bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chimeras of Parvalbumin and Oncomodulin Involving Exchange of the Complete CD Site Show that the Ca2+/Mg2+ Specificity is an Intrinsic Property of the Site
Authors: Durussel Isabelle , Pauls Thomas L. , Cox Jos A. , Berchtold Martin W. 
Published: July 2004

Rat parvalbumin (PV) and oncomodulin (OM) differ in the affinity and selectivity of metal binding to their CD site, which is a high‐affinity Ca2+/Mg2+‐mixed site in PV and a low‐affinity Ca2+‐specific site in OM. To assess to what degree the Ca2+/Mg2+ specificity and affinity of an EF‐hand motif in a protein is intrinsically determined by its sequence, the complete CD sites were exchanged, yielding two chimeras, [S41‐Q71]PV and [D41‐S71]OM. The optical characteristics of a Trp102, inserted in the hydrophobic core of PV, OM and the two chimeras, are very similar in all four proteins, which suggests that the hydrophobic core is qualitatively similar in the chimeras as in the parent proteins. Direct Ca2+ and Mg2+ binding monitored by flow dialysis and gel filtration revealed that [S41‐Q71]PV binds only one Mg2+ with an intrinsic affinity K′Mg2+ of 3.0×104 M−1 and two Ca2+ with an identical K′Ca2+ of 4.4×106 M−1, whereas [D41–S71]OM binds two Mg2+ with a mean K′Mg2+ of 2×104 M−1 and two Ca2+ with a K′Ca2+ of 1.3×104 M−1. K′Ca2+ of the CD site of [S41–Q71]PV was 2.5‐fold higher than of the CD site in [W102]OM, but 5–6‐fold lower than that of the CD site in [W102]PV. In [D41–S71]OM, K′Ca2+ of the CD site was twofold lower than in [W102]PV, but eightfold higher than in [W102]OM. These results indicate that the sequence of the CD site determines its Ca2+/Mg2+‐specificity, whereas its affinity for Ca2+ influenced by the protein into which the CD site is inserted. The inserted CD site in turn influences the affinity of the EF site to which it is paired in the host protein and the paired sites display an equalized affinity for Ca2+.Mg2+ decreases the affinity of the chimeras for Ca2+, but not according to a simple competition model. The Mg2+ antagonism is much more pronounced in [D41–S71]OM than in [S41–Q71]PV, but in each chimera the CD and EF site are quantitatively affected in the same manner. Thus, [S41–Q71]PV which can only bind a single Mg2+ ion, displays a Ca2+/Mg2+‐antagonism for both sites with a K′Mg · compet of 2.3×102 M−1. These results confirm the ‘equalizer’ principle in the cation‐binding parameters of [S41–Q71]PV: both sites display the same Ca2+ affinity and Mg2+ antagonism. In [D41–S71]OM with its two Ca2+/Mg2+ sites the antagonism shows qualitatively the same complexity as in wild‐type PV, although it is somewhat weaker in amplitude.
Phosphorylation of Myeloid‐Related Proteins MRP‐14 and MRP‐8 During Human Neutrophil Activation
Authors: Guignard Florence , Mauel Jacques , Markert Michèle 
Published: July 2004

The myeloid‐related proteins MRP‐14 and MRP‐8 and also p6, three calcium‐binding proteins of the S100 family, translocate to the membrane during human neutrophil activation with stimuli known to require extracellular calcium for activity. When phorbol 12‐myristate 13‐acetate (PMA, an extracellular calcium‐independent stimulus) is used, no translocation is observed. To characterize further the mechanisms involved in their translocation, phosphorylation of these proteins was studied.Three isoforms of MRP‐14 were markedly phosphorylated in the membrane and in the cytosol upon activation with extracellular calcium‐dependent stimuli, such as opsonized zymosan, the calcium ionophore A23187, N ‐formylmethionylleucylphenylalanine in the presence of cytochalasin B and arachidonic acid, or upon extracellular calcium‐independent stimulation (PMA). In no case were p6 and a fourth, more basic isoform of MRP‐14, phosphorylated. In PMA‐activated cells, a phosphorylated acidic isoform of MRP‐8 was detected in the cytosol only. However, phosphorylated MRP‐8 represented only a small fraction of total MRP‐8. Cgp 41251, an inhibitor of protein kinase C (PKC), completely inhibited the phosphorylation of MRP‐8, and decreased cytosolic MRP‐14 phosphorylation. To test whether phosphorylated MRP‐8 could translocate, A23187, which induces translocation of the three S100 proteins, was added after PMA activation. This resulted in translocation of 18%±5% of phosphorylated MRP‐14 and 19%±1% of only nonphosphorylated MRP‐8. However, upon inhibition of PKC, translocation of MRP‐14 and MRP‐8 was increased up to 38%±7% and 34%±3% respectively. This suggests a putative role of phosphorylation and/or of PKC in the modulation of MRP‐14 and MRP‐8 translocation to the membrane.
Rat Pristanoyl‐CoA Oxidase
Authors: Vanhooren Johannes C. T. , Fransen Marc , De Béthune Bernadette , Baumgart Eveline , Baes Myriam , Torrekens Sophie , Van Leuven Fred , Mannaerts Guy P. , Van Veldhoven Paul P. 
Published: July 2004

The composite pristanoyl‐CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5′‐RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS‐gel electrophoresis.The amino acid identity with rat palmitoyl‐CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl‐CoA oxidases (20%), suggesting that the palmitoyl‐CoA oxidase/pristanoyl‐CoA oxidase duplication occurred early in evolution.The carboxy‐terminal tripeptide of pristanoyl‐CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal‐targeting signal‐1 import receptor indicated that SQL functions as a peroxisome‐targeting signal.Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl‐CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl‐CoA oxidase gene, if present, is only poorly or not transcribed.
Characterisation of a Soluble Cytochrome c4 Isolated from Thiobacillus ferrooxidans
Authors: Cavazza Christine , Giudici‐Orticoni Marie‐Thérèse , Nitschke Wolfgang , Appia Corinne , Bonnefoy Violaine , Bruschi Mireille 
Published: July 2004

A soluble c‐type cytochrome was purified to homogeneity from Thiobacillus ferrooxidans. This cytochrome is characterised by an α‐peak wavelength of 552 nm, a molecular mass of 21193 Da (as determined by mass spectroscopy), and a pI value of 9. N‐terminal sequencing yielded the polypeptide sequence up to the 50th residue. The iron content of 1.9 Fe/molecule and the heme/molecule ratio of 2.15 identified this cytochrome as a diheme protein. Optical redox titrations at pH 3.0 revealed the presence of two distinguishable redox species with Em= 385 mV±20 mV and Em= 480 mV±20 mV. EPR spectra recorded on this heme protein showed the presence of two distinct spectral species with gz= 3.1 and gz= 3.35. The gz= 3.35 heme corresponds to the higher potential redox species. In line with the differences in Em values, the two heme species were oxidised by O2 with significantly differing half‐times.All the above mentioned properties demonstrate that this heme protein belongs to the c4 family of diheme cytochromes. The characteristics and functional role of the studied heme protein are discussed with reference to other c‐type cytochromes described in Thiobacilli. Its properties are furthermore compared to other members of the cytochrome c4 family.
Reexamination of Hormone‐Binding Properties of Protein Disulfide‐Isomerase
Authors: Guthapfel Régine , Gueguen Paul , Quemeneur Eric 
Published: July 2004

Protein disulfide‐isomerase (PDI), an abundant multifunctional protein, has been described as a 3,3′,5‐triiodo‐l‐thyronine (T3)‐binding protein. As pointed out by several authors, the physiological significance of this hormone‐binding property has not been fully addressed. To clarify this point, we have analyzed the T3‐binding properties of purified PDI.At equilibrium, T3 binds PDI at two binding sites: first, at a high‐affinity site with a Kd of 21 nM and a Bmax of 1.8×10−3, mol T3/mol PDI monomer, and second at a very low affinity site that is unsaturated up to 100 μM T3. Thus, T3 binding is mainly non‐specific and the specific part represents only about 0.2% of the protein monomer. Cross‐linking experiments at a concentration where mainly specific binding occurs indicate that PDI does not bind l‐T3 exclusively; a wide variety of analogs are also bound. Refolding of reduced denatured ribonuclease A by PDI is inhibited by T3 and analogs, and the inhibition profile reflects the binding properties very closely.Since purified PDI displays neither the specificity expected for a physiological receptor, nor significant T3‐binding activity, results are discussed in terms of a necessary PDI association with another component to form a T3 receptor.
Interaction of Amino Acid Residues at Positions 8–15 of Secretin with the N‐Terminal Domain of the Secretin Receptor
Authors: Gourlet Philippe , Vilardaga Jean‐Pierre , De Neef Philippe , Vandermeers André , Waelbroeck Magali , Bollen Alex , Robberecht Patrick 
Published: July 2004

The ability of secretin, PACAP‐(1–27)‐peptide, and ten hybrid peptides to recognize and activate the rat secretin and vasoactive intestinal polypeptide (PACAP type II VIP1) receptors was tested on recombinant Chinese hamster ovary (CHO) cell lines. PACAP had a 2500‐fold lower affinity than secretin for the secretin receptor, and secretin had a 300‐fold lower affinity than PACAP for the VIP1 receptor. Amino acids 8, 13, and 15 of the PACAP molecule contributed significantly to the low affinity of PACAP for the secretin receptor. The amino acids at positions 5, 9, 10, 15, 16, and unidentified amino acid(s) between positions 17–20 made limited contributions to the low affinity of secretin for the VIP1 receptor.To identify the receptor region that interacts with these amino acids, we constructed chimeric receptors, which consist either of the N‐terminal extracellular part of the secretin receptor and the core of the VIP1 receptor (N‐Sn/VIP1r) or the N‐terminal extracellular part of the VIP1 receptor and the core of the secretin receptor (N‐VIP1/Snr), and tested the ability of the hybrid ligands to activate the adenylate cyclase of CHO cells expressing these chimeric receptors.The N‐Sn/VIP1 receptors had a higher affinity for secretin than for PACAP. The hybrid peptide 6 that consists of the PACAP‐(1–8)‐Sn‐(9–15)‐PACAP‐(16–27)‐peptide sequence had a 30‐fold to 200‐fold higher potency than either parent peptide for the chimeric receptor, which suggests that while the N‐ and/or C‐terminal part of the peptide interact with the transmembrane domain of the receptor, the discriminator region 9–15 recognizes the extracellular N‐terminal domain of the receptor. This was confirmed by the observation that, out of all the peptides tested, hybrid 6 had the weakest potency for activation of the N‐VIP1/Sn chimeric receptors.
Photomodification of Mitochondrial Proteins by Azido Fatty Acids and Its Effect on Mitochondrial Energetics
Authors: Schönfeld Peter , Ježek Petr , Belyaeva Elena A. , Borecký Jiří , Slyshenkov Vyacheslav S. , Więckowski Mariusz R. , Wojtczak Lech 
Published: July 2004

Azido derivatives of long‐chain fatty acids, 12‐(4‐azido‐2‐nitrophenylamino)dodecanoic acid (N3‐NpNH‐Lau) and 16‐(4‐azido‐2‐nitrophenylamino)hexadecanoic acid (N3‐NpNH‐Pam), were used to study the mechanism of the protonophoric function of long‐chain fatty acids in mitochondrial membranes. N3‐NpNH‐Lau was found to increase resting‐state respiration and decrease the membrane potential in a dose‐dependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N3‐NpNH‐Lau as well as of the myristate were reversed or prevented by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carboxyatractyloside. This protective effect of carboxyatractyloside was well expressed in rat heart mitochondria and less so in mitochondria within digitonin‐permeabilized Ehrlich ascites tumour cells. Photomodification of Ehrlich ascites tumour mitochondria by ultraviolet irradiation in the presence of N3‐NpNH‐Lau made them more resistant to the uncoupling effect of myristate, and photomodification of rat heart mitochondria resulted in a strong inhibition of AAC which could not be reversed by serum albumin.Photolabelling of rat heart mitochondria with tritiated N3‐NpNH‐Pam revealed around 10 labelled bands on SDS/polyacrylamide gel electrophoresis. Based on immunodetection with a specific antibody, one of them, corresponding to 30 kDa, was identified as AAC. Specific interaction of AAC with azido fatty acids was confirmed by a high radiolabelling of this band.The role of fatty acids in fine control of the efficiency of oxidative phosphorylation is discussed.
The Lyn‐Catalyzed Tyr Phosphorylation of the Transmembrane Band‐3 Protein of Human Erythrocytes
Authors: Brunati Anna Maria , Bordin Luciana , Clari Giulio , Moret Vittorio 
Published: July 2004

Band‐3 protein (approximately 95 kDa), the major and multifunctional transmembrane protein of human erythrocytes, has been shown to be phosphorylated by endogenous Tyr‐protein kinases on different Tyr residues at its N and C cytoplasmic domains.Both the added p36syk (catalytic domain of 72syk) and Lyn kinases are able to phosphorylate the isolated cytoplasmic domain of band 3 (cdb3), yielded by chymotryptic digestion of band 3 in the isolated membranes (ghosts).However, the two Tyr‐protein kinases exhibited different phosphorylation behaviours when added to the isolated erythrocyte membranes. More precisely, the added p36syk markedly Tyr phosphorylates the band‐3 protein, whereas the added Lyn phosphorylates it very poorly.It is of interest that Lyn can associate with membranes and markedly phosphorylate band 3 when this latter protein has been previously phosphorylated by p36syk, i.e. the p36syk ‐catalyzed phosphorylation is proposed to be a prerequisite for the association of Lyn with the membrane (likely to band 3) and for the Lyn‐catalyzed phosphorylation of different band‐3 Tyr sites.
Purification and Characterisation of Haemolymph 3‐Dehydroecdysone 3β‐Reductase in Relation to Ecdysteroid Biosynthesis in the Cotton Leafworm Spodoptera littoralis
Authors: Chen Jian‐Hua , Webb Tracey J. , Powls Roy , Rees Huw H. 
Published: July 2004

The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littoralis was detected and analysed by HPLC‐RIA. The primary product was identified as 3‐dehydroecdysone (≈ 82%), with lesser amounts of ecdysone (≈ 18%). Interconversion of ecdysone and 3‐dehydroecdysone by prothoracic glands was not detectable.3‐Dehydroecdysone 3β‐reductase activity was demonstrated in the haemolymph. Ecdysone, the end‐product, was characterised by reverse‐phase and adsorption HPLC, chemical transformation into ecdysone 2, 3‐acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and Km values for NADPH and NADH were determined to be 0.94 μM and 22.8 μM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3‐dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (>5 μM). The results suggest that the 3‐dehydroecdysone 3β‐reductase has a high‐affinity (low Km) binding site for 3‐dehydroecdysone substrate, together with a lower‐affinity inhibition site.The 3β‐reductase enzyme was purified to homogeneity using a combination of poly(ethylene glycol) 6000 precipitation and successive FPLC fractionation on Mono‐Q, phenyl Superose (twice), and hydroxylapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDS/PAGE and gel‐filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.
Immunological Demonstration of Protein Kinase Cμ in Murine Tissues and Various Cell Lines
Authors: Rennecke Jörg , Johannes Franz‐Josef , Richter Karl Hartmut , Kittstein Walter , Marks Friedrich , Gschwendt Michael 
Published: July 2004

13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme μ. Using two different PKCμ antibodies (sc‐639 and P26720), PKCμ was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKCμ was recognized only by the antibody P26720 and not by sc‐639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKCμ.Recognition of PKCμ by the antibody sc‐639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), thus mimicking down‐regulation of PKCμ. The lack of a decrease in the PKCμ amount and, thus, the lack of down‐regulation could be proved using the antibody P26720. This antibody was able to recognize PKCμ in extracts of untreated as well as TPA‐treated tissues or cells. Phosphorylation of proteins in a cell‐free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKCμ caused its reduced interaction with the antibody sc‐639. Therefore, this antibody might present a well suited tool for the detection of activated PKCμin vivo. Moreover, our results clearly show that some antibodies, such as sc‐639, might be able to selectively detect non‐phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKCμ.
The O‐Specific Polysaccharide Chain of Campylobacter fetus Serotype B Lipopolysaccharide is a d‐Rhamnan Terminated with 3‐O ‐Methyl‐d‐Rhamnose (d‐Acofriose)
Authors: Senchenkova Sof'ya N. , Shashkov Alexander S. , Knirel Yuriy A. , Mcgovern Joseph J. , Moran Anthony P. 
Published: July 2004

An O‐specific polysaccharide was liberated from Campylobacter fetus subsp. fetus serotype B lipopolysaccharide by mild acid hydrolysis followed by gel chromatography. This polysaccharide was found to contain d‐rhamnose and 3‐O ‐methyl‐d‐rhamnose (d‐Rha3Me, d‐acofriose) in a ratio of approximately 24:1, as well as lipopolysaccharide core constituents. The structure of the polysaccharide was studied by 1H‐NMR and 13C‐NMR spectroscopy, which included two‐dimensional COSY, rotating‐frame NOE spectroscopy (ROESY), and computer‐assisted analysis of the 13C‐NMR spectrum. Methylation analysis using [2H3]methyl iodide and Smith degradation followed by GLC/MS of the derived acetylated oligosaccharide‐alditols was used to determine the location of d‐acofriose. The O‐specific polysaccharide is linear, consists on average of 12 disaccharide repeating units, and is terminated by a residue of d‐acofriose. The following structure of the d‐rhamnan chain was established:
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Reversible Denaturation of Myo ‐Inositol Monophosphatase
Authors: Moreno Fermin , Corrales Susana , Blanco Francisco Garcia , Gore Michael G. , Rees‐Milton Karen , Churchich Jorge E. 
Published: July 2004

The unfolding of bovine brain myo ‐inositol monophosphatase by guanidine · HCl (Gdn · HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn · HCl‐treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn · HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process.The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal‐binding loop. Denaturation of the enzyme by Gdn · HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn · HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1×106M‐1. It is suggested that a residual structure of denatured myo ‐inositol monophosphatase is responsible for the binding of terbium ions.The kinetics of unfolding and refolding as a function of Gdn · HCl concentration were monitored by protein fluorescence in a stopped‐flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80–65 s−1 at 25°C. The refolding kinetics fit monoexponential functions with rate constants in the range 120–65 s−1 depending on the Gdn · HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.
The Effects of Calcium and Other Polyvalent Cations on Channel Formation by Escherichia coliα‐Hemolysin in Red Blood Cells and Lipid Bilayer Membranes
Authors: Döbereiner Andreas , Schmid Angela , Ludwig Albrecht , Goebel Werner , Benz Roland 
Published: July 2004

Channel formation by Escherichia coliα‐hemolysin (HlyA) was studied in lipid bilayer membranes and in erythrocytes as a function of the concentration of divalent and trivalent cations. Hemolysin showed full channel‐forming activity in artificial lipid bilayers, even in the presence of 5 mM EDTA and when the E. coli cells were grown in calcium‐depleted media (< 1 μM Ca2+). The addition of divalent cations decreased the single‐channel conductance by about 50% with half‐saturation constants of S mM and less, while the mean lifetime of the HlyA channel was not affected. The addition of trivalent cations, such as Fe3+ or La3+, had a similar effect on the channel conductance, but the half‐saturation constant was 1 μM or below. These effects may be caused by the binding of the cations to negatively charged groups at the channel mouth and have probably nothing to do with the possible binding of these cations to the repeat domain of the toxin, which is essential for target cell recognition.When cells were grown in calcium‐depleted media, the supernatants showed absolutely no hemolytic activity. Addition of small amounts of Ca2+ to the supernatant led to toxin‐mediated hemolysis. Its half‐saturation constant was 120 μM. Of the other earth alkaline cations only strontium (Sr2+), which has an ion radius similar to Ca2+, led to full activation of HlyA with a Km of 1.5 mM. Ba2+ induced only weak hemolytic activity, while Mg2+ and several heavy metal cations had no effect. These results led to the conclusion that the target cell recognition sites formed by the repeat region of HlyA have defined sizes and bind only ions with defined radii.