bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complexes of the polyamines spermine, spermidine and putrescine with α‐lactalbumins
Authors: MOROZOVA Ludmilla , DESMET Johan , JONIAU Marcel 
Published: March 2005

The effects of polyamines on the spectral properties and thermal stability of different α‐lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca2+‐free (apo) form of bovine or goat α‐lactalbumin resulted in spectral shifts, in both the far‐ and near‐ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield. Also, in the presence of spermine, thermal stability was increased. In contrast, in the case of human and to some extent also equine α‐lactalbumin, the shifts of the CD and fluorescence spectra and the thermal denaturation curves were in the opposite direction.Direct competition of spermine with Mn2+ could be observed for binding to the Ca2+ site of bovine α‐lactalbumin. As the main binding mode of spermine showed a 1:1 stoichiometry [K1= (2±0.5)×104M−1], we have assumed that binding occurs primarily at this site with one of the distal ammonium groups. In order to accommodate the remaining positively charged ammonium groups of spermine, a binding model was constructed by computer modeling, based on the atomic coordinates of both interacting species. This model makes use of the cluster of negatively charged glutamate residues present in the N‐terminal sequence of bovine α‐lactalbumin, sterically close to the Ca2+ site. The spermine molecule could be nicely fitted in a space delineated by the Ca2+ site, on the one hand, and Glu1, Glu7 and Glu11, on the other. Our model, which has been extended to the shorter polyamines spermidine and putrescine, is able to fully account for the observed stabilization of bovine (and goat) α‐lactalbumins against thermal denaturation, and for the observed CD and fluorescence spectral shifts. It also explains why human (and equine) α‐lactalbumin behave differently.
Regulation of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae
Authors: WENZEL Thibaut J. , LUTTIK Marijke A. H. , VAN DEN BERG Johan A. , STEENSMA H. Yde 
Published: March 2005

Expression of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex (PDH complex) and activity of the complex were investigated in cells grown under several conditions. Comparable amounts of PDA1 mRNA and E1α subunit were detected in cells from batch and chemostat cultures grown on various carbon sources, showing constitutive expression of PDA1 at the transcriptional and translational levels. Induction of the regulatory GCN4 mechanism upon histidine starvation, using the anti‐metabolite 3‐amino‐1,2,4‐triazole, increased the levels of PDA1 mRNA by approximately 40%. However, a corresponding increase of E1α concentration or activity of the PDH complex could not be detected. Hence, expression of the PDA1 gene is only regulated to a small extent, if at all, by the GCN4 mechanism.Contrary to the constant levels of PDA1 mRNA and E1α subunit in both batch and chemostat cultures, the specific activity of the PDH complex varied with the culture conditions. The activity of the PDH complex in chemostat cultures was approximately two–threefold higher than in batch cultures grown on the same carbon sources. Overproduction of the E1α subunit in batch cultures resulted in a two–threefold increase in the activity of the PDH complex. Taken together, these results indicate that the activity of the PDH complex is mainly regulated by post‐translational modification of the E1α subunit.Expression of PDA1 and activity of the PDH complex were also detected in cultures grown under conditions where no physiological significance of the PDH complex was expected, i.e. during anaerobic growth on glucose or aerobic growth on ethanol. Apparently, the switch from oxidative growth to fermentation occurs without much effect on the PDH complex. These observations suggest that the PDH complex has an alternative function besides sugar catabolism.
A polymerising Root‐effect fish hemoglobin with high subunit heterogeneity
Authors: FAGO Angela , ROMANO Mario , TAMBURRINI Maurizio , COLETTA Massimo , D'AVINNO Rossana , DI PRISCO Guido 
Published: March 2005

The blood of the teleost Chelodonichthys kumu, living in the temperate waters of New Zealand, contains a single hemoglobin. The complete amino acid sequence of the α and β chain has been established. The presence of a reactive Cys in the external position βCD8(49) causes polymerisation through intermolecular disulfide bridges between β chains, with no alteration of functional features. C. kumu Root‐effect hemoglobin displays very low or no subunit co‐operativity in the physiological pH range. Kinetic experiments on the oxygen dissociation and binding of carbon monoxide show a marked, pH‐dependent functional heterogeneity of the two chains, which contributes to the observed reduction of co‐operativity. In contrast, kinetic heterogeneity of the two chains, which contributes to the observed reduction of co‐operativity. In contrast, kinetic heterogeneity was not observed in the process of CO dissociation, indicating that functional differences between the subunits are detectable only for the dynamic ligand association pathway. The allosteric effector, ATP, seems to increase the pKa of the proton‐linked effect on the slow‐reacting subunit, affecting the quaternary equilibrium through stabilisation of the T state at lower pH, rather than enhancing the functional heterogeneity itself. In position E11 of both chains, Val (usually present at the distal side of the heme), is substituted by Ile. Although this residue has been shown not to significantly alter ligand binding to the α chain, to some extent it can perturb the access of oxygen to the β chain. Thus, this substitution may be the main reason for subunit functional heterogeneity.
Plasminogen‐activator inhibitor type 2 (PAI‐2) is a spontaneously polymerising SERPIN
Authors: MIKUS Peter , URANO Tetsumei , LILJESTRÖM Peter , NY Tor 
Published: March 2005

Plasminogen‐activator inhibitor type 2 (PAI‐2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low‐molecular‐mass form and a secreted, high‐molecular‐mass form that varies with respect to glycosylation. Here we have developed expression systems for both forms of PAI‐2 and biochemically characterised the purified proteins. In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI‐2. With this construct, more than 90% of PAI‐2 was secreted as a glycosylated, 60‐kDa molecular‐mass form, but the level of expression was low and unstable. To obtain higher expression of secreted PAI‐2, a novel expression vector based on the Semliki‐forest‐virus replicon was used. Secreted PAI‐2 was purified to homogeneity and N‐terminal sequence analysis showed that the artificial signal peptide was correctly removed. The intracellular, non‐glycosylated form of PAI‐2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and the intracellular forms of PAI‐2 were found to inhibit plasminogen activators by forming SDS‐resistant complexes and the second‐order rate constants were similar for both forms, ranging over 2.4–2.7×106 M−1s−1 for urokinase‐type PA, 2.5–2.7×105 M−1s−1 for two‐chain tissue‐type PA and 0.8–1.2×104 M−1s−1 for single‐chain tissue‐type PA. None of the purified PAI‐2 forms bound to vitronectin.Circular‐dichroism spectral analysis revealed that PAI‐2 has a CD spectrum that resembles ovalbumin more than PA‐inhibitor type 1, confirming the greater similarity between these two members of the serine‐protease inhibitor family. Similar to what has been described for the Z‐form of α1‐antitrypsin, purified PAI‐2 was found to spontaneuosly form polymers during incubation at room temperature. Attempts to convert PAI‐2 to a stable locked conformation resembling the conformation of latent PAI‐1 by treatment with diluted guanidinium chloride were unsuccessful. Instead, this treatment enhanced the formation of PAI‐2 polymers, possibly by the loop‐sheet polymerisation mechanism described for α1‐antitrypsin.
Glycogenolytic and antiglycogenolytic prostaglandin E2 actions in rat hepatocytes are mediated via different signalling pathways
Authors: PÜSCHEL G. P. , KIRCHNER C. , SCHRÖDER A. , JUNGERMANN K. 
Published: March 2005

Prostaglandin E2 has been reported both to stimulate glycogen‐phosphorylase activity (glycogenolytic effect) and to inhibit the glucagon‐stimulated glycogen‐phosphorylase activity (antiglycogenolytic effect) in rat hepatocytes. It was the purpose of this study to resolve this apparent contradiction and to characterize the signalling pathways and receptor subtypes involved in the opposing prostaglandin E2 actions.Prostaglandin E2 (10 μM) increased glucose output, glycogen‐phosphorylase activity and inositol trisphosphate formation in hepatocyte cell culture and/or suspension. In the same systems, prostaglandin E2 decreased the glucagon‐stimulated (1 nM) glycogen‐phosphorylase activity and cAMP formation.The signalling pathway leading to the glycogenolytic effect of PGE2 was interrupted by incubation of the hepatocytes with 4β‐phorbol 12‐myristate 13‐acetate (100 nM) for 10 min, while the antiglycogenolytic effect of prostaglandin E2 was not attenuated.The signalling pathway leading to the antiglycogenolytic effect of prostaglandin E2 was interrupted by an incubation of cultured hepatocytes with pertussis toxin (100 ng/ml) for 18 h, whereas the glycogenolytic effect of prostaglandin E2 was enhanced.The EP1/EP3 prostaglandin‐E2‐receptor‐specific prostaglandin E2 analogue Sulproston had a stronger glycogenolytic potency than the EP3 prostaglandin‐E2‐receptor‐specific prostaglandin E2 analogue Misoprostol. The antiglycogenolytic potency of both agonists was equal.It is concluded that the glycogenolytic and the antiglycogenolytic effects of prostaglandin E2 are mediated via different signalling pathways in hepatocytes possibly involving EP1 and EP3 prostaglandin E2 receptors, respectively.
Snake‐venom phospholipase A2 neurotoxins
Authors: CHOUMET Valérie , SALIOU Bernard , FIDELER Laure , CHEN Yuang‐Cong , GUBENSEK Franc , BON Cassian , DELOT Emmanuèle 
Published: March 2005

The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (β‐neurotoxins): single‐chain β‐neurotoxins, such as agkistrodotoxin and ammodytoxin‐A, and dimeric β‐neurotoxins, which, in the case of the best studied ones, crotoxin‐like toxins, consist of the non‐covalent association of a phospholipase A2 (CB) and a non‐enzymatic chaperon (CA). Possible evolutionary relationships of these β‐neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex.CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti‐CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone.A 10‐fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA agkistrodotoxin. Ammodytoxin A did not compete with the anti‐CA mAb.These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).
Rat kidney endopeptidase 24.16
Authors: BARELLI Hélène , VINCENT Jean‐Pierre , CHECLER Frédéric 
Published: March 2005

Endopeptidase 24.16 was purified from rat kidney homogenate on the basis of its ability to generate the biologically inactive degradation products neurotensin (1–10) and neurotensin (11–13). On SDS gels of the proteins pooled after the last purification step, the enzyme appeared homogeneous and behaved as a 70‐kDa monomer. The peptidase was not sensitive to specific inhibitors of aminopeptidases, pyroglutamyl aminopeptidase I, endopeptidase 24.11, endopeptidase 24.15, proline endopeptidase and angiotensin‐converting enzyme but was potently inhibited by several metal chelators such as o‐phenanthroline and EDTA and was blocked by divalent cations. The specificity of endopeptidase 24.16 towards peptides of the tachykinin, opioid and neurotensin families was examined by competition experiments of tritiated neurotensin hydrolysis as well as HPLC analysis. These results indicated that endopeptidase 24.16 could discriminate between peptides belonging to the same family. Neurotensin, Lys8‐Asn9‐neurotensin(8–13) and xenopsin were efficiently hydrolysed while neuromedin N and kinetensin underwent little if any proteolysis by the peptidase. Analogously, substance P and dynorphins (1–7) and (1–8) were readily proteolysed by endopeptidase 24.16 while neurokinin A, amphibian tachykinins and leucine or methionine enkephalins totally resisted degradation.By Triton X‐114 phase separation, 15–20% of endopeptidase 24.16 partitioned in the detergent phase, indicating that renal endopeptidase 24.16 might exist in a genuine membrane‐bound form. The equipotent solubilization of the enzyme by seven detergents of various critical miscellar concentrations confirmed the occurrence of a membrane‐bound counterpart of endopeptidase 24.16. Furthermore, the absence of release elicited by phosphatidylinositol‐specific phospholipase C suggested that the enzyme was not attached by a glycosyl‐phosphatidylinositol anchor in the membrane of renal microvilli. Finally, endopeptidase 24.16 could not be released from these membranes upon trypsinolysis.
Interaction of staphylokinase with different molecular forms of plasminogen
Authors: LIJNEN H. Roger , VAN HOEF Berthe , COLLEN Désiré 
Published: March 2005

In order to obtain more information on the mechanism of plasminogen activation by staphylokinase (STA), we have studied the interaction between recombinant STA (STAR) and different molecular forms of human plasminogen, including Glu‐plasminogen (native moiety), Lys‐plasminogen (partially degraded moiety) and low‐molecular‐mass (LMM) plasminogen (moiety lacking kringles 1–4).Addition of 2 μM STAR to 0.4 μM Glu‐plasminogen, Lys‐plasminogen or LMM plasminogen resulted in the generation of proteolytic activity towards the chromogenic substrate D‐Val‐Leu‐Lys‐NH‐PhNO2 (S‐2251) corresponding to the exposure of 1 active center/plasminogen molecule. Complex formation was associated with conversion of the one‐chain plasminogen moieties to two‐chain plasmin, and with quantitative conversion of Glu‐plasminogen to Lys‐plasmin. The stoichiometry of the plasminogen‐STAR complex, determined by binding of the complex to Lys‐Sepharose and measurement of residual STAR, was found to be equimolar. The plasminogen‐STAR complexes were inhibited by α2‐antiplasmin with second‐order rate constants of 2.4 ± 0.17 × 106 M−1 s−1 for Glu‐plasminogen, 2.4 ± 0.21 × 106 M−1 s−1 for Lys‐plasminogen and 9.4 ± 1.5 × 104 M−1 s−1 for LMM plasminogen. Glu‐plasmin‐STAR, Lys‐plasmin‐STAR and LMM plasmin‐STAR had comparable catalytic efficiencies (kcat/Km) for the activation of Glu‐plasminogen (0.24–0.29 μM−1 s−1), Lys‐plasminogen (0.57–0.79 μM−1 s−1) or LMM plasminogen (0.11–0.16 μM−1 s−1).In a human plasma milieu in vitro STAR, Glu‐plasmin‐STAR, Lys‐plasmin‐STAR and LMM‐plasmin‐STAR were equally effective for the lysis of 125I‐fibrin‐labeled human plasma clots [50% clot lysis in 2 h (EC50) with 11–13 nM test compound] and equally fibrin‐selective (residual fibrinogen levels of 72–84% after 2 h at EC50).Our results thus confirm that plasminogen and STAR form a 1:1 stoichiometric complex in which plasminogen is converted to plasmin and Glu‐plasminogen to Lys‐plasmin. The lysine‐binding sites in kringles 1–4 of plasminogen are not required for the complex formation with STAR, nor for the enzyme activity of the complex with STAR in purified systems and in a human plasma milieu. The lysine‐binding sites are, however, important for the rate of the inhibition of the complexes by α2‐antiplasmin.
The reaction mechanism of Ca2+‐ATPase of sarcoplasmic reticulum
Authors: LACAPERE Jean‐Jacques , GUILLAIN Florent 
Published: March 2005

Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5°C, the association and dissociation rate constants of Mg · ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 μM calcium and 5 mM MgCl2 (3–4x 106 M−1· s−1 and 9 s−1 respectively). Therefore, we have determined the true affinity for Mg · ATP (Kd= 3 μM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg · ATP binding is the rate limiting step in the phosphorylation process, and Mg · ATP dissociation is slower than dephosphorylation.The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two‐step mechanism, both steps being accelerated by Mg · ATP. Combining rapid filtration and rapid monitoring of the intrinsic fluorescence of SR Ca2+ ‐ATPase, we showed that rate constants for calcium binding are always lower than those of Mg · ATP binding to an EGTA‐incubated enzyme. We measured dissociation and association rate constants of Mg · ATP binding in the absence of calcium (k‐1= 25 s−1 and k1= 7.5 106 M−1· s−1). This gives a Kd similar to that obtained by equilibrium measurements (3–4 μM).Both non‐phosphorylated conformations of the enzyme have similar affinity for Mg · ATP. Therefore, activation of ATPase activity by an excess of ATP cannot be explained by a change in affinity of the non‐phophorylated enzyme for Mg · ATP. In conjunction with previous results, these data are used to discuss the molecular mechanism for the Ca2+‐ATPase cycle, in which ATP is sequentially substrate and activator on a multiple‐function single site.
High‐resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor
Authors: PAPADOULI Irene , SAKARELLOS Constantinos , TZARTOS Socrates J. 
Published: March 2005

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti‐MIR monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67 – 76 of the AChR α‐subunit. A thorough study of the epitope(s) for anti‐MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data.Use of all possible overlapping hexapeptides within Torpedo and human α40 – 91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide α67 – 71. Systematic screening of peptide analogues, where each amino acid residue within α67 – 76 and α67 – 74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti‐MIR mAb binding, whereas Pro69 and Ala/Asp70 were less but still significantly important. mAb binding to α67 – 76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of α76 or combinations of two substitutions at α73 and α76 were tested. Several of these analogues (mainly His76, Arg76, Val73Ala76, His73Ala76, Val73Arg76) exhibited dramatic mAb binding enhancement. Some anti‐MIR mAbs that do not bind to α67 – 76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.
Modulation of the Intracellular Ca2+‐dependent Proteolytic System is Critically Correlated with the Kinetics of Differentiation of Murine Erythroleukemia Cells
Authors: Sparatore Bianca , Passalacqua Mario , Pessino Anna , Melloni Edon , Patrone Mauro , Pontremoli Sandro 
Published: March 2005

Calpain has been identified as the intracellular proteinase that catalyzes the selective down‐regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti‐calpain antibody resulted in a significant delay in the onset of down‐regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast‐responding clone, calpastatin, the natural calpain‐inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast‐responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti‐calpain antibody.It is proposed that Ca2+‐dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.
Purification and Characterization of the Thyrotropin‐releasing‐hormone‐degrading Ectoenzyme
Authors: Bauer Karl 
Published: March 2005

The membrane‐bound enzyme which catalyzes the degradation of thyrotropin‐releasing hormone (TRH; Glp‐His‐Pro‐NH2) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200000‐fold, with an overall yield of 20%, by conventional chromatographic methods.The enzyme preparation appeared to be electrophoretically homogenous since SDS/PAGE analysis revealed a single band with a molecular mass of 116000 Da. By gel‐filtration chromatography, a molecular mass of 230000 Da was estimated, suggesting that the enzyme consists of two identical subunits.The enzyme could be identified as a glycoprotein by lectin‐binding analysis and by the reduction of the molecular mass to 97000 Da upon treatment of the denatured enzyme with endoglycosidase‐F/N‐glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymic activity.In addition to TRH, the enzyme also hydrolyzed l‐5‐oxoprolyl‐β‐naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis‐Menten kinetics, but considerable differences in the respective Km and Vmax values were noticed.