bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative measurement of the error in the cryptic stereospecificity of methylmalonyl‐CoA mutase
Authors: MICHENFELDER Martina , HULL William E. , RÉTEY János 
Published: March 2005

Samples of methylmalonyl‐CoA and (2H3)methylmalonyl‐CoA were prepared by a combination of chemical and enzymic methods. After ion‐exchange chromatography the unlabelled methylmalonyl‐CoA was pure, the deuterated substance contained 11 – 12% dephospho‐CoA derivative.
The sample of unlabelled methylmalonyl‐CoA was incubated in deuterated buffer with catalytic amounts of methylmalonyl‐CoA mutase, epimerase, and coenzyme B1 2. The progress of the reaction was monitored directly by 1H‐NMR spectroscopy at 500 MHz. After equilibrium was established, a slow mutase‐catalysed deuterium incorporation into migratable positions of succinyl‐CoA was observed.
The sample of (2H3)methylmalonyl‐CoA was incubated in unlabelled buffer with a mixture of methylmalonyl‐CoA mutase, epimerase and coenzyme B1 2. In withdrawn aliquots, the reaction was interrupted by acidification and the lyophilised samples were examined by 1H‐NMR spectroscopy in deuterium oxide. Both rearrangement and protium incorporation into migratable positions of succinyl‐CoA were monitored.
At comparable methylmalonyl‐CoA to succinyl‐CoA conversion rates, deuterium loss from migratable positions was 4–6 times faster than the corresponding protium loss. It is confirmed that the stereochemical error of the mutase is amplified by isotope discrimination when deuterium is in migratable positions, whereas it is diminished when protium is in migratable positions.
Subunit composition of the H+‐ATPase complex from anaerobic bacterium Lactobacillus casei
Authors: MILEYKOVSKAYA Evgenia I. , ABULADZE Anna N. , OSTROVSKY Dmitrii N. 
Published: March 2005

The H+‐ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods.

The crossed‐immunoelectrophoresis of the 14C‐labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1‐ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (α and β), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor Fo of L. casei ATPase complex consists of a 16‐kDa subunit and two subunits with molecular masses less than 14 kDa.
A dicyclohexylcarbodiimide‐sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide‐binding subunit with a molecular mass less than 14 kDa.
Characterization of an arginine‐specific protein kinase tightly bound to rat liver DNA
Authors: LEVY‐FAVATIER Florence , DELPECH Marc , KRUH Jacques 
Published: March 2005

A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit.The main characteristic of this protein kinase is that it is arginine‐specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid‐labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is cAMP‐independent. Its optimal activity requires Mn2+ ions. Vanadate, spermine and heparins have no effect on its activity.
Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate
Authors: KÜHN Hartmut , WIESNER Rainer , ALDER Lutz , FITZSIMMONS Brian J. , ROKACH Joshua , BRASH Alan R. 
Published: March 2005

The pure reticulocyte lipoxygenase converts 15Ls‐hydroxy‐5,8,11,13(Z,Z,Z,E)‐icosatetraenoic acid (15Ls‐HETE) methylester to a complex mixture of products containing 5Ds,14LR,15Ls‐trihydro(pero)xy‐6E,8Z,10E,12E‐icosatetraenoate methyl ester (lipoxin B methyl ester), 5Ds,15Ls‐DiH(P)ETE methyl ester and four 8,15Ls‐DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy‐icosatetraenoic acid].After a short incubation period (15 min) 5Ds,15Ls‐DiH(P)ETE methyl ester was found to be the main product, whereas after a 3‐h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400‐MHz 1H‐NMR.Atmospheric oxygen was incorporated at carbon‐5 and carbon‐14 into the major product. 5Ds,15Ls‐DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15Ls‐HETE and 5Ds,15Ls‐DiHETE.The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.
Lipid interaction of diphtheria toxin and mutants with altered fragment B
Authors: PAPINI Emanuele , COLONNA Raffaele , CUSINATO Federico , MONTECUCCO Cesare , TOMASI Maurizio , RAPPUOLI Rino 
Published: March 2005

The interaction of diphtheria toxin and its cross‐reacting mutants crm 45, 228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing.The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5–6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.
Lipid interaction of diphtheria toxin and mutants with altered fragment B
Authors: PAPINI Emanuele , SCHIAVO Giampietro , TOMASI Maurizio , COLOMBATTI Marco , RAPPUOLI Rino , MONTECUCCO Cesare 
Published: March 2005

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half‐maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate‐controlling step in the process of membrane translocation of diphtheria toxin.crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid‐treated cells, suggesting that the 12‐kDa COOH‐terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin.It is suggested that crm 1001 is non‐toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.
Binding of brain spectrin to the 70‐kDa neurofilament subunit protein
Authors: FRAPPIER Thierry , REGNOUF Françoise , PRADEL Louise Anne 
Published: March 2005

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70‐kDa neurofilament subunit (NF‐L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin‐binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was ‘blotted' with 125I‐fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF‐L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF‐L was reversible and concentration‐dependent. The ability of the pure NF‐L and GFP to form filaments was used to quantify their association with fodrin.

The binding of fodrin to reassembled NF‐L was saturable with a stoichiometry of 1 mol fodrin bound/50±10 mol NF‐L and an apparent dissociation constant Kd= 4.3 × 10−7 M.
The binding involved the N‐terminal domain of the polypeptide chain derived from the [2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine] cleavage of NF‐L.
Binding occurred optimally at physiological pH (6.8‐7.2) and salt concentrations (50 mM).
Interestingly, calmodulin, a Ca2+‐binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF‐L, at Ca2+ physiological concentrations.

The binding of fodrin to pure neurofilaments was not affected by the presence of the 200‐kDa (NF‐H) and the 160‐kDa (NF‐M) subunits. The apparent dissociation constant for the binding of fodrin to NF‐L in the pure NF was 1.0 × 10−6 M with 1 mol fodrin bound/80 ± 10 mol NF‐L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 × 10−7 M and the maximum binding was 1 mol fodrin/55 ± 10 mol GFP.
Properties of the Na+‐dependent Cl−/HCO−3 exchange system in U937 human leukemic cells
Authors: LADOUX Annie , KRAWICE Irène , CRAGOE Edward J. , ABITA Jean Pierre , FRELIN Christian 
Published: March 2005

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride‐sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl− influx and efflux experiments.Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi= 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate‐stimulated uptake pathway for Na+ which was inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate. The bicarbonate‐dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl− and increased by removal of external Cl−. 36Cl− efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl− at a cell pHi of 6.3. Finally a 36Cl− uptake pathway was documented. It was inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (K0.5= 10 μM) and bicarbonate (K0.5= 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+‐independent Cl−/HCO−3 exchange system could be found. The Na+‐dependent Cl−/HCO−3 exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate‐induced swelling of astroglia and of the Li(Na)CO−3/Cl‐ exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.
Prostaglandin release but not superoxide production by rat Kupffer cells stimulated in vitro depends on Na+/H+ exchange
Authors: DIETER Peter , SCHULZE‐SPECKING Agnes , KARCK Ullrich , DECKER Karl 
Published: March 2005

The release of the prostaglandins E2 and D2, induced by zymosan and phorbol ester in cultured rat Kupffer cells, was found to depend on the extracellular concentration of Na+. Eicosanoid formation following the administration of the Ca2+ ionophore A23187 or of arachidonic acid, however, did not require the presence of sodium ions in the medium. A half‐maximal rate of prostaglandin release by zymosan‐treated Kupffer cells was obtained between 4 mM and 5 mM Na+; and a Na+ concentration of ≥ 30 mM was required to maximally stimulate prostaglandin E2 and D2 formation in the cultured liver macrophages. In contrast, the superoxide production following the administration of zymosan or of phorbol ester was quite independent of extracellular Na+.The zymosan and phorbol‐ester‐stimulated release of prostaglandins E2 and D2 was inhibited by amiloride. Artificial intracellular alkalization enhanced the prostanoid production of unstimulated and of zymosan‐stimulated cells whereas artificial intracellular acidification inhibited the zymosan‐elicited prostaglandin synthesis. In contrast, the superoxide formation was independent of the pH changes. The data presented here suggest that the prostaglandin production elicited by zymosan or phorbol ester in cultured rat Kupffer cells requires an activated Na+/H+ exchange.
Relations between plasma membrane and lysosomal membrane
Authors: DRAYE Jean‐Pierre , QUINTART Joël , COURTOY Pierre J. , BAUDHUIN Pierre 
Published: March 2005

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4°C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37°C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell‐associated labels) was rapidly released (half‐life < 1 h), and most label (65%) was released slowly (half‐life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37°C, the patterns of 125I‐polypeptides quantified by autoradiography after SDS‐PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides.The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2 – 4% of cell‐associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H‐labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell‐associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.
Studies of the cellulolytic system of Trichoderma reesei QM 9414
Authors: TOMME Peter , VAN TILBEURGH Herman , PETTERSSON Göran , VAN DAMME Jozef , VANDEKERCKHOVE Joel , KNOWLES Jonathan , TEERI Tuula , CLAEYSSENS Marc 
Published: March 2005

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel).By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43–52].The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N‐terminal region contributes in the binding onto both cellulose types; the homologous C‐terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.
Conformational analysis of the dinucleotides 5′‐methylphospho‐N6‐dimethyladenylyl‐uridine (mpm62A‐U) and 5′methylphospho‐uridylyl‐N6‐dimethyladenosine (mpU‐m62A) and of the trinucleotide U‐m62A‐U
Authors: van den HOOGEN Yvonne T. , LANKHORST Peter P. , GIJSMAN Pieter , HARTEL Arnold J. , van BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

NMR and CD studies were carried out on the dinucleotides 5′‐methylphospho‐N6‐dimethyladenylyl‐uridine (mpm62‐U) and 5′‐methylphospho‐uridylyl‐N6‐dimethyladenosine (mpU‐m62A) and on the trinucleotide U‐m62A‐U. A detailed comparison is given of the conformational features of mpm62A‐U and mpU‐m62A with the corresponding 5′‐nonphosphorylated dinucleotides m62A‐U and U‐m62A, respectively. The behaviour of the trinucleotide U‐m62A‐U is compared with the properties of the constituent dinucleotides U‐m62A and mpm62A‐U.Chemical‐shift and CD data were used to determine the amount of stacking interactions. For each compound NMR spectra were recorded at two or three sample concentrations in order to separate intermolecular and intramolecular base‐base interactions. The coupling constants of the ribose ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles β, γ and ɛ are presented.The combined data indicate a strong similarity between mpm62A‐U and m62A‐U both in degree and in mode of stacking. In contrast, the existence of different types of stacking interactions in mpU‐m62A and U‐m62A is suggested in order to explain the NMR and CD data. It is concluded that dinucleoside bisphosphates serve better as a model for the behaviour of trinucleotides than dinucleoside monophosphates.The trinucleotide U‐m62A‐U adopts a regular single‐stranded stacked RNA structure with preference for N‐type ribose and γ+ and βt backbone torsion angles. The difference in behaviour between the U‐m62A‐ part of U‐m62A‐U and the dimer U‐m62A is seen as a typical example of conformational transmission.
Primary structure of neutral oligosaccharides derived from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500‐MHz 1H‐NMR spectroscopy and quantitative sugar analysis
Authors: BREG Jan , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G. , KLEIN André , LAMBLIN Geneviève , ROUSSEL Philippe 
Published: March 2005

A pool of neutral carbohydrate chains was prepared from respiratory mucins of a patient suffering from bronchiectasis. Fractionation by HPLC led to 35 smaller‐size oligosaccharide‐alditols; the structure of 16 oligosaccharide‐alditols with core type 1 or type 2 has been established (Klein, A., Lamblin, G., Lhermitte, M., Roussel, P., Breg, J., Van Halbeek, H. & Vliegenthart, J. F. G., preceding paper in this journal).In this second part, we identified 19 oligosaccharide‐alditols possessing core types 3 and 4. Nine of the structures (1, 5, 9, 6, 10b, 13, 19, 15b and 18.1) have been described previously to be present in cystic fibrosis mucins [Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J. G. H. M., Van Halbeek, H. & Vliegenthart, J. F. G. (1984) Eur. J. Biochem. 143, 227–234]. The remaining ten are new structures isolated from bronchial mucins; they are all extensions of the above‐mentioned nine oligosaccharides. These compounds are octasaccharide‐alditols containing the Y determinant together with the H determinant of either backbone‐type 1 or 2, and partial structures thereof:

image
and 21b, which is 23c without Fucα(1→3), 18.2, which is 23c without any Fuc in the 6‐branch, and 22b, which is 23c without Fuc in the 3‐branch.
The development of rat α2‐macroglobulin
Authors: FLETCHER Sue , THOMAS Tim , SCHREIBER Gerhard , HEINRICH Peter C. , YEOH George C. T. 
Published: March 2005

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute‐phase proteins. The levels of some acute‐phase proteins have been reported to increase in pregnant and tumour‐bearing animals.Rat α2‐macroglobulin is classified as an acute‐phase protein. In this study we report the expression of α2‐macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, viceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of α2‐macroglobulin expression are localized by in situ hybridization of cDNA for α2‐macroglobulin to mid‐sagittal cryosections of rat embryos.The level of mRNA coding for α2‐macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. α2‐Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. α2‐Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the α2‐macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting α2‐macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.