bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical properties of conjugates of urokinase‐type plasminogen activator with a monoclonal antibody specific for cross‐linked fibrin
Authors: DEWERCHIN Maria , LIJNEN H. Roger , Van HOEF Berthe , De COCK Frans , COLLEN Désiré 
Published: March 2005

Equimolar mixtures of recombinant single chain urokinase‐type plasminogen activator (rscu‐PA) and a murine monoclonal antibody (MA‐15C5) directed against fragment‐D dimer of human cross‐linked fibrin were conjugated, using the cross‐linking agent N‐succinimidyl 3‐(2‐pyridyldithio)propionate (PySSProSu). The conjugate (rscu‐PA/MA‐15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment‐D dimer with a yield of 42 ± 15% (mean ± SD, n= 3), contained an average of 1.2 ± 0.3 IgG molecules/rscu‐PA molecule. On non‐reduced SDS/PAGE it migrated as a main band with apparent Mr of 200000.Specific amidolytic activities expressed/mass of u‐PA were < 250 IU/mg for rscu‐PA/MA‐15C5 and rscu‐PA, 140000 ± 13000 IU/mg and 100000 ± 17000 IU/mg for their plasmin‐generated two chain derivatives rtcu‐PA/MA‐15C5 and rtcu‐PA respectively. Specific activities on fibrin plates were 100000 ± 24000 IU/mg and 130000 ± 49000 IU/mg for rscu‐PA/MA‐15C5 and rtcu‐PA/MA‐15C5 respectively, as compared to 180000 ± 15000 IU/mg for both rscu‐PA and rtcu‐PA. Activation of plasminogen with rscu‐PA/MA‐15C5 (Km= 0.37 ± 0.16 μM, k2= 0.0063 ± 0.0030 s−1) or rtcu‐PA/MA‐15C5 (Km= 19 ± 3.0 μM, k2= 2.0 ± 0.10 s−1) in purified systems followed Michaelis‐Menten kinetics with Km and k2 values comparable to those of rscu‐PA and rtcu‐PA.In an in vitro system composed of a 125I‐fibrin‐labeled whole human plasma clot immersed in citrated human plasma, dose‐ and time‐dependent lysis was obtained; 50% lysis in 2 h required 1.4 μg/ml of rscu‐PA or 0.33 μg/ml of rtcu‐PA, but only 0.22 μg u‐PA/ml of rscu‐PA/MA‐15C5 or 0.15 μg u‐PA/ml of rtcu‐PA/MA‐15C5. Addition of purified fragment‐D dimer reversed the increased fibrinolytic potency of rscu‐PA/MA‐15C5 in a concentration‐dependent way (50% inhibition at 7.2 μg fragment‐D dimer/ml).Thus, conjugation of u‐PA moieties with the fibrin‐specific antibody MA‐15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4‐fold for rscu‐PA and 2.2‐fold for rtcu‐PA.
Catabolic pathway of oligosaccharide‐diphospho‐dolichol
Authors: CACAN René , LEPERS Anne , BELARD Michel , VERBERT André 
Published: March 2005

The degradation of oligosaccharide‐diphospho‐dolichol leads to the release of oligosaccharide material ranging from (Glc)3(Man)9(GlcNAc)2‐P to (Man)3 species and further smaller species. The subcellular location of the glucosidases and mannosidases involved in this catabolic process has been investigated on the basis of their differential sensitivity towards specific inhibitors (castanospermine, deoxymannojirimycin and swainsonine): The results indicate that the first steps of degradation down to the (Man)6 species occurs in the rough endoplasmic reticulum. This result is supported by the fact that the (Man)6 species is the end product when lipid‐intermediate‐derived glucosylated oligosaccharides are incubated with purified rough endoplasmic reticulum membranes.Swainsonine and lysosomotropic agents (chloroquine and ammonium chloride) do not affect the degradation process, thus indicating that neither Golgi apparatus nor lysosomes are involved in this catabolism. The observation of the same degradation pattern of the released oligosaccharide material in mannosidosis fibroblasts, lacking lysosomal mannosidases, confirms these results.Finally, the subcellular distribution of the released oligosaccharide material indicates that the oligomannosides larger than (Man)6 species are sequestered in the particulate fraction whereas, in contrast, oligomannosides smaller than (Man)6 species are found predominantly in the cytosol.Taken altogether, the experiments demonstrate that the first steps of the degradation of oligosaccharide‐diphospho‐dolichol occurs in the rough endoplasmic reticulum producing oligomannosides of the (Man)6 species which are then translocated to the cytoplasm to be further degraded.
Structure of Proteus mirabilis 027 O‐specific polysaccharide containing amino acids and phosphoethanolamine
Authors: VINOGRADOV Evgeny V. , KRAJEWSKA‐PIETRASIK Danuta , KACA Wiestaw , SHASHKOV Alexander S. , KNIREL Yuriy A. , KOCHETKOV Nikolay K. 
Published: March 2005

The following structure of the repeating unit of the O‐specific polysaccharide of Proteus mirahilis 027, containing amides of d‐glucuronic and n‐galacturonic acids with l‐lysine and l‐alanine, respectively, N‐acetyl‐d‐glucosamine and phosphoethanolamine, has been determined:

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The main methods used for structural analysis of the polysaccharide were selective solvolysis with anhydrous hydrogen fluoride and Smith degradation, as well as NMR spectroscopy and mass spectrometry.The immunodominant role of the lateral N‐acetyl‐d‐glucosamine and phosphoethanolamine in manifesting the serological specificity of P. mirubilis 027 has been established.
Conformation of secretin in dimethyl sulfoxide solution
Authors: HOFMANN Mechtild , GONDOL Daniel , BOVERMANN Günter , NILGES Michael 
Published: March 2005

The solution conformation of the 27‐residue polypeptide hormone secretin in dimethyl sulfoxide has been determined on the basis of 1H‐NMR measurements. The experimental data set used in the structure determination consisted of 98 nuclear‐Overhauser‐enhancement‐derived interproton and dihedral angle restraints from coupling constants. The NH‐NH and Hα‐NH NOEs were determined from build‐up rates, while the remaining distances were classified in a qualitative manner.The structure calculations consisted of two phases. First, dynamical simulated annealing calculations were carried out to find conformations of the peptide which satisfy NOE and φ dihedral restraints. The convergence of ten calculated structures was good except for those regions of the molecule where NOE data were not unambiguous. From the calculated set another initial structure was built which was again minimized in several 5‐ps calculations now employing the full empirical energy function. The resulting structures of secretin reveal conformationally well‐defined regions, but not a single uniform secondary structure. The structure is different from the calculated structure from trifluoroethanol/water measurements.
The effect of caldesmon on dynamic properties of F‐actin alone and bound to heavy meromyosin and/or tropomyosin
Authors: GAŁAZKIEWICZ Barbara , BELÁGYI Joseph , DABROWSKA Renata 
Published: March 2005

The effect of caldesmon on the rotational dynamics of actin filaments alone or conjugated with heavy meromyosin and/or tropomyosin has been measured by the electron paramagnetic resonance (EPR) technique using a maleimide spin label rigidly bound to Cys374 of actin. The rotation of actin protomers in filaments and the angular distribution of spin probes on actin were determined by conventional EPR spectroscopy, while torsional motions within actin filaments were detected by saturation transfer EPR measurements.Binding of caldesmon to F‐actin resulted in the reduction of torsional mobility of actin filaments. The maximum effect was produced at a ratio of about one molecule of caldesmon/seven actin protomers. Smooth muscle tropomyosin enhanced the effect of caldesmon, i.e. caused further slowing down of internal motions within actin filaments.Caldesmon increased the degree of order of spin labels on F‐actin in macroscopically oriented pellets in the presence of tropomyosin but not in its absence. Computer analysis of the spectra revealed that caldesmon alone slightly changed the orientation of spin probes relative to the long axis of the filament. In the presence of tropomyosin this effect of caldesmon was potentiated and then approximately every twentieth protomer along the actin filament was affected.Caldesmon weakened the effect of heavy meromyosin both on the polarity of environment of the spin label attached to F‐actin and on the degree of order of labels on actin in macroscopically oriented pellets. Whereas the former effect of caldesmon was independent of tropomyosin, the latter one was observed only in the absence of tropomyosin.
Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small‐angle X‐ray diffraction
Authors: SEYDEL Ulrich , BRANDENBURG Klaus , KOCH Michel H. J. , RIETSCHEL Ernst Th. 
Published: March 2005

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gramnegative bacteria, may assume various three‐dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small‐angle X‐ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia‐coli‐type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (> 90%) at different temperatures (20, 37, and 55°C) and at different lipid/divalent cation molar ratios (20:1 to 1:1).We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20°C, i.e. below the β↔α acyl‐chain‐melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55°C), 4.60 nm for Re lipopolysaccharide (at 37°C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37°C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20°C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1:√2, 1:√3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.
Lactoferrin‐binding sites at the surface of HT29‐D4 cells
Authors: ROIRON Danielle , AMOURIC Michèle , MARVALDI Jacques , FIGARELLA Catherine 
Published: March 2005

The binding of 125I‐lactoferrin to HT29‐D4 cells, a clone of HT29 cells, was studied and compared to the binding of 125I‐transferrin to the same cells. The binding of the two iron‐transport proteins is saturable and reversible suggesting the presence of specific receptors for each protein. Scatchard analysis suggests the existence of binding sites for lactoferrin with the relatively high equilibrium dissociation constant, Kd1 of 408 nM. Additionally, the cell is capable of binding large amounts of lactoferrin with very low affinity, probably in a non‐receptor intermediate fashion. The dissociation constant of transferrin and its receptor was calculated 9.29 nM which corresponds well to values found in the literature. In contrast to lactoferrin, the cell was capable of binding only low amounts of transferrin in a non‐receptor intermediate fashion. After chemical crosslinking of lactoferrin to the cell surface, the radiolabeled lactoferrin was found in a complex of molecular mass 300 kDa. Crosslinking of transferrin resulted in a complex of much higher molecular mass.These data clearly show a binding site for lactoferrin different from the transferrin receptor. Only if competition experiments were performed with a high molar excess of both ligand proteins did a small percentage of either of the two ligands crossreact with the receptor for the other, possibly due to a structural similarity of the two glycoproteins.
Synthetic fragments of β‐casein as model substrates for liver and mammary gland casein kinases
Authors: MEGGIO Flavio , PERICH John W. , MEYER Helmut E. , HOFFMANN‐POSORSKE Edeltraut , LENNON David P. W. , JOHNS R. Basil , PINNA Lorenzo A. 
Published: March 2005

The octapeptide Glu‐Ser‐Leu‐Ser‐Ser‐Ser‐Glu‐Glu, corresponding to the 14–21 sequence of bovine β‐casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi‐enriched fraction of lactating mammary gland (GEF‐casein kinase). Casein kinase‐2 readily phosphorylates the octapeptide at its Ser‐4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 μM and 11 μM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase‐2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu‐Ser‐Ser‐Ser‐Glu‐Glu (Km 210 μM) to the pentapeptide Ser‐Ser‐Ser‐Glu‐Glu (Km 2630 μM). The tetrapeptide Ser‐Ser‐Glu‐Glu is the shortest derivative still phosphorylated by casein kinase‐2, albeit very slowly, and the tripeptides Ser‐Glu‐Glu and Glu‐Leu‐Ser were not substrates at all. Furthermore, the pentapeptide Ser‐Ser‐Ser‐Glu‐Glu was found to be a better substrate than Ser‐Ser‐Ala‐Glu‐Glu, Ser‐Ala‐Ser‐Glu‐Glu and Ser‐Ala‐Ala‐Glu‐Glu by virtue of its lower Km value. These data, while confirming that the motif Ser‐Xaa‐Xaa‐Glu is specifically recognized by casein kinase‐2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine‐containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse‐phase HPLC elution profile data support the hypothesis that a β‐turn conformation is responsible for the remarkable suitability of the octapeptide Glu‐Ser‐Leu‐Ser‐Ser‐Ser‐Glu‐Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase‐2.While neither the peptide Glu‐Ser‐Leu‐Ser‐Ser‐Ser‐Glu‐Glu nor any of its derivatives were affected by casein kinase‐1, a rapid phosphorylation of the octapeptide by GEF‐casein kinase at Ser‐5 (not Ser‐4) was obtained. This phosphorylation site corroborates the concept that, with this protein kinase, the critical acidic residue in a peptide must be located at position +2, instead of +3 as in the case of casein kinase‐2. Consequently the tripeptide Ser‐Glu‐Glu, which is not affected by casein kinase‐2, is still slowly phosphorylated by GEF‐casein kinase.
Characterisation by mass spectrometry and 500‐MHz proton nuclear magnetic resonance spectroscopy of penta‐ and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)
Authors: HOUNSELL Elizabeth F. , LAWSON Alexander M. , STOLL Mark S. , KANE David P. , CASHMORE Geoffrey C. , CARRUTHERS Robert A. , FEENEY James , FEIZI Ten 
Published: March 2005

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500‐MHz 1H NMR are described of the major penta‐ and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio‐Gel P4 chromatography and HPLC. Eleven penta‐ and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397–401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features:Sequences in these oligosaccharides not commonly found in mucins so far studied are chain‐terminating GlcNAα1–4Gal. repeating‐type‐I (Galβ1–3GleNAc) backbones, the backbone branch GlcNAcβ1–6(GlcNAcβ1–3)Gal and the backbone sequence GlcNAcβ1–6Galβ1‐ in the absence of a substitutent at C3 of galactose.
Structure dynamics of the hemoglobin mutants Hb Hôtel Dieu, HbG Philadelphia, HbJ Mexico, Hb St. Mandé and Hb San Diego, studied by nanosecond‐laser‐flash photolysis
Authors: FONTAINE Marie‐Pierre , LINDQVIST Lars , BLOUQUIT Yves , ROSA Jean 
Published: March 2005

The kinetics of the change from the carboxy to the deoxy conformation of the mutated hemoglobins mentioned in the title and of normal human adult hemoglobin were determined from measurements of light absorption changes occurring up to 50 μs after nanosecond‐laser photodissociation of the corresponding CO complexes.The spectral evolution of the mutated hemoglobins was found to be similar in its main features to that of normal hemoglobin. The kinetics could be decomposed into two phases with rates 1.1–1.8 × 106s−1 and 0.17–0.34 × 106s−1 (except Hb St. Mandé which displayed only the faster phase). Study of the mutated subunits of HbJ Mexico (α subunit) and Hb Hôtel Dieu (β subunit) showed that they convert exponentially to the stable deoxy state after photodeligation at the same rates as the corresponding subunits of normal Hb: 1.1 × 106s−1 (α) and 0.3 × 106s−1 (β). The results indicate that there is no direct correlation between the kinetics of spectral relaxation in the time range studied and the oxygenation properties for these hemoglobins. However, there is some indication that the kinetics are dependent upon the region of mutation.
Regulation of protein phosphatase‐1G from rabbit/skeletal muscle
Authors: HUBBARD Michael J. , COHEN Philip 
Published: March 2005

The glycogen‐associated form of protein phosphatase‐1 (PP‐1G) is a heterodimer comprising a 37‐kDa catalytic (C) subunit and a 161‐kDa glycogen‐binding (G) subunit, the latter being phosphorylated by cAMP‐dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C‐terminal to site 1 in the primary structure. The sequence in this region is:

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At physiological ionic strength, phosphorylation of glycogen‐bound PP‐1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP‐1G, while the phospho‐G subunit remained bound to glycogen. Dissociation reflected a ≥ 4000‐fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate‐limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti‐site‐1 Fab fragments to glycogen‐bound PP‐1G.At near physiological ionic strength, PP‐1G and glycogen concentration, site 2 was autodephosphorylated by PP‐1G with a t0.5 of 2.6 min at 30°C, ∼ 100‐fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type‐2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the α subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type‐2A and type‐2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10‐fold less effective than site 2 as a substrate for all four phosphatases.In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP‐1 activity towards the glycogen‐metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen–protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin‐stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP‐1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.
Direct photoaffinity labeling of leukotriene binding sites
Authors: FALK Eugen , MÜLLER Michael , HUBER Michael , KEPPLER Dietrich , KURZ Gerhart 
Published: March 2005

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeiing taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock‐freezing of the samples, irradiation‐induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state.After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time‐dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67000 was labeled. This polypeptide was identified as albumin.Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25000, was identified as subunit 1 of glutathione transferases by immunoprecipitation.The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene‐metabolizing enzymes, and transport systems.