bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholera toxin differentially decreases membrane levels of α and β subunits of G proteins in NG108‐15 cells
Authors: KLINZ Franz‐Josef , COSTA Tommaso 
Published: March 2005

Treatment of NG108‐15 neuroblastoma × glioma cells (24 h) with cholera toxin (0.1 – 10 μg/ml) resulted in a concentration‐dependent reduction of the membrane levels of subunits of GTP‐binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Goα and Gβ1 were reduced by 40 – 50%, whereas those of Gαcommon immunoreactivity and Gi2α were only reduced by 10 – 20% following treatment with 10 μg/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01 – 10 ng/ml) and then decreased at high (0.1 – 10 μg/ml) concentrations. Thus, the down regulation of G‐proteins subunits produced by cholera toxin requires its (ADP‐ribosyl)transferase activity but does not result from a cAMP‐mediated mechanism. The toxin‐mediated decrease of Goα in the membrane was correlated with a diminution of opioid‐receptor‐mediated stimulation of high‐affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108‐15 cells.Northern‐blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for Gβ1 did not change. Thus, the cholera‐toxin‐induced decrease of G‐protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP‐ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Goα and Gβ1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.
Occurrence of tyrosine sulfate in proteins – a balance sheet
Authors: HILLE Annette , HUTTNER Wieland B. 
Published: March 2005

The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins.Upon metabolic labelling of HepG2, Ltk−, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein‐bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein‐bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of trosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane‐spanning proteins contain potential tyrosine sulfation sites.In HepG2 cells, three tyrosine‐sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150‐kDa tyrosine‐sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle.Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes oftrans‐Golgi‐derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine‐sulfated protein or protein class.
The structure of Proteus mirabilis O3 O‐specific polysaccharide containing N‐(2‐hydroxyethyl)‐D‐alanine
Authors: VINOGRADOV Evgeny V. , KACA Wieslaw , SHASHKOV Alexander S. , KRAJEWSKA‐PIETRASIK Danuta , ROZALSKI Antoni , KNIREL Yuriy A. , KOCHETKOV Nikolay K. 
Published: March 2005

O‐Specific polysaccharide was obtained by mild acid degradation of Proteus mirabilis O3 lipopolysaccharide. The polysaccharide was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N‐(2‐hydroxyethyl)‐D‐alanine. The structure of the polysaccharide was determined by methylation, Smith degradation and computer‐assisted analysis of the 13C‐NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was investigated by using 1H and 13C‐NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative). Its absolute configuration was established by comparison of the optical rotation value and CD spectrum of the natural and synthetic product.On the basis of the data obtained, it was concluded that the repeating unit of P. mirabilis O3 O‐specific polysaccharide has the following structure:

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Removal of the amino acid phosphate substituent significantly decreased serological activity of the O‐specific polysaccharide, thus showing the immunodominant role of this group. Serological cross‐reactions between P. mirabilis O3 and O27 were demonstrated and tentatively substantiated.
A hysteretic cycle in glucose 6‐phosphate metabolism observed in a cell‐free yeast extract
Authors: ESCHRICH Klaus , SCHELLENBERGER Wolfgang , HOFMANN Eberhard 
Published: March 2005

The dynamics of a partial glycolytic reaction sequence which converts glucose 6‐phosphate to triose phosphates is described. The study was performed with cell‐free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow‐through reactor supplied with the desalted cell‐free extract as well as glucose 6‐phosphate, ATP and phosphoenolpyruvate.In the reaction system the quasi‐irreversible reactions catalyzed by 6‐phosphofructo‐1‐kinase, pyruvate kinase, and fructose‐1,6‐bisphosphatase are involved. When substrate is supplied continuously, only stable stationary states can be observed. With transient perturbations of the substrate supply, multiple stationary states appear. Cyclic transitions between unique stable stationary states were induced by appropriate changes of the rate of substrate supply. A hysteretic cycle could then be demonstrated when, during reverse transitions, a parameter region of multistability was passed. The presence (in resting yeast) or absence (in growing yeast) of fructose‐1,6‐bisphosphatase did not significantly influence the dynamic capabilities of the investigated reation sequence. The kinetic properties of the cell‐free extracts fit mathematical models developed for in vitro systems reconstituted from purified enzymes.
Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus
Authors: VENTUROLI Giovanni , GABELLINI Nadia , OESTERHELT Dieter , MELANDRI B. Andrea 
Published: March 2005

The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone‐10 two multiprotein complexes: the reaction center and the ubiquinol‐cyctochrome‐c2 reductase (or bc1 complex).
In the presence of cytochrome c2 added externally, at concentrations in the range 10–104 nM, a flash‐induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone‐reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo‐oxidized reaction center. At low ionic strength (10 mM KCI) and at concentrations of cyctochrome c2 lower than 1 μM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half‐time for cytochrome b561 reduction is about 3 ms.
At high ionic stength (200 mM KCI), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome‐b561 reduction. If the absolute values of these rates are compared with those of the cytochrome‐c2‐reaction‐center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate‐limiting and involves electrostatic interactions.
A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second‐order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second‐order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 × 10−11 cm2s−1 can be evaluated.
Structural and immunological properties of the phenolic glycolids from Mycobacterium gastri and Mycobacterium kansasii
Authors: GILLERON Martine , VENISSE Anne , FOURNIE Jean‐Jacques , RIVIERE Michel , DUPONT Marie‐Ange , GAS Nicole , PUZO Germain 
Published: March 2005

Mycobacterial species‐specific antigens belong to the three following classes: phenolic glycolipids (Phe Gl), acyltrehalose‐containing lipooligosaccharides and polar glycopeptidolipids. These antigens have been chemically defined and alkali‐labile epitopes were found to characterize the lipooligosaccharide antigen type.In the present study the major Mycobacterium kansasii phenolic glycolipid epitope namely Phe Gl K‐I was delineated as the distal monoacetylated disaccharidic residue: 2,6‐dideoxy‐4‐O‐methyl‐α‐D‐Arbino‐hexopyranosyl‐(1 → 3)‐2‐O‐methyl‐4‐Oacetyl‐α‐L‐fucopyranose. This acetoxy group is required for K‐I epitope recognition demonstrating that alkali‐labile epitopes also occur in the phenolic glycolipid antigen class. Using immunoelectron microscopy, the Phe Gl K‐I epitope was localized around the electron‐transparent layer on the M. Kansasii cell‐wall surface. Furthermore, two new phenolic glycolipids namely Phe Gl K‐III and Phe Gl K‐IV were discovered in minute amounts. They were purified and characterized by their retention time in direct‐phase column HPLC. These molecules are also M. kansasii antigens, whose epitopes differ from that of Phe Gl K‐I. The complete family of phenolic glycolipids Phe Gl K‐I, K‐II, K‐III and K‐IV was found in both rough and smooth variants of both M. kansasii and Mycobacterium gastri species.
Type 5 acid phosphatase
Authors: LORD Deirdre K. , CROSS Nicholas C. P. , BEVILACQUA Maria A. , RIDER Susan H. , GORMAN Patricia A. , GROVES Ann V. , MOSS Donald W. , SHEER Denise , COX Timothy M. 
Published: March 2005

The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear iron‐containing proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery or iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N‐glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI.Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced > 20‐fold on transformation of normal human monocytes to macrophages by culture in serum‐supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage.
Bacteriophage‐associated glycan hydrolases specific for Escherichia coli capsular serotype K12
Authors: ALTMANN Friedrich , CHRISTIAN Rudolf , CZERNY Thomas , NIMMICH Wolfgang , MÄRZ Leopold 
Published: March 2005

Four bacteriophages were identified, which carry glycan hydrolases specific for the Escherichia coli K12 capsular polysaccharide. All these glycanases catalyze the hydrolysis of the α‐l‐rhamnosyl‐1,5‐β‐3‐deoxy‐d‐manno‐2‐octulosonic acid linkage as demonstrated with a special thiobarbituric acid assay procedure, which discriminates between the C5 substituted and unsubstituted 3‐deoxy‐d‐manno‐2‐octulosonic acid (dOclA). This assay, together with gel filtration, 1H‐NMR and 13C‐NMR spectroscopy showed that depolymerization led to the dimer of the K12 repeating unit, (,5‐β‐dOcl1Ap‐2,3‐α‐lRhap‐1,2‐αlRhap1,)2, as the primary degradation product.The phages (φ12‐W, φ12‐S, φ82‐W1, φ82‐W2) were tested for their ability to infect Escherichia coli strains Su65‐42 (O4:K12:H−) and CDC63‐57 [O139:K82(12):H1]. φ12‐W and φ12‐S, respectively, infected strain Su65‐42 only, φ82‐W2 CDC63‐57 only, and φ82‐W1 both bacterial strains. These distinct host specificities cannot be explained by differences in the action of the glycanases, which depolymerisze the capsules of both strains.
4‐Phenyl‐3‐butenoic acid, an in vivo inhibitor of peptidylglycine hydroxylase (peptide amidating enzyme)
Authors: BRADBURY Alan F. , MISTRY Jashu , ROOS Bernard A. , SMYTH Derek G. 
Published: March 2005

The ability of a series of non‐peptide carboxylic acids to act as substrates or inhibitors of the peptide‐amidating enzyme (peptidyl‐glycine hydroxylase) was assessed by determining their ability to reduce the rate of enzymic conversion of d‐tyrosyl‐valyl‐glycine or d‐tyrosyl‐phenylalanyl‐glycine to the corresponding dipeptide amide. The inclusion of a phenyl substituent in a position distal to the carboxyl group promoted the inhibitory action. The inhibition was found to be irreversible when an olefinic double bond, α or β to the carboxyl group, was present in the molecule; the inhibition appeared to be associated with a covalent interaction between the amidating enzyme and the inhibitor. With 4‐phenyl‐3‐butenoic acid the inhibitory properties were manifest only in the presence of cofactors of the enzyme. When 4‐phenyl‐3‐[2‐14C]butenoic acid was used, the radioactivity was shown to be incorporated into protein that co‐chromatographed with active enzyme.Incubation of rat thyroid carcinoma CA 77 cells in the presence of 4‐phenyl‐3‐butenoic acid led to a decrease in the levels of intracellular amidating activity and of thyrotropin‐releasing hormone, an amidated peptide produced by these cells. The inhibitory effects reached a maximum at approximately 15 h after which the enzyme levels returned to the control values even though the concentration of 4‐phenyl‐3‐butenoic acid in the cells remained unchanged. The results indicate that a mechanism exists in these cells for regulation of amidating activity.
Rates of RNA degradation in isolated rat hepatocytes
Authors: BALAVOINE Sophie , FELDMANN Gérard , LARDEUX Bernard 
Published: March 2005

RNA degradation in isolated rat hepatocytes was measured as the release of radioactive cytidine from fed rats previously labeled in vivo for 60 h with [6‐14C]orotic acid. Rates were determined from the linear accumulation of [14C]cytidine between 30 and 120 min of incubation in the presence of 0.5 mM unlabeled cytidine to suppress reutilization.
In the absence of amino acids, rates of RNA degradation in isolated hepatocytes averaged 3.97%/h. A complete mixture of amino acids added at 10–20 times normal plasma concentration inhibited RNA degradation by 65–70%. However, at physiological concentrations of amino acids, RNA degradation in isolated rat hepatocytes was less responsive as compared to perfused rat livers.
Numerous and large autophagic vacuoles at various stages of digestion were identified throughout the cytoplasm of isolated hepatocytes after 2 h of incubation in the absence of amino acids. The addition of amino acids at 20 times normal plasma concentration abolished almost completely the appearance of autophagic vacuoies. Furthermore, propylamine, which accumulates in lysosomes, suppressed RNA degradation by 65% and the inhibitor of autophagic vacuole formation, 3‐methyladenine, inhibited 70–80% of the degradation. Taken together, these results strongly suggest a contribution of the lysosomal system in the increase of RNA degradation rates in isolated rat hepatocytes.
Vanadate inhibits liver fructose‐2,6‐bisphosphatase
Authors: RIDER Mark H. , BARTRONS Ramon , HUE Louis 
Published: March 2005

Vanadate was found to be a reversible non‐competitive inhibitor of chicken liver fructose‐2,6‐bisphosphatase. The inhibition was best observed in the presence of glycerol 2‐ or 3‐phosphate and half‐maximal effect was obtained with about 0.15 mM vanadate.Vanadate decreased the extent of phosphorylation of the enzyme (E‐P) by fructose 2,6‐[2‐32P]bisphosphate. This did not result from an increased rate of E‐P breakdown, as is the case with phosphoglycerate mutase, an enzyme which shares structural and functional similarity to fructose‐2,6‐bisphosphatase. The data were consistent with the formation of a dead‐end transition state analogue of phosphate in the active site.Inhibition of fructose‐2,6‐bisphosphatase by vanadate offers a likely explanation for the increase in fructose 2,6‐bisphosphate concentration brought about by vanadate in isolated rat hepatocytes.
Studies on the structure and regulation of the human hepatic interleukin‐6 receptor
Authors: ROSE‐JOHN Stefan , SCHOOLTINK Heidi , LENZ Dorothée , HIPP Erich , DUFHUES Gabi , SCHMITZ Hildegard , SCHIEL Xaver , HIRANO Toshio , KISHIMOTO Tadamitsu , HEINRICH Peter C. 
Published: March 2005

Affinity cross‐linking of 125I‐labeled recombinant human interleukin‐6 (IL‐6) to human hepatoma cells (HepG2) allowed the detection of three IL‐6‐containing complexes with molecular masses of 100 kDa, 120 kDa and 200 kDa.Treatment of HepG2 cells with dexamethasone led to a time‐ and dose‐dependent up‐regulation of IL‐6‐receptor mRNA levels. By the use of cross‐linking this effect was also seen at the protein level, where all three IL‐6‐binding complexes increased upon incubation of HepG2 cells with dexamethasone. Under conditions of IL‐6‐receptor up‐regulation by dexamethasone, γ‐fibrinogen mRNA induction by IL‐6 is stronger and occurs earlier than without dexamethasone. We propose therefore that the expression of the IL‐6 receptor might be a rate‐limiting step in acute‐phase‐protein induction.
An NMR study of anion binding to yeast phosphoglycerate kinase
Authors: FAIRBROTHER Wayne J. , GRAHAM Heidi C. , WILLIAMS Robert J. P. 
Published: March 2005

Anion binding to yeast phosphoglycerate kinase has been investigated using 1H‐NMR spectroscopy. The use of anionic paramagnetic probes, [Cr(CN)6]3− and [Fe(CN)6]3−, has enabled the location of the primary anion binding site in the ‘basic‐patch’ region of the amino‐terminal domain. The anions interact most closely with Arg‐65 and Arg‐168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3‐phosphoglycerate. Binding of 3‐phosphoglycerate and 1,3‐bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity.Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.
Protein‐decorated micelle structure of sodium‐dodecyl‐sulfate–protein complexes as determined by neutron scattering
Authors: IBEL Konrad , MAY Roland P. , KIRSCHNER Kasper , SZADKOWSKI Halina , MASCHER Erik , LUNDAHL Per 
Published: March 2005

The structure of the complex between sodium dodecyl sulfate (SDS) and a deuterated bifunctional enzyme, N‐5′‐phosphoribosylanthranilate isomerase/indole‐3‐glycerol‐phosphate synthase (Mr 49484), has been studied in dilute solution by small‐angle neutron scattering. The complex nearly acquired its final size, as shown by molecularsieve chromatography, at the chosen SDS concentration of 1.6 mM, which is slightly below the critical micelle concentration of 1.8 mM (at the ionic strength of 0.1 M). The 452 amino‐acid residues of the bifunctional enzyme were combined with 216 detergent molecules. The complex was found to be composed of three protein‐decorated SDS micelles of unequal size, connected by short flexible polypeptide segments. The largest of the three micelles was the middle one. The SDS‐protein complex contained the dodecyl hydrocarbon moieties in three globular cores. Each core was surrounded by a hydrophilic shell, formed by the hydrophilic and amphiphilic stretches of the polypeptide chain, and by the sulfate head groups of the detergent. The average thickness of these shells was 0.7–0.8 nm.The three‐micelle complex was cleaved with trypsin at a single site, possibly in a micelle‐connecting segment, into a single‐micelle fragment at the carboxyl‐terminal which comprised 73 SDS molecules and 163 amino‐acid residues, and a dual‐micelle fragment. One of the micelles within this larger fragment contained 42 SDS molecules and about 90 amino‐acid residues; the other micelle contained 101 SDS molecules and about 190 amino‐acid residues. The individual micelle sizes seemed to be determined by the amino‐acid sequence.
Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b2) by product binding to the semiquinone transient
Authors: TEGONI Mariella , JANOT Jean‐Marc , LABEYRIE Françoise 
Published: March 2005

Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (Fs) to the cytochrome b2 moiety of flavocytochrome b2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39–45] and to stabilize markedly the Fs state of the prosthetic flavin, relative to the oxidized (Fo) and the reduced (Fh) states [Tegoni, M., Janot, J.‐M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, 491–503].In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the Fs‐pyruvate complex is actually much more stable than the Fo‐pyruvate and Fh‐pyruvate complexes.The inhibition produced by pyruvate has been characterized under steady‐state conditions using both ferri‐cytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to Fo and an uncompetitive effect due to binding to the Fs intermediate in a dead‐end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.