bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of the Gene Encoding Pyrococcus Furiosus Ornithine Carbamoyltransferase and Study of its Expression Profile In Vivo and In Vitro
Authors: Roovers Martine , Hethke Carina , Legrain Christianne , Thomm Michael , Glansdorff Nicolas
Published: July 2004

The gene coding for ornithine carbamoyltransferase (OTCase, argF) in the hyperthermophilic archaea Pyrococcus furiosus was cloned by complementation of an OTCase mutant of Esckerickia coli. The cloned P. furiosus argF gene also complemented a similar mutant of Sacckaromyces cerevisiae. Sequencing revealed an open reading frame of 314 amino acids homologous to known OTCases and preceded by a TATA box showing only limited similarity with the Euryarchaeota consensus sequence. This is in accordance with the comparatively low in vitro promoter activity observed in a cell‐free purified transcription system. Transcription initiates in vivo as well as in vitro at a guanine, 22 nucleotides downstream of the TATA box. Upstream from argF is a putative gene for diphthine synthetase, a eukaryotic enzyme assumed to occur also in archaea but not in bacteria.
Biochemical Characterisation of Ornithine Carbamoyltransferase from Pyrococcus Furiosus
Authors: Legrain Christianne , Villeret Vincent , Roovers Martine , Gigot Daniel , Dideberg Otto , Piérard André , Glansdorff Nicolas
Published: July 2004

Ornithine carbamoyltransferase (OTCase) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is a 400±220‐kDa polymer of a 35‐kDa subunit, in keeping with the corresponding gene sequence [Roovers, M., Hethke, C., Legrain, C., Thomm, M. & Glansdorff, N. (1997) Isolation of the gene encoding Pyrococcus furiosus ornithine cabamoyltransferase and study of its expression profile in vivo and in vitro, Eur: J. Biochem. 247, 1038–10451. In contrast with the dode‐cameric catabolic OTCase of Pseudomonas aeruginosa, P furiosus OTCase exhibits no substrate cooperativity. In keeping with other data discussed in the text, this suggests that the enzyme serves an anabolic function. Half‐life estimates for the purified enzyme ranged over 21–65 min at 100°C according to the experimental conditions and reached several hours in the presence of ornithine and phosphate. The stability was not markedly influenced by the protein concentration. Whereas comparative examination of OTCase sequences did not point to any outstanding feature possibly related to thermophily, modelling the enzyme on the X‐ray structure of P aeruginosa OTCase (constituted by four trimers assembled in a tetrahedral manner) suggests that the molecule is stabilized, at least in part, by a set of hydrophobic interactions at the interfaces between the trimers. The comparison between P aeruginosa and P furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starting from a similar quaternary structure of high internal symmetry. Recombinant P furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme. In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained.
A Reciprocal Allosteric Mechanism for Efficient Transfer of Labile Intermediates between Active Sites in CAD, the Mammalian Pyrimidine‐Biosynthetic Multienzyme Polypeptide
Authors: Irvine Heather S. , Shaw Seán M. , Paton Ann , Carrey Elizabeth A.
Published: July 2004

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240‐kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate.

Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition‐state analogue phosphonacetyl‐L‐aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5′‐phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell.

We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition‐state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.
Expression and Characterization of a Lactosaminoglycan‐Carrying Glycoprotein of Zajdela Hepatoma Cell Surface
Authors: Goulut‐Chassaing Chantal , Bourrillon Roland
Published: July 2004

In poorly differentiated hepatoma cells, a glycoprotein carrying lactosaminoglycans is identified, and the structure of its glycan moiety is proposed. After membrane solubilization, protein fractionation by gel filtration, and electroelution, this glycoprotein (GPIII) was identified by its affinity for Datura stramonium lectin and its content in large glycopeptides. As shown by PAGE, GPIII has an apparent molecular mass of 100 kDa and is highly glycosylated (36%). It appears as an integral membrane glycoprotein. It is absent from normal hepatocytes, in that no heavy glycopeptides could be detected that bound to Datura lectin or to specific antiserum. The glycan moiety of GPIII has been analyzed according to carbohydrate composition, glycosidase treatment, affinity chromatography on immobilized pokeweed, Datura and Griffonia lectins, and by NMR and methylation analyses. The glycan is a N‐linked tetraantennary lactosami‐noglycan of 6.6 kDa, containing Gal, GlcNAc, Man, and NeuNAc in a 16:14:3:4 molar ratio, with an average of three repeating unitshranch. Its β‐Gal residues are in the penultimate position and are linked in β1–4 at least in four structural elements (three peripheral and one internal). It contains a very branched structure with Galα1–3Galβ1–4GlcNAc side chains linked in the C6 position to an inner Gal residue in a main branch. α‐Gal and NeuNAc residues [mainly NeuNAcα(2–3) linkage] are expressed as the non‐reducing terminal groups. A possible structural model is proposed for this heterogeneous lactosaminogly‐can, although no definitive structure can be established. That this lactosaminoglycan‐carrying glycoprotein GPIII is not expressed in hepatocytes suggests its expression to be linked to the undifferentiated and/or malignant state of this hepatoma.
Hypoxanthine Uptake through a Purine‐Selective Nucleobase Transporter in Trypano Soma Brucei Brucei Procyclic Cells is Driven by Protonmotive Force
Authors: De Koning Harry P. , Jarvis Simon M.
Published: July 2004

The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22°C was rapid and saturable, exhibiting an apparent Km, of 9.3±2.0 μM and a Vmax of 4.5±0.8 pmol · (107cells)−1· s−1. All the natural purine nucleobases tested (Km 1.8–7.2 μM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high‐affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N‐ethylmaleimide and N, N′‐dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2′, 7′‐bis‐(carboxyethyl)‐5, 6‐carboxy‐fluorescein to determine membrane potential and intracellular pH (pHi, the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH7 after base‐loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N‐ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electro‐genic. These data show that hypoxanthine uptake in T b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+‐symporter model for this transporter.
Purification, Characterization and Partial Amino Acid Sequencing of Hydroxycinnamoyl‐CoA: Tyramine N‐(hydroxycinnamoyl)transferase from Tobacco Cell‐Suspension Cultures
Authors: Negrel Jonathan , Javelle Francine
Published: July 2004

We report the purification of hydroxycinnamoyl‐CoA: tyramine N‐(hydroxycinnamoy1)transferase (THT) to apparent homogeneity in 12% yield from tobacco (Nicotiana tabacum L. cv. Xanthi) cell‐suspension cultures elicited with a commercial preparation of pronase. The purification procedure employs only four chromatography steps and takes advantage of the fact that the transferase binds tightly both to phenyl‐Sepharose and to hydroxyapatite. The native enzyme has a PI of 5.2 and consists of two identical or very similar subunits of approximately 24 kDa. The purified enzyme can synthesise a wide range of amides due to its relatively low specificity for cinnamoyl‐CoA derivatives and hydroxyphenethylamines, but its best substrates are tyramine and feruloyl‐CoA. THT follows Michaelis‐Menten kinetics in the presence of low concentrations of feruloyl‐CoA but negative cooperativity occurs when this concentration increases above 2.5 μM, resulting in a marked decrease of the affinity for tyramine. Large deviations from Michaelis‐Menten kinetics are also observed when 3‐methoxytyramine is used as acyl acceptor. The activity of tobacco THT is not affected by the addition of CaCl2, or MgCl2, but its maximal velocity is increased up to twofold by addition of ethanol to the assay mixture. It is inhibited in vitro by L‐tyrosine benzyl ester, which binds reversibly to the tyramine‐binding site. Experiments performed using L‐tyrosine benzyl ester and caffeoyl‐CoA as inhibitors confirm that feruloyl‐CoA is the first substrate to add to the transferase in an ordered bi‐bi mechanism. Part of the amino acid sequence of the transferase, elucidated by microsequencing of tryptic peptides, is also described.
The Heat‐Shock Protein HslVU from Escherichia Coli is a Protein‐Activated ATPase as well as an ATP‐Dependent Proteinase
Authors: Seol Jae Hong , Yoo Soon Ji , Shin Dong Hun , Shim Yoon Kyung , Kang Man‐Sik , Goldberg Alfred L. , Chung Chin Ha
Published: July 2004

HslVU in Escherichia coli a new two‐component ATP‐dependent protease composed of two heat‐shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome β‐type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B‐chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250‐kDa complex) to HslU (a 450‐kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20‐fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2–3‐fold in the presence of HslV. Concentrations of insulin B‐chain that maximally stimulated the ATPase allowed maximal rates of the B‐chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N‐ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein‐activated ATPase as well as an ATP‐dependent proteinase, and these processes appear linked.

Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP‐dependent hydrolysis of peptides and proteins appear different.
Phosphorylation of Human General Transcription Factors TATA‐Binding Protein and Transcription Factor IIB by DNA‐Dependent Protein Kinase
Authors: Chibazakura Taku , Watanabe Fumiaki , Kitajima Shigetaka , Tsukada Kinji , Yasukochi Yukio , Teraoka Hirobumi
Published: July 2004

DNA‐dependent protein kinase (DNA‐PK) has been known to catalyze phosphorylation of a number of regulatory factors involved in DNA replication and transcription such as simian virus 40 T antigen, p53, c‐Myc, Spl, and RNA polymerase II (Pol II). We examined the possibility that DNA‐PK phosphory‐lates the general transcription factors TATA‐binding protein (TBP) and transcription factor (TF) IIB, which play key roles in the formation of transcription initiation complex with Pol II. By using a highly purified preparation of DNA‐PK from Raji cells, both TBP and TFIIB were shown to be phosphorylated in vitro by DNA‐PK. We then investigated the effect of the phosphorylation of these factors on Pol II basal transcription. Stepwise analysis of preinitiation complex formation by electrophoretic mobility shift assay revealed that the phosphorylation of TBP and TFIIB by DNA‐PK did not affect the formation of promoter (P)‐TBP and P‐TBP‐TFIIB complexes but synergistically stimulated the formation of P‐TBP‐TFIIB‐TFIIF‐Pol II complex. Similarly, combination of the phosphorylated TBP and TFIIB synergistically stimulated Pol II basal transcription from adenovirus major late promoter. These observations suggest that DNA‐PK could positively regulate the Pol II basal transcription by phosphorylating TBP and TFIIB.
The Biochemical Effect of the Naturally Occurring Trp644→Arg Mutation on Human β3‐Adrenoceptor Activity
Authors: Piétri‐Rouxel France , St John Manning Brian , Gros Jérôme , Strosberg A. Donny
Published: July 2004

A Trp→Arg mutation at amino acid position 64 in the human β3‐adrenoceptor is reportedly associated with morbid obesity; carriers suffer from increased gain in mass, early‐onset diabetes, insulin resistance, and an increased waist‐to‐hip ratio [Clément, K., Vaisse, C., Manning, B. S., Basdevant, A., Guy‐Grand, B., Ruiz, J., Silver, K. D., Shuldiner, A. R., Froguel, R & Strosberg, A. D. (1995) N. Engl. J. Med. 333, 352–3541. Here, we report the stable expression of the genes encoding the wild‐type or the [Arg64]β3‐adrenoceptor in two different cell types: hamster CHO‐K1 and human HEK293. The mutated receptor displayed unchanged pharmacological values compared to the wild type for the binding inhibition (Ki) and adenylyl cyclase activation constants (Kact) in two independent clones of both cell lines. However, maximal cAMP accumulation was significantly reduced in response to various β3‐adrenergic agonists, including endogenous catecholamines, (−)‐epinephrine and (–)‐norepinephrine, the non‐selective agonist (−)‐isoproterenol, and the β3‐adrenergic selective agonist CGP 12177A. Treatment with Pertussis toxin did not restore the adenylyl cyclase activity to that of the wild type, suggesting that the reduction in cAMP accumulation observed in cells expressing [Arg64]β3‐adrenoceptor is not due to enhanced interaction of the β3‐adrenoceptor with the inhibitory Gi protein.
Participation of a Stress‐Activated Protein Kinase Cascade in the Activation of Tyrosine Hydroxylase in Chromaffin Cells
Authors: Thomas Gareth , Haavik Jan , Cohen Philip
Published: July 2004

Sodium arsenite and osmotic shock both stimulated stress‐activated protein kinase‐2 (SAPK2, also termed RK, β38, CSBP and Mxi2) and its downstream target mitogen‐activated protein kinase (MAP kinase)‐activated protein kinase‐2 (MAPKAP‐K2) in bovine adrenal chromaffin and rat PC12 cells. The same stimuli also increased tyrosine hydroxylase activity 2–3‐fold and induced its phosphorylation at Serl9, a residue phosphorylated by MAPKAP‐K2 in vitro. The arsenite‐induced activation of tyrosine hydroxylase and its phosphorylation at Ser19 were prevented by SB 203580 at concentrations similar to those that inhibited SAPK2 in vitro. These results indicate that MAPKAP‐K2 mediates the stress‐induced activation of tyrosine hydroxylase. SB 203580 had no effect on the phosphorylation or activation of tyrosine hydroxylase induced by nerve growth factor or forskolin, which trigger the phosphorylation of Ser31 and Ser40, respectively. Stimulation of bovine adrenal chromaffin cells with acetylcholine activated SAPK2 and MAPKAP‐K2, as well as p42/p44 MAP kinases and their downstream target MAPKAP‐K1. The half‐times for activation of MAPKAP‐K1 and MAPKAP‐K2 (1 min) were similar. In contrast, the activation of tyrosine hydroxylase by acetylcholine peaked within 1 min and gradually declined thereafter. Neither SB 203580 (which blocked the activation of MAPKAP‐K2 by acetylcholine) nor PD 98059 (which prevented the activation of p42/p44 MAP kinases by acetylcholine) affected tyrosine hydroxylase activation after 1 min, but these compounds inhibited activation by 40–50% after 5 min. PD 98059 prevented the acetylcholine‐induced phosphorylation of tyrosine hydroxylase at Ser31, the residue targetted by p42Ip44 MAP kinases in vitro, but did not inhibit the phosphorylation of Ser40 (which is phosphorylated by MAPKAP‐K1 in vitro). Our results establish that p42/p44 MAP kinases mediate the acetylcholine‐induced phosphorylation of tyrosine hydroxylase at Ser31. SB 203580 did not suppress the phosphorylation of Serl 9 or Ser40 by acetylcholine but, like PD 98059, this drug decreased the phosphorylation of Ser31. SAPK2 may therefore contribute to the acetylcholine‐induced activation of tyrosine hydroxylase by facilitating (in an unknown way) its phosphorylation by MAP kinases.
Identification of an Ubiquitin‐Ligation System for the Epidermal‐Growth‐Factor Receptor
Authors: Mori Seijiro , Tanaka Keiji , Kanaki Harumi , Nakao Mitsuyoshi , Anan Tadashi , Yokote Koutaro , Tamura Ken , Saito Yasushi
Published: July 2004

Some receptor tyrosine kinases such as the receptors for epidermal‐growth factor (EGF) and platelet‐derived growth factor undergo polyubiquitination as a consequence of ligand binding. The EGF receptor is also ubiquitinated by treatment with herbimycin A, an ansamycin antibiotic widely used as a tyrosine kinase inhibitor. To investigate the mechanism of the receptor ubiquitination, we have established an assay system in which herbimycin‐A‐induced ubiquitination processes can be analyzed in vitro. We now show that herbimycin A treatment of the purified EGF receptor induces polyubiquitination of the receptor in rabbit‐reticulocyte lysate. Both DEAE unadsorbed material (fraction I) and high salt eluate (fraction II) of the reticulocyte lysate are involved cooperatively in the ubiquitination process, where the ubiquitin‐conjugating enzyme UBC4 can functionally substitute for fraction I. A ubiquitin‐protein ligase‐like activity, partially purified from fraction II by DEAE anion‐exchange chromatography, also functions in concert with UBC4. The precise mechanism of herbimycin A‐induced ubiquitination of the EGF receptor is not fully understood, however, our present findings suggest that direct interaction with herbimycin A results in some modification of the receptor which is recognized by the ubiquitin‐conjugating system in rabbit‐reticulocyte lysate.
The Structure of the Carbohydrate Backbone of the Lipopolysaccharide from Acinetobacter Strain ATCC 17905
Authors: Vinogradov Evgeny V. , Bock Klaus , Petersen Bent O. , Holst Otto , Brade Helmut
Published: July 2004

The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter strain ATCC 17905 was studied. After deacylation of the lipopolysaccharide, a mixture of two compounds (ratio approximately 2:1) was isolated by high‐performance anion‐exchange chromatography, the structures of which were determined by NMR spectroscopy and electrospray‐mass spectrometry as

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[Sug, 3‐deoxy‐d‐manno‐2‐octulopyranosonic acid (Kdo) in oligosaccharjde 1 (major portion) and d‐glyc‐ero‐d‐talo‐2‐octulopyranosonic acid (Ko) in oligosaccharide 2 (minor portion)]. All monosaccharide residues also possess the d‐configuration and are present in the pyranose form.
The Effect of Platelet‐Derived Growth Factor and Adipogenic Hormones on the Expression of CCAAT/Enhancer‐Binding Proteins in 3T3‐L1 Cells in Serum‐Free Conditions
Authors: Bachmeier Manfred , Löffler Georg
Published: July 2004

In the absence of serum and serum substitutes, insulin at high doses together with platelet‐derived growth factor BB (PDGF BB), corticosterone, and 3‐isobutyl‐1‐methyl‐xanthine is required to stimulate differentiation of 3T3‐L1 preadipocytes. Under these conditions the differentiating cells express the CCAAT/enhancer‐binding proteins (C/EBP) C/EBPδ, C/EBPβ, and C/EBPα with a similar sequence as described for differentiating cells in the presence of serum. The major differences detected under serum‐free conditions are as follows: (a) PDGF BB is the major stimulator of the expression of the C/EBPβ isoform liver activator protein (LAP). (b) The expression of LAP is also increased in preconfluent, proliferating cells due to the treatment with PDGF BB. (c) A small protein of 20 kDa (p20C/EBPβ) is detected with the anti‐C/EBPβ antibody. It is synthesized at high levels in such cells, which subsequently express high levels of the differentiation markers C/EBPα and glycerol‐3‐phosphate dehydrogenase. (d) Treatment of cells with fibroblast growth factor‐2 (bFGF) in addition to adipogenic hormones results in differentiation and C/EBPα expression only to a very moderate extent as compared to treatment with PDGF BB but leads to a strong expression of both C/EBPβ and C/EBPδ.
Distinct Efficacies for Two Endogenous Ligands on a Single Cognate Gonadoliberin Receptor
Authors: Tensen Cornelis , Okuzawa Koichi , Blomenröhr Marion , Rebersi Frank , Leurs Rob , Bogerd Jan , Schulz Rüdiger , Goos Henk
Published: July 2004

A cDNA encoding a putative gonadoliberin receptor was cloned from the pituitary of the African catfish. Conceptual translation predicts a protein of 379 amino acids which shows typical characteristics of GTP‐binding‐protein‐coupled receptors. The isolated cDNA was stable expressed in human embryonic kidney (HEK) 293 cells which were used for studies on gonadoliberin‐activated second messenger systems (inositol phosphate production; increase in CAMP andor intracellular Ca2+). The isolated cDNA encoded a functional receptor, designated catfish gonadoliberin receptor (cfGnRH‐R), which had an amino acid sequence similarity of 38% with mammalian gonadoliberin receptors. In contrast to its mammalian counterparts which lack an intracellular carboxy‐terminal domain, the cfGnRH‐R contains an additional 49 amino acid residues. From the two endogenous gonadoliberins in African catfish, chicken gonadoliberin‐11 had a several hundredfold higher potency than catfish gonadoliberin to activate cfGnRH‐R‐associated second messenger systems in transfected HEK 293 cells. This is in line with the previously determined higher gonadotropin‐release capacity of chicken gonadoliberin‐I1 in catfish. Stimulation of second messenger systems with chicken gonadoliberin‐11, but not with catfish gonadoliberin, resulted in a biphasic effect and chicken gonadoliberin‐II led to a higher maximum stimulation than catfish gonadoliberin. Challenging cfGnRH‐R simultaneously with chicken gonadoliberin‐II and catfish gonadoliberin did not lead to additive effects. In contrast, two types of mutual inhibitory effects were recorded. These data indicate that a single cognate cfGnRH‐R couples with distinct efficacies to signal transduction systems upon stimulation by the two endogenous gonadoliberins which, in addition, may interact negatively.
Seryl‐tRNA Synthetase from the Extreme Halophile Haloarcula marismortui
Authors: Taupin Claire M.‐J. , Härtlein Michael , Leberman Reuben
Published: July 2004

The seryl‐tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortul, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2, solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol, there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2, stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme.

The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl‐tRNA synthetase. The wild‐type gene and a mutant gene with a deletion of the halophile‐specific insertion were expressed in E. coli using the T7 RNA polymerase and the ThiofusionTM expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl‐tRNA synthetases which illustrates the high negative‐charge density of the surface of the hyperhalophilic enzyme.
Structural and Serological Characterisation of the O‐Specific Polysaccharide from Lipopolysaccharide of Acinetobacter calcoaceticus Strain 7 (DNA Group 1)
Authors: Vinogradov Evgeny V. , Pantophlet Ralph , Haseley Simon R. , Brade Lore , Holst Otto , Brade Helmut
Published: July 2004

S‐form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1). The structure of the O‐antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de‐O‐acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure:

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in which Pyr is pyruvate. The O‐acetyl substitution of D‐Gal was non‐stoichiometric. The O‐antigen was specifically recognised in western blots by polyclonal rabbit antisera.
Modulation of the Metarhodopsin I/Metarhodopsin II Equilibrium of Bovine Rhodopsin by Ionic Strength
Authors: Delange Frank , Merkx Maarten , Bovee‐Geurts Petra H. M. , Pistorius Arthur M. A. , Degrip Willem J.
Published: July 2004

The effects of ionic strength on formation and decay of metarhodopsin II (MII), the active photointer‐mediate of bovine rhodopsin, were studied in the native membrane environment by means of ultraviolet/visible and Fourier‐transform infrared (FTIR) spectroscopy. By increasing the concentration of KCI in the range from hypotonic to 4 M, the apparent pKa of the metarhodopsin I(MI)/MII equilibrium is shifted by approximately pH three, in favor of the MII intermediate. In addition, the apparent rate of MII formation is enhanced by an increase in ionic strength (about twofold in the presence of 2 M KCI). MII decay is independent of the salt concentration. Attenuated‐total‐reflectance/FTIR data show that the high‐salt conditions have no effect on the rigidity of the membrane matrix and do not induce structural changes in the intermediates themselves. Different salts were tested for their ability to shift the MI/MII equilibrium; however, no clear ion dependence was observed. We interpret these results as an indication for direct involvement of the cytosolic surface charge in the regulation of the photochemical activity of bovine rhodopsin.
Biosynthesis of Renin in Mouse Kidney Tumor As4.1 Cells
Authors: Jones Craig A. , Petrovic Nenad , Novak Edward K. , Swank Richard T. , Sigmund Curt D. , Gross Kenneth W.
Published: July 2004

As4.1, a renin‐expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42–47 kDa. The predominant intracellular renin was processed into two chains, of 33–34 and 5 kDa. N‐glycanase treatment removed N‐linked oligosaccha‐rides and yielded products of 41 kDa for prorenin and 31–32 kDa for the heavier chain of two‐chain renin. The N‐terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N‐terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 Z1.4% (mean percentage of total precipitable radioactivity2SD) of de‐novo‐synthesized protein secreted into the medium and 0.2±0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell−1 h−1. Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, β‐glucuronidase, by double‐fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin‐1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.
Purification, Structure and In vitro Molecular‐Chaperone Activity of Artemia P26, a Small Heat‐Shockh/α‐Crystallin Protein
Authors: Liang Ping , Amons Reinout , Macrae Thomas H. , Clegg James S.
Published: July 2004

Encysted brine‐shrimp gastrulae bring their metabolism to a reversible standstill during diapause and quiescence, demonstrating a remarkable resistance to unfavourable environmental conditions. For example, mortality of Artemia embryos under normal temperature and hydration is very low, even after two years of anoxia, and embryos commonly experience complete desiccation as part of their developmental program. Previous evidence from our laboratories indicated that p26, an abundant low‐molecular‐mass cyst‐specific protein capable of translocation into the nucleus, may have a protective function in Artemia cysts. p26 was purified to apparent homogeneity and a continuous sequence of 141 of its amino acids was determined by peptide sequencing, revealing that it is a member of the small‐heat‐shock/α‐crystallin family of proteins. As determined by molecular‐sieve chromatography and sucrose‐density‐gradient centrifugation, native p26 is a multimer of about 27 monomers with a molecular mass of approximately 700 kDa. Inactivation of citrate synthase was less when the enzyme was heated in the presence rather than the absence of p26. Additionally, the renaturation of heat‐inactivated citrate synthase was promoted by p26. These results indicated that p26 possesses molecular‐chaperone activity, a property of other small heat‐shock/u‐crystallin proteins. Our findings demonstrate that p26 has the potential to protect the macromolecular components of Artemia embryos, either as they encyst or upon exposure to environmental extremes. Protection may depend upon the ability of p26 to function as a molecular chaperone.
Metabolic Response in Arenicola marina to Limiting Oxygen as Reflected in the 1H‐NMR Oxymyoglobin Signal
Authors: Kreutzer Ulrike , Jue Thomas
Published: July 2004

Many intertidal animals can endure prolonged periods of environmental stress and have developed strategies to preserve a functioning energy state in the cell. Recent 1H/31P‐NMR techniques have allowed investigators to monitor directly mammalian tissue metabolism in vivo. In particular, the signals of myoglobin (Mb) offer a unique opportunity to explore the intracellular oxygen‐partial‐pressure [p(O2)] interaction in Arenicolu marina, a standard model to study hypoxia tolerance in invertebrates. The present study reveals that the 1H‐NMR MbO, signal at ‐2.9 ppm is detectable in tissue and reflects directly the oxygenated state. As the p (02) declines, MbO2, saturation and oxygen consumption decrease. However, phosphotaurocyamine concentration remains unaltered until the MbO2, saturation falls below 33%. The extracellular to intracellular p (O2) gradient appears substantial. The study establishes the 1H‐NMR technique as an approach to measure the intracellular p (O2) with an oxygenated state marker and presents the interrelationship between oxygen and the metabolic adaptation during hypoxic stress.
Effects of Spermine and Its Cytotoxic Analogs on Nucleosome Formation on Topologically Stressed DNA In vitro
Authors: Basu Hirak S. , Smirnov Ivan V. , Peng HongFan , Tiffany Karen , Jackson Vaughn
Published: July 2004

We investigated the effects of the polyamine spermine and two of its cytotoxic analogs 1,11‐bis(ethyl‐amino)‐4,8‐diazaundecane (BE‐3‐3‐3) and 1,19‐bis(ethylamino)‐S,10,15‐tirazanonadecane (BE‐4‐4‐4‐4) on the formation of nucleosomes on negatively and positively supercoiled DNA in vitro. Histones H2A, H2B, H3 and H4 were reconstituted onto DNA to form nucleosomes and the polyamines were added either before or after histone addition. The structural state of the nucleosome was monitored by analyzing the DNA topoisomers that were present after topoisomerase I treatment. Although polyamines induced DNA aggregation to various degrees, high concentrations of topoisomerase I were able to relax the aggregated DNA and the helical pitch was found to be unaltered in the aggregates. When histones were associated with negatively coiled DNA, the polyamine‐induced aggregation did not alter nucleosome structure. The induced aggregate did inhibit nucleosomal transitions when examined on positively coiled DNA. BE‐4‐4‐4‐4 was most effective and BE‐3‐3‐3 least effective. These analogs were also extremely effective in inhibiting histone deposition onto DNA. A potential mechanism for the action of these analogs is both to inhibit histone deposition during DNA replication and also disrupt nucleosomal dynamics due to aberrant chromatin condensation. These results also suggest that BE‐4‐4‐4‐4 and BE‐3‐3‐3 may produce their cytotoxic effect through slightly different mechanisms.
Mitochondria1 Asparaginyl‐tRNA Synthetase Encoded by the Yeast Nuclear Gene YCR24c
Authors: Landrieu Isabelle , Vandenbol Micheline , Härtlein Michael , Portetelle Daniel
Published: July 2004

One of the open reading frames located on yeast Saccharomyces cerevisiae chromosome III, YCR24c, appeared to code for a protein of unknown function, but the predicted sequence showed similarity with asparaginyl‐tRNA synthetase from Escherichiu coli, with 3896 amino acid identity. There is a putative mitochondrial targeting signal at the N‐terminus of the YCR24c product. Northern blot analysis of total RNA from a wild‐type strain Σ1278b confirmed that YCR24c was transcribed. Disruption of the chromosomal copy of YCR24c in a respiratory‐competent haploid cell induced a petite phenotype, but did not affect cell viability. This respiratory‐defective phenotype is typical for a mutation in a nuclear gene that induces a non‐functional mitochondrial protein synthesis system.

The protein encoded by YCR24c was expressed in Escherichia coli in a histidine‐tagged form and isolated. The enzyme aminoacylated unfractionated Escherichiu coli tRNA with asparagine. These results identified YCR24c as the structural gene for yeast mitochondrial asparaginyl‐tRNA synthetase.
Study of Non‐Covalent Enzyme‐Inhibitor Complexes of Aldose Reductase by Electrospray Mass Spectrometry
Authors: Potier Noelle , Barth Patrick , Tritsch Denis , Biellmann Jean‐François , Van Dorsselaer Alain
Published: July 2004

Specific non‐covalent interactions between aldose reductase (AR), its NADP+ cofactor and five inhibitors have been characterized by electrospray mass spectrometry (ES‐MS). These results indicated that the protein could be desorbed and maintained in the gas phase in a form very close to its native conformation. Collisionally induced dissociation (CID)‐MS and CID‐MS‐MS showed that the adenosine diphospbate part of the cofactor interacts strongly with AR. The relative stability of the ternary AR NADP+ inhibitor complexes was established and successfully correlated with the IC50, values. All inhibitors were shown to only bind to AR holoenzyme. These results are important for the field of drug development insofar as ES‐MS might provide a rapid and very sensitive method for the screening of potential drugs or for the identification of compounds displaying high binding affinity to a target biomolecule.
1H‐NMR Study of Inter‐Segmental Hydrogen Bonds in Sperm Whale and Horse Apomyoglobins
Authors: Yamamoto Yasuhiko
Published: July 2004

NMR signals for HisB5 NδH and HisEF5 NɛH protons of sperm whale and horse apomyoglobins were assigned and compared with the corresponding signals of the holoproteins in terms of pH and temperature dependence behaviors of their shifts and line widths in order to gain insight into structural difference between the apoproteins and the holoproteins. Since these protons are involved in internal hydrogen bonds at the interfaces between the B helix and the GH corner and between the EF corner and the H helix, local structures of the interfaces in these proteins have been inferred from the analyses of these signals. A large difference in the line width of HisEF5 NɛH proton signal between the apoproteins and the holoproteins strongly suggested that a sizable structural alteration is induced in the EF‐H interface by the removal of heme. However, the results for HisBS NδH proton resonance indicated the absence of a significant structural alteration in the B‐GH interface by heme extraction. These results are consistent with the data obtained from mutation [Hughson, F. M. & Baldwin, R. L. (1989) Biochemistry 28, 4425–44221 and amide‐proton‐exchange kinetic [Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544–15481 studies, which indicated that the A, B, G and H helices in apomyoglobin maintain the same packing as they do in holoprotein.
Investigation of Two Glycosylated Forms of Bile‐Salt‐Dependent Lipase in Human Pancreatic Juice
Authors: Mas Eric , Franc Jean‐Louis , Lecestre Dominique , Crotte Christian , Lombardo Dominique , Sadoulet Marie‐Odile
Published: July 2004

Pure human pancreatic bile‐salt‐dependent lipase, devoid of its oncofetal glycoform [Mas, E., Abouakil, N., Roudani, S., Miralles, F., Guy‐Crotte, O., Figarella, C., Escribano, M. J. & Lombardo, D. (1993) Biochem. J. 289, 609–6151, was analyzed on immobilized concanavalin A (ConA). Two variants were separated: an unabsorbed ConA‐unreactive fraction; and an absorbed ConA‐reactive fraction. Carbohydrate compositions of ConA‐reactive and ConA‐unreactive fractions were not significantly different, and analysis of 3H‐labelled oligosaccharides liberated from these fractions on the ConA‐Sepharose column indicated that the fractionation of the bile‐salt‐dependent lipase on this column depends upon oligosaccharide structures. The activity of the ConA‐reactive fraction was however much lower, independent of the substrate (4‐nitrophenyl hexanoate or cholesteryl esters), than that of the ConA‐unreactive fraction. Therefore, catalytic constants for the hydrolysis of 4‐nitrophenyl hexanoate were determined; both fractions had quite similar Km, while the kcat, for the ConA‐unreactive fraction was 3–4‐fold higher than that of the ConA‐reactive fraction. ConA‐reactive and ConA‐unreactive fractions were shown to have slightly different molecular masses and different amino acid compositions. Cleavage patterns after cyanogen bromide treatment of the ConA‐reactive and ConA‐unreactive fractions suggested that the ConA‐reactive (high Mr, form) and ConA‐unreactive (low Mrform) forms could be different isoforms of the bile‐salt‐dependent lipase secreted by the human pancreas.
Changes in Cellular and Plasma Membrane Phospholipid Composition after Lipopolysaccharide Stimulation of Human Neutrophils, Studied by31P NMR
Authors: Wright Lesley C. , Nouri‐Sorkhabi M. Hossein , May George L. , Danckwerts Laura S. , Kuchel Philip W. , Sorrell Tania C.
Published: July 2004

Lipopolysaccharide (endotoxin, LPS) exerts potent proinflammatory effects on neutrophils which may involve membrane phospholipid metabolism. The cellular and plasma membrane phospholipid composition of resting neutrophils and those stimulated with 50 μg ml‐1LPS were studied by31P NMR and chemical analysis. A rapid new method for plasma membrane purification was employed, involving the direct lysis of cytoplasts. Chemical analyses showed that, although total cellular phospholipid content did not change with LPS stimulation, there was twice the amount of phospholipid present in plasma membranes isolated from stimulated cells, resulting in a lowered cholesterol/phospholipid ratio. Since internal membranes have lower cholesterol content this result is consistent with an origin from insertion of these membranes (most probably from the endoplasmic reticulum) into the plasma membrane, thereby increasing its fluidity. The individual phospholipid classes of both cells and membranes were quantified by31P‐NMR spectroscopy after dissolution in sodium cholate without prior extraction of lipids, allowing partial resolution of the major phospholipid classes and ether‐linked phospholipids. Ether‐linked lipids were distinguished froin diacyl phospholipids by hydrolysis of lipid extracts with HC1 and phospholipase A1, There was a significant increase in phosphatidylserine in both cells and plasma membranes after stimulation, with a decrease in the phosphatidylethanolamine (diacyl and plasmalogen) content in the cells. Plasma membranes from stimulated cells exhibited a significant decrease in a phospholipid tentatively identified as 2‐arachidonoyl‐1‐alkyl‐sn‐glycero‐3‐phosphocholine, a precursor of the lipid inflammatory mediator, platelet‐activating factor. This report is the first to elaborate the changes in phospholipid composition in human neutrophils as a whole, and in plasma membranes separated from them, before and after stimulation by the physiological activator, LPS.
NMR Solution Structure of an Oxidised Thioredoxin h from the Eukaryotic Green Alga Chlamydomonas reinhardtii
Authors: Mittard Virginie , Blackledge Martin J. , Stein Mariana , Jacquot Jean‐Pierre , Marion Dominique , Lancelin Jean‐Marc
Published: July 2004

NMR solution structures of a cytosolic plant thioredoxin h (112 amino acids, 11.7 kDa) from the green alga Chlamydomonas reinhardtii have been calculated on the basis of 1904 NMR distance restraints, which include 90 distances used to restrain 45 hydrogen bonds, and 44 φ dihedral restraints. The structure of C. reinhardtii thioredoxin h was solved in its oxidised form, and the ensemble of 23 converged structures superpose to the geometric average structure with an atomic rmsd of 0.080 nm ± 0.016 for the (N, Cα, C) backbone atoms of residues 4–110. Comparisons with other thioredoxins, such as thioredoxin from the bacterium Escherichia coli, thioredoxin 2 from a cyanobacterium of the Anahaena genus, and human thioredoxin, showed that thioredoxin h models share more structural features with human thioredoxin than with other bacterial thioredoxins. Examination of the accessible surface around the redox‐active peptide sequence indicates that a potent thioredoxin‐h–substrate interaction could be similar to the vertebrate thioredoxin–substrate interactions.
Characterisation of the Isolated Che Y C‐terminal Fragment (79–129)
Authors: Bruix Marta , Muñoz Victor , Campos‐Olivas Ramón , Del Bosque José Ramón , Serrano Luis , Rico Manuel
Published: July 2004

To gain insight into how the three‐dimensional structure, stability and folding of the protein Che Y are related to one another, we have performed a conformational analysis of a long fragment of this protein, encompassing its C‐terminal 51 residues (79–129). This fragment consists of residues in the β‐strands 4 and 5 and α‐helices 4 and 5 of native Che Y. The study has been performed by two‐dimensional NMR and far‐ultraviolet circular dichroism in aqueous solution and in 30% (by vol.) trifluoroethanol/water at 273 K and 298 K. We observe little structure for this fragment in aqueous solution which could be due to low helical populations in the regions corresponding to helices 4 and 5. Within the limits of the residual helical structure experimentally detected, helix 4 appears to extend beyond the N‐terminus observed in the native structure by over four residues belonging to the preceding loop. In 30% trifluoroethanol the helical content of both helices increase and helix 4 extends further to include the preceding β‐strand 4. None of the long‐range NOES present in native Che Y are observed under the explored experimental conditions. The conformational shifts of the Hα protons within the α‐helices of fragment 79–129 are identical to those of shorter synthetic peptides corresponding to the isolated a‐helices. Thus, the fragment 79–129 appears to behave as an open chain with low local helical populations. The very low intrinsic ability for structure formation displayed by this region of Che Y at pH 2.5 suggests that in the folded protein this region could be mainly stabilised by interactions with the N‐terminal Che Y region. This is in accordance with the contact map of Che Y, which shows that the strongest non‐local contacts of C‐terminal residues are with residues of the N‐terminal region, while those within the C‐terminal region are very weak. More importantly, the relationship appears to be possibly extended to the folding properties of the protein, since the C‐terminal region is not structurally formed in the folding transition state of Che Y but in the final steps of the folding.