bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipid interaction of diphtheria toxin and mutants
Authors: DEMEL Rudy , SCHIAVO Giampietro , KRUIJFF Ben de , MONTECUCCO Cesare 
Published: March 2005

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface‐pressure‐increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm).DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm).The low‐pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.
Primary structure of a 15‐kDa protein from rat intestinal epithelium
Authors: KANDA Tatsuo , ODANI Shoji , TOMOI Masaaki , MATSUBARA Yoichi , ONO Teruo 
Published: March 2005

An abundant and novel cytosolic protein was purified from the rat intestinal epithelium by gel filtration, ionexchange and hydroxylapatite chromatography. The protein was eluted into two different positions (fractions 1 and 2) on DEAE‐cellulose chromatography. We have completed the primary structure of the protein of fraction 1 by Edman degradation. The protein (144 565 Da) contains 127 amino acid residues and has an acetylated alanine at its NH2‐terminus. Comparison of the primary structure of the protein with porcine gastrotropin [Walz, A. D., Wider, M. D., Snow, J. W., Dass, C. & Desiderio, D. M. (1988) J. Biol. Chem. 263, 14189–14195] and rat hepatic fatty‐acid‐binding protein revealed that identical residues within these proteins are found in 90 and 54 out of a total of 127 positions, respectively.Bioactivity studies demonstrated that neither the protein nor liver and intestinal fatty‐acid‐binding proteins influence gastric acid secretory activity in rats with gastric fistulas compared to pentagastrin. The protein showed very low affinity for palmitic‐acid‐binding in vitro assay system and only trace amounts of endogenous fatty acids were detected from the protein.The protein, rat intestinal 15‐kDa protein is considered to be a new member of the fatty‐acid‐binding protein family based on its structural features.
On the FAD‐induced dimerization of apo‐lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens
Authors: van BERKEL Willem J. H. , BENEN Jacques A. E. , SNOEK Marja C. 
Published: March 2005

The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197–207].The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochem. Biophys. Acta 159, 244–256].The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of startng material.Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two‐step sequential process. Initial flavin‐binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum.The kinetics of FAD‐induced dimerization are strongly dependent on the apoenzyme used. At 0°C, the monomeric apoenzyme‐FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH.
Differentiation of ovine brown adipocyte precursor cells in a chemically defined serum‐free medium
Authors: CASTEILLA Louis , NOUGUÈS Jean , REYNE Yves , RICQUIER Daniel 
Published: March 2005

The rapid apparent conversion of brown adipose tissue into white adipose tissue in newborn offspring of large mammals, such as sheep and cattle is not explained at the cellular level. To study the differentiation of lamb brown adipocyte, a genomic fragment corresponding to the uncoupling protein was cloned from an ovine DNA library. Stromal vascular fibroblasts isolated from the perirenal adipose tissue of newborn lambs completely differentiated into brown adipocytes expressing the uncoupling protein gene, in a chemically defined serum‐free medium. Dexamethasone was necessary for the expression of the uncoupling protein gene. When stromal vascular fibroblasts were isolated from 3‐week‐old lambs, the glucocorticoid analog still promoted in vitro differentiation of adipocytes. However those adipocytes were unable to express uncoupling mRNA and could be considered as white adipocytes.The data indicate that dexamethasone is necessary but not sufficient clone for the complete differentiation of brown adipocytes, and that the preadipocytes are committed to differentiation into brown or white adipocytes before culture.
Genetic engineering of Escherichia coli inorganic pyrophosphatase
Authors: LAHTI Reijo , SALMINEN Tiina , LATONEN Sari , HEIKINHEIMO Pirkko , POHJANOKSA Katariina , HEINONEN Jukka 
Published: March 2005

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766–772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases.Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338–345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild‐type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9‐(diethylamino)‐5H‐benzo(α) phenoxazine‐5‐one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.
Characterization of a novel type of chain‐terminator Galβ1–6Galβ1–4)GlcNAc in an oligosaccharide related to N‐glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis
Authors: MICHALSKI Jean Claude , LEMOINE Jérôme , WIERUSZESKI Jean Michel , FOURNET Bernard , MONTREUIL Jean , STRECKER Gérard 
Published: March 2005

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical‐ionization mass spectrometry and 400‐MHz 1H‐NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N‐acetyllactosamine‐type glycan:

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The second is unusual with a terminal disaccharide Galβ1‐6Gal, which had not yet been described for glycans of the N‐acetyllactosamine type:
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Cytosolic l‐alanine:4,5‐dioxovalerate transaminase differs from the mitochondrial form
Authors: SINGH Nishi Kant , TYAGI Rakesh Kumar , DATTA Kasturi 
Published: March 2005

l‐Alanine:4,5‐dioxovalerate transaminase was detected in the kidney cytosolic fraction with a lower specific activity than the mitochondrial enzyme. The enzyme was purified from the cytosol to homogeneity with a yield of 32%, and comparative analysis with the mitochondrial form was performed. Both forms of the enzyme have identical pH and temperature optima and also share common antigenic determinants. However, differences in their molecular properties exist. The molecular mass of the native cytoplasmic enzyme is 260 kDa, whereas that of the mitochondrial enzyme is 210 kDa. In addition, the cytoplasmic l‐alanine:4,5‐dioxovalerate transaminase had a homopolymeric subunit molecular mass of 67 kDa compared to a subunit molecular mass of 50 kDa for the mitochondrial l‐alanine:4,5‐dioxovalerate transaminase. This is the first report of two forms of l‐alanine:4,5‐dioxovalerate transaminase. The different responses of cytosolic and mitochondrial l‐alanine:4,5‐dioxovalerate transaminases to hemin supplementation both in vitro and in vivo was demonstrated. Maximum inhibition of mitochondrial l‐alanine:4,5‐dioxovalerate transaminase activity was demonstrated with hemin injected at a dose of 1.2 mg/kg body mass, whereas the same dose of hemin stimulated the cytosolic enzyme to 150% of the control. A one‐dimensional peptide map of partially digested cytosolic and mitochondrial l‐alanine:4,5‐dioxovalerate transaminase shows that the two forms of the enzymes are structurally related. Partial digestion of the cytosolic form of the enzyme with papain generated a fragment of 50 kDa which was identical to that of the undigested mitochondrial form (50 kDa). Moreover, papain digestion resulted in a threefold increase in cytosolic enzyme activity over the native enzyme, and such enhancement was comparable to the activity of the mitochondrial form of the enzyme. Therefore, we conclude that the cytosolic form of l‐alanine:4,5‐dioxovalerate transaminase is different from the mitochondrial enzyme.Furthermore, immunoblot analysis indicated that the mitochondrial enzyme has antigenic similarity to the cytosolic enzyme as well as to the papain‐digested cytosolic enzyme 50‐kDa fragment.
Modulation by oxygen of the glucagon‐dependent activation of the phosphoenolpyruvate carboxykinase gene in rat hepatocyte cultures
Authors: HELLKAMP Josef , CHRIST Bruno , BASTIAN Helge , JUNGERMANN Kurt 
Published: March 2005

In liver phosphoenolpyruvate carboxykinase (PCK) activity, protein and mRNA are localized predominantly in the periportal zone. The activation of the PCK gene by glucagon was studied in primary rat hepatocyte cultures under physiological arterial and venous oxygen tensions [16% and 8% (by vol.)]. PCK gene expression was monitored on the level of transcription, mRNA abundance and enzyme activity as well as enzyme synthesis and degradation.

Transcription of the PCK gene was increased by 10 nM glucagon maximally after 0.5 h; it reached nearly basal levels again after 2 h. The increase in transcription was 45% lower under 8% oxygen than under 16% oxygen.
PCK mRNA was maximally increased after 2 h under 16% oxygen and after 4 h under 8% oxygen; it subsequently declined to twice the basal values after 8 h. The maximal increase after 2 h was 50% lower under 8% oxygen than under 16% oxygen.
PCK enzyme activity was maximally increased after 4–6 h. The maximal enhancement after 4 h was 50% lower under 8% oxygen than under 16% oxygen.
The increase in PCK enzyme activity was due to an enhanced synthesis rate of PCK protein. The rate increase after 3 h was 35% lower under 8% oxygen than under 16% oxygen.
The degradation of PCK protein was equal under both oxygen tensions.

The results show that in cultured rat hepatocytes the induction of PCK gene expression is modulated by physiological concentrations of oxygen. The modulation occurred at the level of gene transcription, mRNA abundance, enzyme protein synthesis and enzyme activity. The periportal to perivenous oxygen gradient could be the major factor responsible for the predominant expression of the PCK gene in the periportal zone.
Sphingolipid biosynthesis in cultured neurons
Authors: MANDON Elisabet C. , van ECHTEN Gerhild , BIRK Rolf , SCHMIDT Richard R. , SANDHOFF Konrad 
Published: March 2005

Addition of exogenous sphingosine homologues (D‐erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time‐ and concentration‐dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half‐maximal reduction of enzyme activity occurred after a 4‐h treatment with 25 μM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4–8 h with 50 μM long‐chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10–50 μM), was fed to the cells, decrease of 3‐ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D‐erythro‐sphingosine, L‐threo‐C18‐sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions.Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long‐chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long‐chain acyl‐CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.
Purification and properties of the phosphofructokinase from Lactobacillus bulgaricus
Authors: LE BRAS Gisèle , DEVILLE‐BONNE Dominique , GAREL Jean‐Renaud 
Published: March 2005

Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6‐phosphate into fructose 1,6‐bisphosphate in Lactobacillus bulgaricus (Lactobacillus delbrueckii, subspecies bulgaricus) has been purified to homogeneity and some of its structural and functional properties have been studied. The enzyme is a tetramer composed of four 35‐kDa subunits. Its N‐terminal sequence determined on 38 residues is homologous to those of the major allosteric enzymes from Escherichia coli and Bacillus stearothermophilus, suggesting that the three proteins have closely related structures.The maximum velocity of the enzyme from L. bulgaricus increases with pH according to the ionization of a group with a pK of 6.2. At all pH values, the saturation by fructose 6‐phosphate is hyperbolic. At the optimum pH of 8.2, the maximum velocity and the affinities for the ATP and fructose 6‐phosphate substrates are not modified by the presence of ADP or GDP nor by phosphoenolpyruvate. Partial inhibition by phosphoenolpyruvate exists at acidic pH, but is not related to an allosteric mechanism similar to that in E. coli. This inhibition results from a shift from 6.2 to 7.1 of the pK of an ionizable group which controls Vmax. Protection against thermal denaturation shows that the enzyme binds phosphoenolpyruvate and not GDP. The phosphofructokinase from L. bulgaricus appears as a structural analog of the E. coli enzyme which does not undergo an allosteric transition between two states R and T, but instead remains in a unique conformational state, intermediate between the R and T states; the active sites have an R‐like conformation since they bind fructose 6‐phosphate, whereas the regulatory sites have a T‐like conformation since they bind phosphoenolpyruvate and not GDP.
Ribosome‐bound EF‐1α‐like protein of yeast Saccharomyces cerevisiae
Authors: DIDICHENKO Svetlana A. , TER‐AVANESYAN Michael D. , SMIRNOV Vladimir N. 
Published: March 2005

The SUP2 (SUP35) omnipotent suppressor gene encodes the EF‐1α‐like polypeptide, intimately involved in the control of translational ambiguity in the yeast Saccharomyces cerevisiae. The present study is devoted to the immunological characterization of the Sup2 protein.The SUP2 gene was fused to the Escherichia coli lacZ gene and a polyclonal antibody against the corresponding Sup2–β‐galactosidase hybrid protein was obtained. This antibody identified a 79‐kDa protein that was absent in those cells where the SUP2 gene was disrupted, and an abundance of this protein was observed in cells overexpressing the SUP2 gene. The localization of this protein was studied in subcellular fractionation experiments. The SUP2 gene product proved to be uniformly distributed throughout ribosome‐enriched samples, i.e. free polysomes, crude microsomes and rough endoplasmic reticulum. It was not found in the cytoplasm and smooth endoplasmic reticulum. The SUP2‐encoded protein was fully ribosome associated and less abundant than the ribosomal protein L3. Also, in a sucrose gradient, Sup2 preferentially cosedimented with the 40S ribosomal subunit, but not with the 60S subunit. The functional significance of this association is discussed.
Inhibitors of metabolic reactions
Authors: LILL Ute , KOLLMANN‐KOCH Angelika , BIBINGER Angelika , EGGERER Hermann 
Published: March 2005

Substrate and intermediate analogue inhibitors of enzymes were prepared in which the thioester oxygen of acyl‐CoA substrates is replaced by hydrogen with formation of CoA‐thioethers.Experiments performed with ATP citrate lyase and S‐(3,4‐dicarboxy‐3‐hydroxybutyl)‐CoA are consistent with citryl‐CoA but not with citryl‐enzyme being the direct precursor of the products acetyl‐CoA and oxaloacetate. Consistent with these results, a previously described isotopic exchange between acetyl‐CoA and [3H]CoASH, indicating the formation of an acetyl‐enzyme in the reaction pathway, could not be confirmed.Substrate analogue CoA‐thioethers of malate synthase are inhibitors endowed with the affinity of the substrates. Acetyl carboxylase and fatty acid synthetase are not inhibited by the substrate analogue S‐ethyl‐CoA; S‐carboxyethyl‐CoA, which could substitute for malonyl‐CoA, is likewise not inhibitory. An explanation is proposed.Previously suggested roles of S‐carboxymethyl‐CoA, an acetyl‐CoA‐related inhibitor of citrate synthase, are discussed in the light of new experimental data. S‐Acetyl, S‐propionyl and S‐carboxymethyl derivatives of 1,N6‐etheno‐CoA loose the high affinity of their CoA‐counterparts to citrate synthase, probably because the ethylene group prevents proper binding to the enzyme.
Ligand binding at membrane mimetic interfaces
Authors: DESFOSSES Bernard , CITTANOVA Nicole , URBACH Wladimir , WAKS Marcel 
Published: March 2005

The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenyl‐butazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2‐ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane‐water interface. Upon micellar incorporation, the dansylsarcosine‐albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin‐albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 × 104 M−1 cm−1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Kaη 1.0 × 105 M−1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant.These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2‐ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N‐bromosuccinimide, indicate perturbations of the sole tryptophan‐214 microenvironment. However, the distance between the indole residue and tyrosine‐411 does not seem substantially modified by the 15% decrease affecting the α helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane‐like interface which generates two protein subpopulations differing in their membrane‐surface and ligand affinities. Overall and local conformational changes, originating from this surface‐induced effect, may thus constitute a ligand‐release facilitating mechanism acting at cellular membrane levels.
Purple acid phosphatase from bovine spleen
Authors: DIETRICH Markus , MÜNSTERMANN Dieter , SUERBAUM Hildegard , WITZEL Herbert 
Published: March 2005

Oxidation of the reduced (pink) phosphate‐free bovine spleen acid phosphatase with 1.5 mol H2O2 or sodium peroxodisulfate/mol, in the presence of Mes or Bistris pH 5, leads to a species with an absorption maximum at 558 nm. Addition of acetate or oxidation in the presence of acetate buffer engenders a species with a maximum at 550 nm. Addition of phosphate to both species shifts the maximum immediately to 540 nm; this is the species also found after preparation from the spleen. The assumption that these species represent strongly bidentate‐binding hydroxo, acetato and phosphato complexes of the Fe(III)‐Fe(III) system is supported by replacement reactions with other ligating oxoanions followed by their typical spectral shifts. These oxoanion complexes cannot be dissociated by gel filtration; this is possible only after reduction to the Fe(II)‐Fe(III) system. The oxidized species without EPR signals below g values of 2 still reveals 5% activity which cannot be reduced to zero even in the presence of higher concentrations of peroxodisulfate. The pH optimum of the reaction with α‐naphthyl phosphate shifts from 5.9 to 5.3 in the oxidized species.The apparent pK values around 4.5 as derived from the pH dependence of activity, of the EPR spectra, and the spectral shifts of the phosphate‐saturated reduced and oxidized species are assigned to an aquo/hydroxo equilibrium at the Fe(III) or an equilibrium, where the phosphato ligand is replaced by a hydroxo ligand. A reaction mechanism is proposed in which a hydroxo ligand at the chromophoric Fe(III) attacks the phosphoric acid ester group only when that is monoprotonated and pre‐oriented by electrostatic interaction with the nonchromophoric metal ion. Binding and inhibition studies with the oxoanions indicate that they compete with the catalytically active hydroxo group of the reduced and oxidized enzyme with nearly the same inhibition constants. Catalysis is not affected by the oxoanions which replace the additional μ‐hydroxo ligand in the 558‐nm‐absorbing Fe(III)‐Fe(III) species. In contrast to hemerythrin and ribonucleotide reductase, a binuclear iron center is proposed for the purple acid phosphatase, which is bridged by a carboxylato and two aquo/hydroxo groups, but without a μ‐oxo bridge.