bluuebunny/crossref_metadata_2025_split huggingface dataset:
A family of bombinin‐related peptides from the skin of Bombina variegata
Authors: SIMMACO Maurizio , BARRA Donatella , CHIARINI Fernanda , NOVIELLO Lia , MELCHIORRI Pietro , KREIL Günther , RICHTER Klaus 
Published: March 2005

A peptide fraction was isolated from the skin of Bombina variegata that showed antimicrobial activity. This fraction contained several molecular species, all of them consisting of 27 amino acid residues, with a constant C‐terminal region (from residues 14–27), including an amidated carboxyl end and a variable N‐terminal segment. These peptides are related but not identical to bombinin [Csordas, A. & Michl, H. (1970) Monatsh. Chem. 101, 182–189].By using synthetic oligonucleotides corresponding to the C‐terminal region of the peptides, a cDNA library from the skin of B. variegata was screened and several positive clones coding for the corresponding peptide precursors were isolated and sequenced. Each clone contained the genetic information for a different bombinin‐like peptide. The antimicrobial activity towards different bacterial species of a synthetic peptide corresponding to one of the variants deduced from cDNA sequences was tested. This peptide was found to be mainly active against different isolates of Staphylococci and Escherichia coli.
Effect of the cobalt‐N coordination on the cobamide recognition by the human vitamin B12 binding proteins intrinsic factor, transcobalamin and haptocorrin
Authors: STUPPERICH Erhard , NEXØ Ebba 
Published: March 2005

The binding of several corrinoids to the binding site of human intrinsic factor, transcobalamin or haptocorrin was investigated. p‐Cresolyl cobamide and 2‐amino‐vitamin B12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were ≥ 103 times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B12, which contains a Co‐coordinated nucleotide. Pseudovitamin B12, with a weak Co‐N coordination bond, revealed only moderate affinity to intrinsic factor. From these findings it is concluded that the cobamide binding to intrinsic factor and transcobalamin is strongly affected by the Co‐N coordination bonds of their lower cobalt nucleotide ligands. We suggest that the Co‐N coordination bond positions the nucleotide at a critical distance to the corrin ring, which is recognized by the binding proteins.Human haptocorrin, however, disclosed to distinctive selectivity regarding the different corrinoid structures. The protein bound all corrinoids with similar efficiency, independent of the strength of their Co‐N coordinations, or the structures of their lower Coα ligands. Hence, the corrin ring, rather than a structural feature induced by the Co‐N coordination, has to be considered responsible for the corrinoid binding to haptocorrin.
Peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1 → 3 to the asparagine‐linked N‐acetylglucosamine residue
Authors: TRETTER Verena , ALTMANN Friedrich , MÄRZ Leopold 
Published: March 2005

The ability of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N‐glycopeptides and N‐glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase‐C glycoprotein, which contain xylose linked β1 → 2 to β‐mannose and fucose linked α1 → 3 to the innermost N‐acetylglucosamine, were used as substrates.In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100‐fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1 → 3‐linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations.Hence we conclude that α1 → 3 fucosylation of the inner N‐acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.
The plasma membrane H+ ‐ATPase from yeast
Authors: WACH Achim , GRÄBER Peter 
Published: March 2005

The H+ ‐ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated.The pH dependence of the rate of ATP hydrolysis forms a bell‐shaped curve with a maximum at pH 6 and half‐maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding.Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.
The 5′ untranslated regions of acetyl‐coenzyme A carboxylase mRNA provide specific translational control in vitro
Authors: LÓPEZ‐CASILLAS Fernando , KIM Ki‐Han 
Published: March 2005

Acetyl‐coenzyme A carboxylase (ACC) catalyzes the rate‐limiting step in the biosynthesis of long‐chain fatty acids. Transcription of the single‐copy ACC gene from two independent promoters, together with the differential splicing of the transcripts, gives rise to mature ACC mRNA having the same open reading frame (ORF), but exhibiting heterogeneity in their 5′ untranslated region (5′‐UTR). Class 1 ACC mRNA are transcribed from the inducible promoter 1 and their 5′‐end leading sequences are provided by exon 1. Class 2 ACC mRNA are transcribed from the constitutively expressing promoter 2 and their leading sequences are derived from exon 2.In order to understand the role of different 5′ UTR of ACC mRNA we have synthesized in vitro transcripts with defined ACC mRNA 5′ UTR and examined their relative translational efficiencies in rabbit reticulocyte lysates. The major translation product of both forms of ACC mRNA was initiated at the first AUG of the ORF. Class 1 transcripts had a 6–9‐fold better translational efficiency than class 2 transcripts, based on the quantity of major peptide produced by a given amount of transcript. The poor translational efficiency of class 2 transcripts can be improved by the removal of sequences contributed by exon 2, suggesting that they play an inhibitory role in the translation of class 2 types of ACC mRNA. In addition to their higher translational efficiency, the class 1 transcripts can also initiate translation at in‐frame non‐AUG codons, located in exon 1, i.e. upstream to the starting AUG of the common ACC mRNA ORF. This results in novel ACC peptides with extended N termini. These observations are consistent with the hypothesis that the 5′ UTR heterogeneity in the ACC mRNA may be involved in post‐transcriptional control, at the level of translation, of the ACC gene expression.
Molecular symmetry and arrangement of subunits in extracellular hemoglobin from the nematode Ascaris suum
Authors: DARAWSHE Saleh , DANIEL Ezra 
Published: March 2005

The arrangement of subunits and molecular symmetry of extracellular hemoglobin from the nematode Ascaris suum, an 11.7S molecule mass 332 kDa and composed of eight identical subunits, was studied.Dissociation of the molecule at alkaline and acid pH yielded 4.6S and 2.7S components, identified as polypeptide‐chain dimers and monomers, respectively. Cross‐linking with glutardialdehyde followed by SDS/PAGE resulted in a maximum number of eight bands identified in order of decreasing mobility as monomeric and 2–8 cross‐linked‐polypeptide‐chain species. Comparison with values predicted from theory shows that the distribution of protein among the various cross‐linked species, obtained after different extents of exposure to cross‐linker, is consistent with a two‐layered arrangement of subunits involving one type of interaction between subunits from different layers and another between subunits within the same layer. Electron micrographs of the molecule showed two profiles, a square and a rectangle. We propose a model for the molecule which is eight subunits arranged in two layers, stacked in an eclipsed orientation. The proposed model is consistent with the results from sedimentation, cross‐linking and electron microscopy. Taken together, our findings indicate D4 symmetry for Ascaris hemoglobin.
Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium‐transport enzyme
Authors: RONZANI Nelly , STEPHAN Lore , HASSELBACH Wilhelm 
Published: March 2005

The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium‐transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding.The vanadate‐free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7–14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure‐binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure‐binding relationship indicates that the description of vanadate binding requires at least a two‐step reaction sequence. The volume increments which predominate at lower pressure values, range from 200–400 ml · mol−1 depending on the composition of the reaction medium containig 5 μM and 20 μM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20–40 ml · mol−1. Vanadate binding is reduced in the presence of 30 μM calcium, and simultaneously both binding volumes are diminished by 100 ml · mol−1 and 20 ml · mol−1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.
Effect of N‐glycan removal on the enzymatic activity of porcine thyroid peroxidase
Authors: LONG Yannick , FRANC Jean‐Louis , KANIEWSKI Jacques , LANET Jeanne , GIRAUD Annie 
Published: March 2005

Active porcine thyroid peroxidase (pTPO) has been purified either by deoxycholate extraction followed by immunoaffinity purification (pTPO A) or by trypsin/digitonin extraction followed by ion‐exchange and gelfiltration chromatography (pTPO B); pTPO A appeared as a full‐length molecule, while pTPO B appeared as peptide fragments. Purified pTPO were deglycosylated either by peptide N‐glycosidase F (PNGase F) or by endo‐β‐N‐acetylglucosaminidase H (endo H) treatment. Electrophoretic controls and affinity blotting with concanavalin A indicated that deglycosylation was not total and that pTPO was more efficiently deglycosylated by endo H than by PNGase F.The enzymatic activity of pTPO A, checked by guaiacol and iodide oxidation, was inhibited by PNGase F and endo H deglycosylation, while that of pTPO B was not. After deglycosylation, the apparent Km of pTPO A for guaiacol and iodide increased, while the Vmax for both substrates decreased. The state of aggregation of pTPO A before and after deglycosylation was checked by sucrose density‐gradient centrifugation. Results indicated that this inhibition was not due to a loss of pTPO A solubility. These observations suggest that deglycosylation induced a modification of the tertiary structure of pTPO A which affected the active‐site domain of the enzyme.
Protein stability and molecular adaptation to extreme conditons
Authors: JAENICKE Rainer 
Published: March 2005

Proteins, due to the delicate balance of stabilizing and destabilizing interactions, are only marginally stable. Adaptation to extreme environments tends to shift the ‘mesophilic’ characteristics of proteins to the respective extremes of temperature, hydrostatic pressure, pH and salinity, such that, under the mutual physiological conditions, the molecular properties are similar regarding overall topology, flexibility and solvation. Enhanced intrinsic stability requires only minute local structural changes so that general strategies of stabilization cannot be established. Apart from mutative changes of amino‐acid sequences, extrinsic factors (or cellular components) may be involved in ‘extremophilic adaptation’.The molecular basis of acidophilic, alkalophilic and barophilic adaptation is still obscure. Mechanisms of enhanced thermal stability involve improved packing density, as well as specific local interactions. In halophiles, water and salt binding of the intrinsically stable protein inventory is accomplished by favoring acidic over basic amino acid residues and decreased hydrophobicity.General limits of viability are: (a) the susceptibility of the covalent structure of the polypeptide chain toward hydrolysis or hydrothermal degradation; (b) the competition of extreme solvent parameters with the weak electrostatic and hydrophobic interactions involved in protein stabilization; (c) perturbations of the folding and assembly of proteins; and (d) ‘dislocation’ of biochemical pathways due to effects of extreme conditions on the intricate network of metabolic reactions.
Site‐directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: SCHULZE Egbert , WESTPHAL Adrie H. , BOUMANS Hans , de KOK Arie 
Published: March 2005

Site‐directed mutagenesis was performed in the protease‐sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex.Upon binding of peripheral components, the 24‐subunit core of A. vinelandii wild‐type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers.Large deletions of the protease‐sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (Δ P361‐R362) or the point mutation K367Q in the protease‐sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N‐terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli.Deletion of the complete Ala + Pro‐rich sequence between the protease‐sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N‐terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild‐type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes.It is concluded that the binding site for E3 is located on the N‐terminal part of the protease‐sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease‐sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.
Synthesis and evaluation of (+) and (−)‐2,2‐difluorocitrate as inhibitors of rat‐liver ATP‐citrate lyase and porcine‐heart aconitase
Authors: SAXTY Barbara A. , NOVELLI Riccardo , DOLLE Roland E. , KRUSE Lawrence I. , REID David G. , CAMILLERI Patrick , WELLS Timothy N. C. 
Published: March 2005

The enantiomers (+) and (−)‐2,2‐difluorocitrate have been synthesized. Both are good inhibitors of ATP‐citrate lyase, showing competitive inhibition against citrate, with Kis= 0.7 μM for (+)‐2,2‐difluorocitrate and 3.2 μM for (−)‐2,2‐difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon‐carbon bond cleavage as a substrate and there is no evidence of irreversible time‐dependent inactivation. When ATP‐citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate‐induced rate for the (+)‐enantiomer and 2% for the (−)‐enantiomer. 19F‐NMR studies confirm that only the (+)‐enantiomer is chemically processed.The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (−)‐2,2‐Difluorocitrate is a competitive inhibitor against citrate (Kis= 1.5 μM), whereas the (+)‐enantiomer is a relatively poor mixed inhibitor (Ki > 300 μM). The (−)‐enantiomer irreversibly inactivates aconitase at 1.1 min‐1· mM‐1 at 25°C and pH 7.4, whereas no irreversible inhibition is seen with the (+)‐enantiomer. Therefore, it would be expected that the (+)‐enantiomer would slow the rate of acetyl‐CoA synthesis in vivo, without inhibiting the citric acid cycle.
Combined glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase in catecholamine‐stimulated guinea‐pig cardiac muscle
Authors: BÜNGER Rolf , MUKOHARA Naoki , KANG Young‐Hee , MALLET Robert T. 
Published: March 2005

The steady‐state reactant levels of triose‐phosphate isomerase and the glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea‐pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3‐phosphate were directly measured and compared with those of creatine kinase. Nonworking Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near‐equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP]·[Pi]) was derived from creatine kinase and the free [NAD+]/(NADH]·[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near‐zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose‐phosphate isomerase was near‐equilibrium only in control or potassium‐arrested Langendorff hearts as well as in postischemic ‘stunned’ hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure‐volume work the enzyme was displaced from equilibrium.The alternative phosphorylation ratio [ATP]‘/([ADP]’·[Pi]) was derived from the magnesium‐dependent glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1–2.0 mM). As predicted, [ATP]/([ADP]·[Pi]) and [ATP]'/([ADP]'·[Pi]') were in excellent aggreement when glycolysis was virtually halted by hyperkalemic arrest (flux ∼ 0.2 μmol C3· min‐1· g dry mass‐1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux ∼ 5–14 μmol C3· min‐1· g dry mass‐1). In contrast, when flux increased due to transition from no‐work to physiological pressure‐volume work (rate increase from ∼ 3 to 11 μmol C3· min‐1· g dry mass‐1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial ‘stunning,’ not due to hydraulic work load per se, glyceraldehyde‐3‐phosphate levels increased from about 4 μM to > 16 μM. Thus the guinea‐pig cardiac glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near‐equilibrium catalysis at significant flux provided glyceraldehyde‐3‐phosphate levels rise, e.g., due to ‘stunning’ or adrenergic hormones.
Transcriptional and post‐transcriptional effects of glucose on liver phosphoenolpyruvate‐carboxykinase gene expression
Authors: MEYER Silke , HÖPPNER Wolfgang , SEITZ Hans J. 
Published: March 2005

The present stdy investigates the effect fo glucose on the gene expression of the hepatic glucoregulatory enzyme, phosphoenolpyruvate carboxykinase (PPrvck). By use of hepatocytes in culture and FAO hepatoma cells it could be demonstrated that glucose suppressed the effect of dibutyrul cyclic AMP (Bt2cAMP), glucocorticoids or both, to increase PPrvck mRNA and consequently PPrvck enzyme activity. Glucose had a dual effect; it reduced PPrvck gene transcription and it accelerated the rate of Pprvck mRNA degradation.The effect wa sspecific for glucose, as glucose‐related carbohydrates such as manose, galactose and sorbitol were without effect on PPrvck mRNA. The repressive effect of glucose was limited to certain proteins; glucose had no effect on Bt2cAMP and glucocorticoid provoked induction of tyrosine aminotransferase (TAT). Also the pattern of mRNA in vitro translation products was virtually unaffected when FAO hepatoma cells were incubated either in the presence or absence of glucose, demonstrating gthe specificity of the effect of glucose on gene expression of selected proteins.In FAO hepatoma cells and in hepatocytes in culture, insulin, like glucose, also decreased PPrvck mRNA. While the effect of glucose and insulin was additive in FAO hepatoma cells, in primary hepatocytes in culture an effect of glucose by itself on PPrvck mRNA could only be demonstrated in the absence of insulin. Correspondingly also in vivo, the effect of glucose was demonstrated in the absence of insulin (provoked by streptozotocin diabetes); glucose application reduced the amount of hepatic PPrvck mRNA.To summarize, glucose is capable of suppressing the effect of glucocorticoids and Bt2cAMP on increasing the PPrvck mRNA level. The carbohydrate reduces the rate of PPrvck geen transcription and accelerates the rate of PPrvck mRNA degradation. While in FAO hepatoma cells the effect is evident in the presence of insulin, in hepatocytes in culture the effect of glucose cannot be demonstrated in the presence of insulin, questioning its role under physiological conditions.