined conditions which lead to this inhibition.Isolated rat hepatocytes were incubated in the perifusion system in which the cells can be maintained at a steady state in the presence of low amino acid concentrations.Combinations of leucine (0.5 mM) with either alanine, glutamine, asparagine or proline (2 mM) inhibited proteolysis by 40–50%. Under these conditions, both in the absence and presence of the transaminase inhibitor, aminooxyacetate, a correlation was found between the extent of inhibition of proteolysis and the sum of the total intracellular amounts of asparate and glutamate.Inhibition of proteolysis by leucine and leucine analogues did not correlate with their ability to activate glutamate dehydrogenase.
Serum albumin binding of palmitate and stearate
Authors: BRODERSEN Rolf , VORUM Henrik , SKRIVER Elisabeth , PEDERSEN Anders Overgaard 
Published: March 2005

In usual studies of ligand binding to a carrier, free and bound ligand concentrations are measured in equilibrium mixtures with varying carrier and ligand concentrations. The observed data are then analyzed by a binding equation such as Scatchard's or the general binding equation. With palmitic, stearic and oleic acids as ligands we found that the aqueous solubility is too low to allow this procedure. We have consequently transformed the general binding equation so that it does not contain parameters related to aqueous solutions of the ligand. While the classical binding equations describe affinities of transfer of a ligand from an aqueous solution to the carrier, the new equation is valid for transfer of a ligand from one bound state to another, i.e. for relative binding description. The relative binding constants, L1, L2, L3…Li, in the new equation thus define the transfer affinity for the ligand from a 1:1 complex with a standard carrier to an i:1 complex of the ligand with the carrier investigated.Binding of palmitate and stearate to human serum albumin was studied by determination of dialytic exchange rates between identical fatty acid/albumin solutions. The results were analyzed by the new equation without reference to ligands in aqueous solution.
Circular dichroic spectroscopy of membrane haemoproteins
Authors: DEGLI ESPOSTI Mauro , PALMER Graham , LENAZ Giorgio 
Published: March 2005

The circular dichroism (CD) of dihaem cytochrome b from mitochondrial and bacterial ubiquinol:cytochrome‐c reductase (bc1 complex) has been characterized. The dichroic properties of the yeast purified cyt b are very similar to those of the native cyt b within the mitochondrial bc1 complex. The CD spectra in the Soret region of the native cytochrome b present in all species studied show an intense bisignate Cotton effect having a zero‐crossing wavelength close to the absorbance maximum.In preparations partially or completely depleted of the low‐potential b haem (b1) the CD spectra exhibit a single positive Cotton effect resembling the corresponding absorption spectrum. This is particularly evident in the purified cytochrome b‐562 from Rhodobacter sphaeroides R26, which contains only the high‐potential b haem (bh). These spectral features together with the reconstitution of the cytochrome b1 haem have been used to resolve the CD contribution of each haem to the CD spectra of cytochrome b.The mechanisms which might be responsible for the optical activity have been examined. It appears that the CD spectra of cytochrome b derive from both the mutual interaction of its two haems (giving rise to exciton coupling) and to the interaction of each haem with nearby aromatic residues, other than the pairs of histidines which coordinate the iron. The dipole coupling between haem and aromatic residues appears to be more important than exciton coupling in the CD spectra of oxidized b cytochromes and correlations have been made between the CD features and the proposed structure of cytochrome b.
Méthionyl‐tRNA synthétase du germe de blé
Authors: ARCHAMBAULT de VENÇAY Jacques 
Published: March 2005

Methionyl‐tRNA synthetase from wheat germ. – Effect of an endogenous protease and correlations between structural characteristics and catalytic properties. Methionyl‐tRNA synthetase (MetRS) has been described as a free monomeric or oligomeric enzyme, or included in a multienzyme complex. Moreover, on limited tryptic digestion, it can generate shorter forms.So, when purified from wheat‐germ lysate, the possible presence of proteases able to hydrolyse this enzyme was investigated.When extraction was performed with sulfhydryl‐blocking reagents, an active monomeric MetRS of Mr 105000 was purified. This enzyme form was identical to the structure exhibiting methionyl‐tRNA synthetase activity in multisynthetic complexes. Without this inhibitor, MetRS was purified as an active dimeric form of Mr 165000 with identical subunits of Mr 82000.A protease inhibited by sulfhydryl‐blocking reagents and included in a complex of Mr 2 · 106 was isolated from this wheat‐germ lysate. This protease was able to hydrolyse different proteins (albumin, casein), but was without activity for a trypsin substrate, such as N‐α‐benzoyl‐dl‐arginine p‐nitroanilide. When added to a solution of Mr‐105000 MetRS, it yielded an inactive peptide of Mr 20000, containing numerous charged amino acids and a protein of Mr 82000, able to give an active dimeric enzyme of Mr 165000. Amino acid analysis of this last form, indicated an identical structure with the active dimeric MetRS of Mr 165000, purified in the absence of protease inhibitors. Moreover, the affinity for methionine was the same for the monomeric enzyme of Mr 105000 and the dimeric form of Mr 165000, probably because proteolysis did not affect the catalytic domain.When enzymic activity of the proteolysed form (Mr 2×82000) was studied versus enzyme concentration, a decrease in specific activity, at low concentrations, was seen. This phenomenon was analysed on the basis of the existence of an equilibrium between an active dimer and two inactive monomers. With the active monomeric form of Mr 105000, no change in specific activity with decreasing enzyme concentration occurred.
Structures of O‐glycosidically linked oligosaccharides isolated from human meconium glycoproteins
Authors: CAPON Calliope , LEROY Yves , WIERUSZESKI Jean‐Michel , RICART Guy , STRECKER Gérard , MONTREUIL Jean , FOURNET Bernard 
Published: March 2005

The structure of major O‐glycosidicall linked neutral and acidic oligosaccharides isolated from human meconium glycoproteins were established. Neutral and acidic oligosaccharides were released by alkaline borohydride treatment, purified by Biogel P‐6 and fractionated by high‐performance liquid chromatography. This approach resulted in 50 neutral and 30 acidic oligosaccharides. The present study reports the primary stuctural analysis of five neutral oligosaccharides, ten monosialylated oligosaccharides, one monosialylated monosulfated oligosaccharide and three disialylated oligosaccharides, by permethylation, fast‐atom‐bombardment mass spectrometry analysis and 400‐MHz 1H‐NMR spectroscopy.The following structure have not been described previously:
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Phosphorylation/dephosphorylation of the β light chain of clathrin from rat liver coated vesicles
Authors: LOEB Jacques E. , CANTOURNET Benoit , VARTANIAN Jean‐Pierre , GORIS Jozef , MERLEVEDE Wilfried 
Published: March 2005

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin β light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(l‐lysine) which acts through binding to the β light chain. The phosphorylation of other proteins is also increased in the presence of poly(l‐lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50‐kDa protein doublet from rat liver coated vesicles is inhibited.Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin β light chain. This activity is different from the protein phosphatase which dephosphorylates the 50‐kDa protein. This enzyme seems to be unrelated to the ATP/Mg‐dependent protein phosphatase, or the polycation‐stimulated protein phosphatases, which dephosphorylate the 50‐kDa protein and β light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the β‐light‐chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 μM orthovanadate and 5 mM p‐nitrophenyl phosphate but not by 10 mM EDTA.
Biochemical and theoretical approach to localization of metal‐ion‐binding sites in the actin primary structure
Authors: STRZELECKA‐GOŁLASZEWSKA Hanna , BOGUTA Grzegorz , ZMORZYŃSKI Stainslaw , MORACZEWSKA Joanna 
Published: March 2005

The number of Ca2+ ions bound at sites other than the single high‐affinity site in CaCl2‐induced polymers of rabbit skeletal muscle, chicken gizzard, and bovine aorta actin was determined. The polymer of skeletal muscle and aorta actin contained 4 mol Ca2+/mol, whereas gizzard actin only 3 mol weakly bound Ca2+/mol monomer. This difference correlates with the deletion in smooth muscle γ‐actin of one out of four NH2‐terminal acidic residues typical of skeletal and smooth muscle α‐actin isoforms, suggesting that this additional acidic residue in α‐actins is involved in the weak binding of cations which is essential for polymerization. This experimental result, as well as a theoretical analysis of the actin primary structure, argue against the implication of the NH2‐terminal acidic residues in the high‐affinity site for divalent cation.The analysis of the actin primary structure aimed at identification of sequences resembling the known Ca2+‐binding patterns has revealed the absence of an EF‐hand Ca2+ ‐binding site. The best match was obtained between the sequence of the 292–301/segment and that of Ca2+ site in lectins. However, in the light of experimental data discussed, it is more plausible that the actual high‐affinity Ca2+ site in actin involves sequentially distant residues from the NH2‐ and COOH‐terminal portions of the polypeptide chain.
Monoclonal antibodies to protein kinase Cγ
Authors: CAZAUBON Sylvie , MARAIS Richard , PARKER Peter , STROSBERG A. Donny 
Published: March 2005

Four monoclonal antibodies (mAbs), derived from high‐responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype γ1, K and 15G4 is of isotype γ2b,K. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific of the bovine γ isoform of protein kinase C and cross‐react with protein kinase Cγ from a variety of animal species.Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase Cγ cofactor‐dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor‐independent catalytic activity of protein kinase Cγ. Competition analysis between these mAbs and phosphatidylserine, 12‐O‐tetradecanoylphorbol 13‐acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase Cγ. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase Cγ.
Expression of muscle‐specific proteins necessary to regulate translation of the mRNA for a 40‐kDa housekeeping polypeptide in rat L6 cells
Authors: PRAMANIK Saroj K. , BAG Jnanankur 
Published: March 2005

Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells. This mRNA was translated in the proliferating myoblasts but not in the non‐proliferating myotubes. A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle‐specific genes in mitogen‐poor differentiation medium. In the absence of any detectable accumulation of muscle‐specific α‐actin mRNA, the P40 mRNA remained in the translated state. A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle specific actin and tropomyosin mRNAs. On the other hand, it showed no effect on the translation of P40 mRNA. Addition of Ca2+ to the EGTA‐treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days. Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non‐translated state. An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle‐specific mRNAs.The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA‐dependent micrococcal‐nuclease‐treated homologous cell‐free extracts. The extracts from myoblasts and myotubes were able to translate P40 mRNA. Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs. Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA. The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing thdse ribosomes with a high‐ionec‐strength buffer. In order to elucidate the role of interaction between mRAN and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA‐protein(mRNA) complexes were also examined. Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of mNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells. Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein then polysomal P40 mRNP. Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNA complexes showed that they were composed of identical polypeptides. The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP.
Purification of cytochrome‐c oxidase retaining its pulsed form
Authors: BRANDT Ulrich , SCHÄGGER Hermann , von JAGOW Gebhard 
Published: March 2005

A new purification procedure for cytochrome‐c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X‐100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady‐state kinetics were found to be the same as thoes reported for other preparations.In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide‐binding kinetics.The formation of resting kinetics biphasic cyanide‐binding kinetics was shown to be induced by a short incubation at pH 5.0.
Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity
Authors: HECHT Katrin , WRBA Alexander , JAENICKE Rainer 
Published: March 2005

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus sterothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off.Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optmum curve for Km, oxaloacetate while Km, nadh remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I 1 M. Non‐halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentration is caused by the salt‐induced increase in rigidity of the enzyme, rather than gross structural changes.