bluuebunny/crossref_metadata_2025 huggingface dataset:
Further Characterisation of Fast, Slow and Cardiac Muscle Tropomyosins from Salmonid Fish
Authors: Jackman Donna M. , Waddleton Deena M. , Younghusband Banfield , Heeley David H.
Published: July 2004

Separate cDNA libraries were constructed from cardiac muscle and slow myotomal muscle of mature brown trout (Salmo trutta). The complete sequence of tropomyosin (TM) that is specific to these muscles was determined from full‐length transcripts isolated from the corresponding library. The identity of the sequences was supported by protein data. When compared to the sequence of Atlantic salmon fast myotomal TM [Heeley, D. H., Bieger, T., Waddleton, D. M., Hong, C., Jackman, D. M., McGowan, C., Davidson, W. S. & Beavis, R. C. (1995) Characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish, Eur J. Biochem. 232, 226–234], the main difference in the N‐ and C‐terminal sequences comprising the site of end‐to‐end overlap occurs at residue 276 where an asparagine in fast TM is replaced by a histidine in both cardiac and slow TM. Trout cardiac TM exhibited greatest similarity to chicken cardiac TM while trout slow TM exhibited greatest similarity to skeletal α‐TMs. Thus, none of the three salmonid TM sequences corresponds to a β‐type TM. In calorimetry experiments (0.1 M salt, pH 7.00, t= 10–60°C), in the presence of dithiothreitol, differences were observed in the thermal unfolding profiles of the purified isoforms. A single endotherm [tm= 39.5°C) was noted for cardiac TM. Two endotherms were observed for fast TM [tm= 26.5°C and 39.8°C (main)] and slow TM [tm= 37.4°C and 46.9°C (main)]. Fast TM was cloned and over expressed in the bacterial cell lines JM105 and BL21. Upon cell lysis, recombinant TM (rc TM) made in JM105 was rapidly and quantitatively cleaved between residues 6 and 7. Intact rc TM was produced by using BL21, as shown by Edman‐based sequencing, carboxypeptidase digestion and mass analysis. In viscometry assays, performed at low ionic strength (pH 7.00, t= 5°C) the full‐length rc TM exhibited markedly lower relative viscosity values than the corresponding wild type.
In vitro Activation of the NADPH Oxidase by Fluoride
Authors: Wölfl Jutta , Dagher Marie‐Claire , Fuchs Alexandra , Geiszt Miklós , Ligeti Erzsébet
Published: July 2004

The possible mechanism of activation of the NADPH oxidase by fluoride was investigated in the cell‐free system. It is shown that the stimulatory effect of fluoride is inhibited by guanosine 5′‐O ‐(2‐thiodiphosphate) (GDP[S]) and potentiated by GTP. The effect of fluoride is not additive with GTP[S]. Fluoride activation requires the presence of Mg2+ in millimolar concentration but is independent of Al3+. The activating effect of fluoride is preserved in solubilized membrane extract after removal of the majority of heterotrimeric GTP‐binding proteins by immunoadsorption. Fluoride has no direct action either on the nucleotide exchange or GTP hydrolysis of the isolated Rac protein. In contrast, fluoride effectively inhibits Rac‐GTPase activity enhanced by a membrane component. In this way, fluoride could prolong the prevalence of Rac in the GTP‐bound state and, as a consequence, activate NADPH oxidase. The possibility of the involvement of a membrane‐bound Rac GTPase‐activating protein activity in the physiological regulation of the enzyme is raised.
Conformational and Associative Behaviours of the Third Helix of Antennapedia Homeodomain in Membrane‐Mimetic Environments
Authors: Berlose Jean‐Philippe , Convert Odile , Derossi Daniele , Brunissen Alié , Chassaing Gérard
Published: July 2004

The third helix of antennapedia homeodomain pAntp‐(43–58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp‐(43–58) and [Pro50]pAntp‐(43–58) has been analyzed by different biophysical methods. pAntp‐(43–58) adopts an amphipathic helical structure in 30% (by vol.) hexafluoroisopropanol, in perfluoro‐tert‐butanol and in the presence of SDS micelles. CD spectra indicate that the conformation of [Pro50]pAntp‐(43–58) in contrast to pAntp‐(43–58) is independent of the media used. 1H‐NMR spectroscopy in SDS micelles or in perfluoro‐tert‐butanol allows detection of aggregated peptides probably in a ribbon 27 type conformation. These conformations became the predominant structure when Gln50 was replaced by Pro50. Interproton‐distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: α‐helix and essentially extended structures. Consecutive CHα(i)/CHα(i+1) NOES are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with φ and ψ angles in the β‐sheet and γ‐turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 μM). Similar interaction was observed with reverse‐phase micelles made of bis(2‐ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 31P‐NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.
Purification, Molecular Cloning and Expression in Escherichia coli of Homospermidine Synthase from Rhodopseudomonas viridis
Authors: Tholl Dorothea , Ober Dietrich , Martin William , Kellermann Josef , Hartmann Thomas
Published: July 2004

Homospermidine synthase (HSS) catalyzes the synthesis of the polyamine homospermidine from 2 mol putrescine in an NAD+‐dependent reaction. In this study, the enzyme was purified from anaerobically grown cultures of the photosynthetic bacterium Rhodopseudomonas viridis to electrophoretic homogeneity using a three‐step procedure. The enzyme was shown to be a homodimer of 52‐kDa subunits. Six endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers designed against these peptides, specific PCR products from R. viridis DNA were obtained that were used as hybridization probes to isolate the hss gene from a library constructed in λEMBL4. The hss gene and flanking regions were sequenced and were shown to exist as a single copy in the R. viridis genome. HSS is translated from a monocistronic mRNA and possesses no detectable similarity to previously sequenced gene products. Escherichia coli, which lacks HSS activity, was transformed with an expression plasmid containing the hss coding region under the control of a bacteriophage T7 promoter. Upon induction, transformed E. coli cells accumulate enzymatically active and highly stable R. viridis HSS at levels corresponding to 40–50% of the soluble protein in crude extracts.
A 30‐kDa Host Protein Binds to Two Very‐Late Baculovirus Promoters
Authors: Jain Anjali , Hasnain Seyed E.
Published: July 2004

A 30‐kDa host factor (polyhedrin‐promoter‐binding protein; PPBP) specifically binds to sequences critical for transcription from the baculovirus polyhedrin (p29) gene initiator promoter [Burma, S., Mukherjee, B., Jain, A., Habib, S. & Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750–2757; Mukherjee, B., Burma, S. & Hasnain, S. E. (1995) J. Biol. Chem. 270, 4405–4411]. A host factor also binds, in gel shift assays, to the very‐late p10 gene promoter through DNA sequence motifs similar to the PPBP p29 interaction. The p10 host factor complex was specifically competed out with oligonucleotides containing p29 cognate sequence motifs AATAAA and TAAGTATT, but this did not occur when these motifs were replaced with random sequences. From ultraviolet cross‐linking analysis, the molecular mass of this host factor was estimated to be approximately 30 kDa. Experiments were performed to investigate if this host factor displayed any differences in affinity and turnover with respect to the p29 and p10 untranslated leader sequences known to be important for temporal fine tuning and the late burst of transcription. Half‐life determination of the p10‐binding protein revealed similar binding affinities for the initiator elements of both the promoters, but higher affinity for the p10 5′‐untranslated region (≈30 min versus ≈10 min). The involvement of a similar host factor binding to both the p10 and p29 promoters indicates the possibility of a similar mode of transcription initiation from these two very‐late promoters.
An Essential Lysine in the Substrate‐Binding Site of Ornithine Carbamoyltransferase
Authors: Valentini Giovanna , De Gregorio Ambra , Di Salvo Cinzia , Grimm Rudi , Bellocco Ersilia , Cuzzocrea Giovanni , Iadarola Paolo
Published: July 2004

Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4, resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2‐aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2‐aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed Km, values for substrates equivalent to those of native enzyme and lowered kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.
The Spleen Protein‐Tyrosine Kinase TPK‐IIB is Highly Similar to the Catalytic Domain of p7Psyk
Authors: Brunati Anna Maria , James Peter , Guerra Barbara , Ruzzene Maria , Donella‐Deana Arianna , Pinna Lorenzo A.
Published: July 2004

TPK‐IIB is a protein kinase that is predominant in the cytosol of spleen and is characterized by a high specific activity toward acidic peptide substrates and a low auto‐phosphorylation activity. A prominent 52‐kDa component purifies with the kinase [Marin, O., Donella‐Deana, A., Brunati, A. M., Fischer, S. & Pinna, L. A. (1991) J. Biol. Chem. 266, 17798–17803]. Here we demonstrate that the 52‐kDa protein displays sequence identity with the Miller‐Dieker lissencephaly protein (LIS 1). The protein is not related to any known protein kinase and lacks an ATP‐binding motif. The ATP binding and phosphotransferase activities of TPK‐IIB can be fully accounted for by a minor 38‐kDa protein band (p38/TPK‐IIB) which can be separated from the 52‐kDa protein by Mono‐Q/FPLC in the presence of EDTA. Sequence analysis of p38/TPK‐IIB reveals a high level of similarity, if not identity, with the catalytic domain of p72syk, a protein‐tyrosine kinase implicated in the activation of hematopoietic cells. Antibodies raised against the catalytic domain of p72syk, but not antibodies raised against its N‐terminal segment, cross‐react with p38/TPK‐IIB. The peptide substrate specificity of p72syk is almost identical to that of p38/TPK‐IIB, which also supports the classification of TPK‐IIB as a close relative (possibly a proteolytic or alternative spliced form) of p72syk. p38/TPK‐IIB, however, exhibits a specific activity which is sixfold higher than that of p72syk and appears to be 50‐fold more sensitive to inhibition by heparin. Thus, the observation that Ca2+‐dependent degradation of p72syk by particulate fraction of rat spleen is accompanied by increased catalytic activity and increased sensitivity to heparin would be consistent with the possibility that hyperactive p38/TPK‐IIB might be proteolytically generated from p72syk in response to an increase of intracellular Ca2+.
The Structure of the Keratan Sulphate Chains Attached to Fibromodulin Isolated from Articular Cartilage
Authors: Lauder Robert M. , Huckerby Thomas N. , Nieduszynski Ian A.
Published: July 2004

Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion‐exchange chromatography. Their structures have been determined by high‐field 1H‐NMR spectroscopy and high‐pH anion‐exchange chromatography.

Both α(2‐6)‐ and α(2‐3)‐linked N‐acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is α(1‐3)‐linked to N‐acetylglucosamine has been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N‐linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both α(2‐6)‐linked N‐acetylneuraminic acid and α(1‐3)‐linked fucose.

It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non‐reducing end of the chain. The galactose residue adjacent to the non‐reducing cap is sulphated in only 1–3% of chains, compared with a sulphation level of over 40% closer to the reducing end. This highlights the difference between the chain termini and the repeat region in terms of structure and points to the potential for functional importance.

The repeat region and capping fragments of the N‐linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have the following general structure: NeuAc‐(α2–3/6)Gal[6SO3−](β1–4)GlcNAc6SO−3(β1–3)Gal[6SO3−](β1‐4)‐[Fuc(α1‐3)]0–1GlcNAc6SO−3(β1‐3)Gal[6SO3−](β1–4))6–7GlcNAc6SO−3.
Probing Electron Transfer in Flavocytochrome P‐450 BM3 and Its Component Domains
Authors: Munro Andrew W. , Daff Simon , Coggins John R. , Lindsay J. Gordon , Chapman Stephen K.
Published: July 2004

Rapid events in the processes of electron transfer and substrate binding to cytochrome P‐450 BM3 from Bacillus megaterium and its constituent haem‐containing and flavin‐containing domains have been investigated using stopped‐flow spectrophotometry. The formation of a blue semiquinone flavin form occurs during the NADPH‐dependent reduction of the flavin domain and a species with a similar absorption maximum is also seen during reduction of the holoenzyme by NADPH. EPR spectroscopy confirms the formation of the flavin semiquinone. The formation of this semiquinone is transient during fatty acid monooxygenation by the holoenzyme, but in the presence of excess NADPH the species reforms once fatty acid is exhausted. Electron transfers through the reductase domain are too rapid to limit the fatty acid monooxygenation reaction. The substrate‐binding‐induced haem iron spin‐state shift also occurs much faster than the kcat at 25°C. The rate of first electron transfer to the haem domain is also rapid; but it is of the order of 5–10‐times larger than the kcat for the enzyme (dependent on the fatty acid used). Given that two successive electron transfers to haem iron are required for the oxygenation reaction, these rates are likely to exert some control over the rate of fatty acid oxygenation reactions. The presence of large amounts of NADPH also results in decreased rates of electron transfer from flavin to haem iron. In the difference spectrum of the active fatty acid hydroxylase, features indicative of a high‐spin iron haem accumulate. These are in accordance with the presence of large amounts of an Fe3+‐product bound enzyme during turnover and indicate that product release may also contribute to rate limitation. Taken together, these data suggest that the catalytic rate is not determined by the accumulation of a single intermediate in the reaction scheme, but rather that it is controlled in a series of steps.
Synthetic Phospho‐Oligosaccharide Fragments of Lipophosphoglycan as Acceptors for Leishmania majorα‐d‐Mannosylphosphate Transferase
Authors: Brown Gavin M. , Millar Alan R. , Masterson Claire , Brimacombe John S. , Nikolaev Andrei V. , Ferguson Michael A. J.
Published: July 2004

Protozoan parasites of the genus Leishmania synthesise lipophosphoglycans, phosphoglycans and proteophosphoglycans that contain phosphosaccharide‐repeat units of [‐6Galβ1‐4Manα1‐P‐]. In this study, a GDP‐Man‐dependent α‐mannosylphosphate‐transferase activity was detected in washed Leishmania major membranes using synthetic phospho‐oligosaccharide fragments of lipophosphoglycan as acceptor substrates. The divalent‐cation‐dependent α‐mannosylphosphate‐transferase activity had an apparent Km for GDP‐Man of about 15–20 μM and a pH optimum of 7.0. The activity showed a requirement for a non‐reducing terminal βGal residue and for one or more phosphodiester units preceding the acceptor site. Based on these results, the activity may be defined as a GDP‐Man:Galβ1‐4Manα1‐P‐R α‐mannosylphosphate‐transferase. This acceptor specificity is consistent with a role for the α‐mannosylphosphate transferase in the elongation of phosphosaccharide‐repeat domains of Leishmania glycoconjugates rather than in the priming of these domains. An identical or similar activity must exist in the amastigote forms of the Leishmania that produce and secrete proteophosphoglycan material and the activity therefore represents a feasible target for the development of chemotherapeutics.
Protein Engineering Studies of Dichloromethane Dehalogenase/Glutathione S‐Transferase from Methylophilus Sp. Strain DM11
Authors: Vuilleumier Stéphane , Leistnger Thomas
Published: July 2004

The structural gene for dichloromethane dehalogenase/glutathione S‐transferase (GST, EC 2.5.1.18) from Methylophilus sp. strain DM11 was subcloned into a multicopy plasmid under the control of the T7 polymerase promoter, allowing expression in Escherichia coli and easy purification of the enzyme in good yield. Several point mutations leading to amino acid changes at residues Tyr6, His8 and Ser12 of the protein were introduced in this gene. Mutations at Tyr6, the N‐terminal tyrosine known to be essential for enzymatic activity in glutathione S‐transferases of the α, μ and π classes, had little effect on the activity of dichloromethane dehalogenase. The same applied for mutations at residue His8, which from multiple alignments of GST sequences may also correspond to the conserved N‐terminal tyrosine residue of GST enzymes. The higher turnover rate of the wild‐type enzyme with dibromomethane compared with dichloromethane was lost in mutants with amino acid replacements at residue Hiss, but retained in mutant proteins at Tyr6. Mutations at Ser12 led to mutants with drastically reduced enzymatic activity, pinpointing this residue as an essential determinant of catalytic efficiency.
Structure/Activity Relationships of Polyfunctional Diterpenes of the Tigliane Type
Authors: Krauter Goetz , Von Der Lieth Claus‐W. , Schmidt Rainer , Hecker Erich
Published: July 2004

For protein kinase C (PKC), a family of isoenzymes with serine/threonine‐kinase activity identified as the major cellular receptor for certain skin irritants and tumor promoters, a new pharmacophore model is presented. By structure/activity relationship studies of naturally occurring and synthetic diterpene esters of the tigliane type (PKC activators) it is demonstrated that in addition to the oxygen at C20 it is the O‐acyl function in position C13 which is critically essential for skin‐irritant and tumor‐promoting bioactivities rather than other oxygen atoms. This result is confirmed and extended by computer‐assisted molecular modeling of tigliane‐type and ingenane‐type tumor promoters. It is contrasted to certain features attributed to the pharmacophore based upon the recently determined crystallographic structure of the effector‐binding domain Cys2 of PKCδ complexed with the pseudo‐agonist phorbol 13‐acetate.
l‐Aspartate Oxidase from Escherichia coli
Authors: Tedeschi Gabriella , Negri Armando , Mortarino Michele , Ceciliani Fabrizio , Simonic Tatjana , Faotto Ludovica , Ronchi Severino
Published: July 2004

l‐Aspartate oxidase is a monomeric flavoprotein that catalyzes the first step in the de novo biosynthetic pathway for pyridine nucleotide formation under both aerobic and anaerobic conditions. In spite of the physiological importance of this biosynthesis in particular in facultative aerobic organisms, such as Escherichia coli, little is known about the electron acceptor of reduced l‐aspartate oxidase in the absence of oxygen. In this report, evidence is presented which suggests that in vitro fumarate can play such a role. l‐Aspartate oxidase binds succinate and fumarate with Kd, values of 0.24 mM and 0.22 mM, respectively. A competitive behaviour was observed for these two dicarboxylic acids towards iminoaspartate and sulfite ions. Photoreduction experiments suggest that fumarate and succinate bind at or close to the active site of the molecule. A new fumarate reductase activity of l‐aspartate oxidase is reported using benzylviologen or l‐aspartate as reductants and fumarate as oxidant. Steady‐state kinetics for the oxidase and the fumarate reductase activity of l‐aspartate oxidase were obtained using either fumarate or oxygen as electron acceptor and l‐aspartate as electron donor. Finally, succinate was identified as the product of the l‐aspartate:fumarate oxidoreductase activity using radiolabeled fumarate under anaerobic conditions. The results suggest that fumarate can be a valuable alternative to oxygen as a substrate for l‐aspartate oxidase.
In vivo Phosphorylation of the Purine/Cytosine Permease from the Plasma Membrane of the Yeast Saccharomyces cerevisiae
Authors: Pinson Benoît , Pillois Xavier , Brèthes Daniel , Chevallier Jean , Napias Christian
Published: July 2004

The purine/cytosine permease, encoded by the FCY2 gene, is a carrier located in the plasma membrane of the yeast Saccharomyces cerevisiae. Polyclonal antibodies were raised against two peptides that corresponded to the sub‐N‐terminal and C‐terminal sequences of the putative protein deduced from the FCY2 gene. Immunoprecipitation experiments performed with protein extracts labelled in vivo with 35S showed that purine/cytosine permease is specifically detected as a broad and diffuse band. The apparent molecular mass of this protein was 45–50 kDa. By means of in vivo pulse/chase 35S‐labelling experiments, we observed a slight increase in the apparent molecular mass of purine/cytosine permease during the chase. This shift in electrophoretic mobility of the protein suggested a post‐translational modification. This molecular mass increase was eliminated by alkaline phosphatase treatment of the immunoprecipitate, which strongly suggested phosphorylation of the carrier. This proposal was confirmed by in vivo [32P]Pi labelling and immunoprecipitation of purine/cytosine permease with purified anti‐(sub‐N‐terminal peptide) IgG or anti‐(C‐terminal peptide) IgG. Phosphoamino acid analysis indicated that phosphorylation occurred on seryl residues of purine/cytosine permease. By means of thermosensitive secretory‐pathway‐mutant strains, we demonstrated that purinekytosine permease phosphorylation occurred either between the Golgi apparatus and the plasma membrane or in the plasma membrane itself.
Identification of Functional Domains and Critical Residues in the Adenosinetriphosphatase Inhibitor Protein of Mitochondrial F0F1 ATP Synthase
Authors: Papa Sergio , Zanotti Franco , Cocco Tiziana , Perrucci Cecilia , Candita Cosimo , Minuto Michele
Published: July 2004

Peptide segments of the inhibitor protein (IF1) of the F0F1 ATP synthase complex from bovine‐heart mitochondria have been constructed by chemical synthesis. The IF1‐(42–58)‐peptide was equally effective as IF1 in inhibiting the ATPase activity of both the F0F1 complex in the mitochondrial membrane deprived of IF1 (SMP) and soluble F1. The IF1‐(22–46)‐peptide inhibited the ATPase activity in the soluble F1 but had no effect on either the ATPase activity or H+ conduction in SMP. Substitution of the His or Lys residues with Ala in the IF1‐(42–58)‐peptide decreased the inhibition of ATP hydrolysis. The inhibition exerted by the IF1‐(42–58)‐peptide on ATP hydrolysis in SMP exhibited a pH dependence, similar to that observed with IF1, which was lost upon replacement of His or Lys with Ala. In soluble F1, inhibition of ATP hydrolysis by IF1, the IF1‐(42–58)‐peptide and the IF1‐(22–46)‐peptide was pH dependent when F1 was first incubated with ATP. The IF1‐(42–58)‐peptide also caused inhibition of passive H+ conduction in SMP. This activity of the synthetic peptide was weaker, as compared to that of IF1, and practically unaffected by substitution of His or Lys with Ala. An antibody against the IF1‐(42–58)‐synthetic peptide stimulated ATP hydrolysis in the membrane‐bound F0F1 complex with associated IF1 but was without effect on H+ conduction. An antibody against IF1 stimulated both processes.
Identification of a Yeast Peroxisomal Member of the Family of AMP‐Binding Proteins
Authors: Blobel Fabian , Erdmann Ralf
Published: July 2004

We established a reverse‐genetic approach to identify peroxisomal proteins involved in peroxisomal fatty acid metabolism of Saccharomyces cerevisiae. Putative peroxisomal peripheral membrane proteins were isolated by successive extraction of purified peroxisomes and purified by HPLC and SDS/PAGE. Six proteins were identified by peptide sequence analysis, including acyl‐CoA oxidase and a trifunctional enzyme of the peroxisomal β‐oxidation system as well as peroxisomal malate dehydrogenase 3 and carnitine acetyltransferase. In addition two previously unknown putative peroxisomal proteins were identified, an unknown 40‐kDa protein and a protein which we named Pcs60p, both major constituent of the isolated protein fraction. Pcs60p is encoded by ORF Z36091 of chromosome II from S. cerevisiae and consists of 543 amino acids with a molecular mass of 60.5 kDa. Biochemical, immunofluorescence microscopy and immunocytochemical data confirmed that Pcs60p is a peroxisomal peripheral membrane protein but the protein is also localized in the peroxisomal matrix. Consistent with the intraperoxisomal localization, the consensus sequence for a peroxisomal‐targeting signal 1 (PTS1) is present at the extreme C‐terminus of Pcs60p. Deletion studies revealed that the peroxisomal localization of the protein depends on the presence of this signal sequence. Expression of Pcs60p is highly inducible by oleic acid, however, the protein is dispensable for growth on oleic acid as single carbon source. Pcs60p belongs to the family of proteins which act via an ATP‐dependent covalent binding of AMP to their substrates and shows the highest degree of similarity to the Escherichia coli long chain acyl‐CoA synthetase.
4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3
Authors: Seibold Birgit , Matthes Martina , Eppink Michel H. M. , Lingens Franz , Van Berkel Willem J. H. , Müller Rudolf
Published: July 2004

4‐Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50‐fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44‐kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4‐hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45°C. Chloride ions were inhibitory, and competitive with respect to NADH.

4‐Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4‐hydroxybenzoate to 3,4‐dihydroxybenzoate, the enzyme converted 2‐fluoro‐4‐hydroxybenzoate, 2‐chloro‐4‐hydroxybenzoate, and 2,4‐dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed.

The gene encoding 4‐hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promotor was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4‐hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three‐dimensional structure is known. The active‐site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three‐dimensional fold. Based on an isolated region of sequence divergence and site‐directed mutagenesis data of 4‐hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.
Biochemical Evidence for Membrane‐Bound Endoderm‐Specific Alkaline Phosphatase in Larvae of the Ascidian, Halocynthia roretzi
Authors: Kumano Gaku , Yokosawa Hideyoshi , Nishida Hiroki
Published: July 2004

An endoderm‐specific alkaline phosphatase with a molecular mass of 86 kDa has been found in tadpole larvae of the ascidian, Halocynthia roretzi. The histochemical staining of sections of the larvae revealed that the enzyme is a membrane‐associated protein. Two distinct bands were detected by zymographic technique on SDS/PAGE of the membrane preparation of the larvae. The two enzymes can be solubilized from the membrane preparation by sodium cholate and separated from each other by Cibacron blue‐3GA affinity chromatography. The 86‐kDa enzyme is likely to be the endoderm‐specific one because the susceptibilities to alkaline phosphatase inhibitors coincide between the 86‐kDa enzyme and that found in the endoderm of larvae by histochemical staining. The endoderm‐specific alkaline phosphatase was purified and its N‐terminal amino acid sequence was found to show little similarity to those of other proteins.
Intermolecular and Intramolecular Interactions of the 33‐kDa Protein in Photosystem II
Authors: Seidler Andreas
Published: July 2004

Intermolecular and intramolecular interactions of the extrinsic 33‐kDa protein in photosystem II were investigated by cross‐linking with a water‐soluble carbodiimide as cross‐linking agent. This zero‐length cross‐linker is known to cross‐link the 33‐kDa protein to the chlorophyll‐α‐binding protein CP47 [Bricker, T. M., Odom, W. R. & Queirolo, C. B. (1988) FEBS Lett. 231, 111–117; Enami, I., Kaneko, M., Kitamura, N., Koike, H., Sonoike, K., Inoue, Y. & Katoh, S. (1991) Biochim. Biophys. Acta 1060, 224–2321. In this work, cross‐linking was observed not only to CP47 but also to a small intrinsic subunit. In addition, through the use of a high‐resolution SDS‐gel system, three intramolecular cross‐linked products of the 33‐kDa protein were detected. To search for additional cross‐linking sites that might not be accessible to the cross‐linker in intact photosystem II, the isolated 33‐kDa protein was activated for cross‐linking and subsequently bound to CaCl2‐washed photosystem II. In the complementary experiment, CaCl2‐washed photosystem II was activated, then reconstituted with the 33‐kDa protein. The results of the cross‐linking reactions demonstrated that all carboxylic acid groups involved in cross‐linking were located on the 33‐kDa protein and all primary amines were located on intrinsic membrane proteins. No cross‐linking other than those observed in cross‐linking experiments with intact photosystem II were observed. This indicated that the 33‐kDa protein is bound to CP47 and a small subunit but not to the photosystem II reaction centre. This observation is consistent with the finding that cross‐linking was independent of the presence or absence of the manganese cluster. Possible residues on the 33‐kDa protein and CP47 involved in cross‐linking are suggested.
Chemical Synthesis and Characterization of Maurotoxin, a Short Scorpion Toxin with four Disulfide Bridges that Acts on K+ Channels
Authors: Kharrat Ryadh , Mabrouk Kamel , Crest Marcel , Darbon Hervé , Oughideni Razika , Martin‐Eauclaire Marie‐France , Jacquet Guy , El Ayeb Mohammed , Van Rietschoten Jurphaas , Rochat Hervé , Sabatier Jean‐Marc
Published: July 2004

Maurotoxin is a toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus. It is a 34‐amino‐acid peptide cross‐linked by four disulfide bridges. Maurotoxin competes with radiolabeled apamin and kaliotoxin for binding to rat‐brain synaptosomes. Due to its very low concentration in venom (0.6% of the proteins), maurotoxin was chemically synthesized by means of an optimized solid‐phase technique. The synthetic maurotoxin was characterized. It was lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). The synthetic maurotoxin competed with 125I‐apamin and 1251‐kaliotoxin for binding to rat‐brain synaptosomes with half‐maximal effects at concentrations of 5 nM and 0.2 nM, respectively. Synthetic maurotoxin was tested on K+ channels and was found to block the Kv1.1, Kv1.2, and Kv1.3 currents with half‐maximal blockage (IC50) at 37, 0.8 and 150 nM, respectively. Thus, maurotoxin is a scorpion toxin with four disulfide bridges that acts on K+ channels. The half‐cystine pairings of synthetic maurotoxin were identified by enzymatic cleavage. The pairings were Cys3–Cys24, Cys9–Cys29, Cys13–Cys19 and Cys31–Cys34. This disulfide organization is unique among known scorpion toxins. The physicochemical and pharmacological properties of synthetic maurotoxin were indistinguishable from those of natural maurotoxin, which suggests that natural maurotoxin adopts the same half‐cystine pairing pattern. The conformation of synthetic maurotoxin was investigated by means of circular dichroism spectroscopy and molecular modeling. In spite of its unusual half‐cystine pairings, the synthetic‐maurotoxin conformation appears to be similar to that of other short scorpion toxins.
Brain Capillary Endothelial Cells Express two forms of Erythropoietin Receptor mRNA
Authors: Yamaji Ryoichi , Okada Tadayuki , Moriya Maki , Naito Mikihiko , Tsuruo Takashi , Miyatake Kazutaka , Nakano Yoshihisa
Published: July 2004

To study the existence of the erythropoietin receptor (Epo‐R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT‐PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo‐R mRNA as a minor form and an intron‐5‐inserted form of Epo‐R mRNA, thus a soluble form of Epo‐R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed, rHuEpo showed a dose‐dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with 125I‐rHuEpo showed that RBECs had a single class of receptors with low‐affinity (Kd= 860 pM) and that the number of sites/cell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo‐R but also a soluble form of Epo‐R and that erythropoietin acts directly on brain capillary endothelial cells as a competence factor.
In vivo Modulation of Annexins I, II and V Expression by Thyroxine and Methylthiouracil
Authors: El Btaouri Hassan , Claisse Didier , Bellon George , Antonicelli Frank , Haye Bernard
Published: July 2004

Regulation of annexin concentration and localization were investigated in thyroid tissues of hypothyroid [methylthiouracil (MeSur) treatment], euthyroid (control) and hyperthyroid [thyroxine (T4) treatment] rats.

A low level of circulating thyroid hormones induces a decrease of total thyroid calcium‐binding protein concentration when compared with the concentration in unstimulated animals. Conversely, concentrations of annexins I, II and V increase. The accumulation of these proteins in two subcellular compartments (cytosolic and particulate fractions) can be reversed by addition of thyroid hormones. The finding of a specific increase in annexins concentration in thyroid‐hormone‐deficient rats, with a general decrease of the total calcium‐binding protein content points to a very important role of these proteins in the cells. Furthermore, hyperthyroidism gives opposite results. To investigate the transduction pathway of annexins I‐, II‐ and V‐induced biosynthesis by thyroid hormones in thyroid glands, we used cultured pig thyroid cells as in vitro model system. In previous work [16], we have shown that annexin concentrations and localization are under TSH control via the adenylate cyclase pathway. In the presence of MeSur (in the culture medium), the protein‐binding iodine remains low, indicative of weak thyroid hormone synthesis (data not shown) and that the annexins content is unchanged.

These results suggest that, in thyroid tissue, an indirect mechanism links thyroid hormones to annexin expressions via the TSH feed‐back loop, and excludes autocrine regulation.
The Assisted Self‐Association of ATP4‐ by a Poly(Amino Acid) [Poly(Lys)] and Its Significance for Cell Organelles That Contain High Concentrations of Nucleotides
Authors: Sigel Helmut , Corfù Nicolas A.
Published: July 2004

The occurrence of high concentrations of ATP in certain cell organelles prompted us to study the self‐association of ATP via the concentration dependence of the 1H‐NMR chemical shifts for H2, H8 and H1′ in D2O at pD 8.4 (25°C) in the range 0.0025–0.4 M in the presence and absence of poly(α,l‐lysine), where [Lys units] was 0.4 M. The experiment in the presence of poly(Lys) was repeated at pD 12.1. At pD 8.4, the poly(amino acid) is protonated, i.e. poly(H·Lys)n+, whereas at pD 12.1 only approximately 10% of the ɛ‐amino groups are positively charged. The results in all three systems are consistent with the isodesmic model of indefinite non‐cooperative stacking. The stacking tendency follows the series: ATP4− (K= 1.3 M−1; pD 8.4)< ATP4−/poly(H·Lys)n+ (K= 11.5 M−1; pD 8.4)> ATP4−/90% poly(Lys)/10% poly(H·Lys)n+ (K= 3.1 M−1; pD 12.1). It is evident that poly(H·Lys)n+ assists the association of ATP by a factor of approximately 10, and it is suggested that, via its positively charged ɛ‐ammonium groups, poly(H·Lys)n+ acts as a matrix by aligning ATP4 ions via ionic interactions with the negatively charged phosphate residues. The intragranular concentrations of various constituents of several storage or secretory cell organelles, as reported in the literature, are tabulated. The chromaffin granules of the adrenal medulla and the dense granules of blood platelets contain particularly high concentrations of nucleotides ([ATP] is approximately 0.14 M in the chromaffin granules and 0.5 M in the dense granules of rabbit blood platelets) and amines, such as epinephrine or 5‐hydroxytryptamine. These granules, and probably also the storage vesicles of certain neurons (which seem to have a similar composition), appear, if the total concentrations of the various solutes are considered, to be osmotically unstable, which means that the intragranular solutes must be associated. This aggregation is discussed, especially with regard to the nucleotides.
The Recombinant α Isoform of Protein Kinase CK1 from Xenopus laevis can Phosphorylate Tyrosine in Synthetic Substrates
Authors: Pulgar Victor , Tapia Claudio , Vignolo Pamela , Santos João , Sunkel Claudio E. , Allende Catherine C. , Allende Jorge E.
Published: July 2004

The cDNA coding for protein kinase CK1α has been cloned from a Xenopus laevis cDNA library. The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38 874 Da. The sequence is identical to that of the human CK1α and to the bovine CK1α, except that it is 12 amino acids longer than the latter protein. Southern blotting with a 264‐bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X. laevis possesses a family of related CK1 genes. CK1α was expressed in Escherichia coli as a glutathione transferase fusion protein (GT‐CK1α) and certain of its characteristics were determined. The recombinant GT‐CK1α fusion protein was found to have apparent Km values for ATP (12 μM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 μM) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1α activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80: Tyr20). This inhibition is competitive and shows an approximate Ki of 5 μM. CK1α can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.
Triiodothyronine Regulates β3‐Adrenoceptor Expression in 3T3‐F442A Differentiating Adipocytes
Authors: El Hadri Khadija , Pairault Jacques , Fève Bruno
Published: July 2004

The effect of thyroid hormones on the β3‐adrenoceptor expression was studied in the pseadipose β3‐F442A cell line. As assessed by molecular and pharmacological analyses, triiodothyronine addition to differentiating β3‐F442A cells caused a 2.3‐fold increase in β3‐adrenoceptor mRNA levels, which was correlated with a parallel induction of β3‐adrenoceptor number and of β3‐adrenoceptor coupling to the adenylate cyclase system. Nuclear transcription experiments showed that triiodothyronine did not significantly alter the transcription rate of the β3‐adrenoceptor gene. By contrast, the hormone increased by 36% the half‐life of β3‐adrenoceptor mRNA. Triiodothyronine exhibited a discrete effect on β3‐adrenoceptor expression when added to mature β3‐F442A adipocytes. This study indicates that thyroid hormones exert a differentiation‐dependent and post‐transcriptional regulation of β3‐adrenoceptor expression in adipocytes.
Glutamine Inhibits the Lowering Effect of Glucose on the Level of Phosphoenol pyruvate Carboxykinase mRNA in Isolated Rat Hepatocytes
Authors: Lavoinne Alain , Husson Annie , Quillard Muriel , Chédeville Arlette , Fairand Alain
Published: July 2004

The expression of phosphoenolpyruvate carboxykinase (P‐pyruvate CK) was shown to be decreased by hypoosmolarity and increased by glutamine in perfused liver from fed rats [Newsome, W. P., Warskulat, U., Noe, B., Wettstein, M., Stoll, B., Gerok, W. & Häussinger, D. (1994) Biochem. J. 304, 555–560]. This work was undertaken to specify the mechanisms of glutamine action, using isolated hepatocytes from rats that had been starved for 24 h. At low concentrations (up to 5 mM), glutamine elicited a decrease in the level of P‐pyruvate CK mRNA through cell swelling and, at higher concentrations, an increase in the mRNA level was observed. Experiments with combinations of glucose and glutamine or glucose and various amino acids demonstrated that glutamine counteracted the inhibitory effect of glucose on P‐pyruvate CK mRNA at a transcriptional level, and strongly suggested that the amide group of glutamine was involved in this effect. The metabolism of glucose was required for the reinforcement of the apparent stimulatory effect of glutamine, as demonstrated by the use of various sugars. Glucosamine, but not mannosamine, increased the level of P‐pyruvate CK mRNA, as did glucose plus glutamine. These results suggest that the pathway leading from glucosamine‐6‐phosphate production might be responsible, at least partly, for the effect observed on P‐pyruvate CK mRNA.
The Emerging Three‐Dimensional Structure of a Receptor
Authors: Hucho Ferdinand , Tsetlin Victor I. , Machold Jan
Published: July 2004

The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most‐characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nm×8 nm) were deduced from electron‐microscopy studies. Its subunits are arranged around a five‐fold axis of pseudosymmetry in the order (clockwise) αHγαLδβ. Its two agonist/competitive‐antagonist‐binding sites have been localized by photola‐belling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane‐spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand‐gated ion channels. The binding site for regulatory non‐competitive antagonists has been localized by photolabelling and site‐directed‐mutagenesis studies within this ion channel.

Therefore a three‐dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/competitive‐antagonist‐binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.
Conformational Study of [Met5]Enkephalin‐Arg‐Phe in the Presence of Phosphatidylserine Vesicles
Authors: D'alagni Maria , Delfini Maurizio , Di Nola Alfredo , Eisenberg Moises , Paci Maurizio , Roda L. Giorgio , Veglia Gianluigi
Published: July 2004

The interaction of [Met5]enkephalin‐Arg‐Phe with phosphatidylserine (PtdSer) was studied by circular dichroism (CD), two‐dimensional nuclear magnetic resonance spectroscopy, hybrid distance geometry simulated annealing (DG–SA) and molecular dynamics (MD) calculations.

The very low solubility of [Met5]enkephalin‐Arg‐Phe and the instability of the solution containing PtdSer vesicles at low pH values did not allow us to observe the amide proton resonances in the usual two‐dimensional NMR work. NOESY cross‐peaks of protons of side chains from two‐dimensional NMR were converted into distances which were used as restraints for modelling with DG‐SA and MD. Our results indicate that, in aqueous solutions at pH 7.68 [Met5]enkephalin‐Arg‐Phe exists in the absence of PtdSer as a random distribution of conformers, whereas in the presence of PtdSer it adopts conformations containing a common orientation of the bonds of Cα2, Cα3, Cα4 and Cα5, although different orientations of the peptide planes are consistent with the results.

Two of the reported conformers from MD simulations are characterized by the presence of a 2←4 γ and inverse γ turns centered on Gly3. A gradual decline of order was observed when moving from the central moiety of the peptide to both the N‐terminus and C‐terminus. Finally, the DG‐SA and MD calculations resulted in a structure such that the orientation of the Phe4 and Met5 side chains favours hydrophobic interactions with the apolar portion of the PtdSer vesicle to form a hydrophobic cluster. These data support the hypothesis of a role of lipids to modify the conformation of [Met5]enkephalin‐Arg‐Phe to permit the interactions with the receptor site.
Interleukin‐1β Induces Nuclear Factor κ B in Epithelial Cells Independently of the Production of Reactive Oxygen Intermediates
Authors: Bonizzi Giuseppina , Dejardin Emmanuel , Piret Bernard , Piette Jacques , Merville Marie‐Paule , Bours Vincent
Published: July 2004

A large body of work has been devoted to tumor necrosis factor α or interleukin‐1β (IL‐1β) signaling leading to the activation of the transcription factor nuclear factor‐κ B (NF‐κ B) in various cell types. Several studies have indicated that NF‐κ B activation depends strictly on the production of reactive oxygen intermediates. In this report, we first demonstrated that IL‐1β is a potent activator of NF‐κ B in various epithelial transformed ccll lines (OVCAR‐3, SKOV‐3, MCF7 A/Z). In these cells, IL‐1β rapidly induces NF‐κ B through a complete degradation of Iκ B‐α while H2O2 activates NF‐κ B with slower kinetics through a partial degradation of IkB‐α, p100 and p105. We showed that IL‐1β‐mediated induction of NF‐κ B in OVCAR‐3 and in other epithelial cell lines does not proceed through the production of reactive oxygen intermediates, while the same cytokine activates NF‐kB in lymphoid cells through the intracellular generation of H2O2. Our study demonstrated that several signaling pathways lead to the activation of NF‐κ B, following IL‐1β treatment in different cell types.