bluuebunny/crossref_metadata_2025 huggingface dataset:
A Mammalian Tryptophanyl‐tRNA Synthetase is Associated with Protein Kinase Activity
Authors: Paley Elena L.
Published: July 2004

Bovine Trp‐tRNA synthetase is a dimer with subunit molecular mass of 60 kDa (p60) which catalyzes ATP‐dependent formation of tryptophanyl‐tRNA. Evidence is presented that Trp‐tRNA synthetase whose homogeneity had been proven by SDS/PAGE and silver staining of the gel is autophosphorylated in vitro. Anti‐(Trp‐tRNA synthetase) antibodies, whose specificity was verified by using a combination of different approaches, were able to effectively inhibit and immunoprecipitate the Trp‐tRNA‐synthetase‐associated kinase activity. The two‐dimensional tryptic phosphopeptide map of autophosphorylated p60 Trp‐tRNA synthetase was found to be similar to that of its major 40–kDa degradation fragment bearing resemblance to previously demonstrated unlabeled peptide patterns of the Trp‐tRNA synthetase forms. Trp‐tRNA synthetase which had undergone denaturation during SDS/PAGE, regained serine/threonine specific protein kinase activity (PK 60) after guanidine treatment. Trp‐tRNA synthetase induced phosphorylation of specific substrate such as 100–kDa protein in non‐immune but not in anti‐(Trp‐tRNA synthetase) sera which distinguishes Trp‐tRNA‐synthetase‐associated kinase from other protein kinases. Sequence analysis permitted the identification of regions of bovine Trp‐tRNA synthetase sharing similarity with the catalytic domains of known protein kinases. These findings suggest that PK 60 and Trp‐tRNA synthetase (p60) are either closely related or identical.
Isolation and Amino Acid Sequence Analysis Reveal an Ancient Evolutionary Origin of the Cleavage Stage (CS) Histones of the Sea Urchin
Authors: Brandt Wolf E. , Schwager Sylva U. L. , Rodrigues Jerry A. , Busslinger Meinrad
Published: July 2004

The cleavage stage (CS) H1, H2A, and H2B histones of the sea urchin, which have previously been identified by their distinct electrophoretic mobility on Tritonlacidurea gels, are known to be maternally expressed during oogenesis and have been implicated in chromatin remodeling of the male pronucleus following fertilization. Here, we describe the isolation of these three CS histones by reverse‐phase HPLC chromatography. Moreover, a novel CS H3 protein was identified by the same purification procedure. A low incorporation of radioactive amino acids into the CS histones during early development revealed that the bulk of these proteins in the blastula embryo are derived from the maternal pool of the egg. Amino acid analysis, together with the previously described electrophoretic mobilities, unequivocally identified the purified proteins as CS histones. Peptide sequence analysis confirmed the novel nature of the CS variants as they are distantly related to the early, late, and sperm histone subtypes of the sea urchin. The CS H1 protein displays highest sequence similarity with the H1M (B4) histone of Xenopus laevis, indicating that the frog H1M protein may be a vertebrate homologue of the CS HI histone. These data suggest an ancient evolutionary origin and wide distribution of the CS histone variants.
Post‐Translational Processing of Rat Ribosomal Proteins
Authors: Williamson Nicholas A. , Raliegh Jeanette , Morrice Nicholas A. , Wettenhall Richard E. H.
Published: July 2004

The complete amino acid sequences of rat and yeast (Saccharomyces cerevisiae) ribosomal proteins derived from precursors containing an N‐terminal ubiquitin or ubiquitin‐like sequence (C‐terminal extension proteins or CEPs) were determined and investigated for any post‐translational modifications by reverse‐phase HPLC purification, direct amino acid sequence and mass spectrometric analyses. Covalent modifications were detected in the rat liver proteins RS27a (CEP‐80), RL29, RL37 and RL40 (CEP‐52), while RS30 (CEP), RL36a, RL39 and RL41 were unmodified. Heterogeneity of RS27a was due to C‐terminal truncations, with Lys80 missing from about 20% of the liver RS27a population; C‐terminal processing was also detected with RL29 and RL37. No other covalent modifications of liver, brain or thymus RS27a were detected. The rat RL40 structure was identical to the cDNA‐predicted sequence except for complete stoichiometric Nε‐trimethylation of Lys22 within its zinc‐finger motif; this modification occurred in the ribosomes of all three rat tissues investigated but not in yeast ribosomes. The methylation characteristics of RL40 were distinct from those of ribosomal protein RL29 in the rat, which was differentially monomethylated at Lys4 in the liver, brain and thymus (27%, >99% and 95% methylation, respectively). In the case of liver, there was no appreciable difference in the RL29 methylation status of free and membrane‐bound ribosomes. The possibilities of an essential role for RL40 methylation in the formation of rat ribosomes, and a distinct regulatory role for RL29 methylation in the rat, are discussed.
Purification and Characterization of Flavoproteins and Cytochromes from the Yellow Bioluminescence Marine Bacterium Vibrio Fischeri Strain Y1
Authors: Petushkov Valentin N. , Lee John
Published: July 2004

Several flavoproteins and cytochromes that occur as major components in extracts of the yellow bioluminescence Y1 strain of the marine bacterium Vibrio fischeri have been purified and characterized with respect to their mass (SDSPAGE and matrix‐assisted laser‐desorption/ionization MS), chromatographic properties, N‐terminal sequence, and spectroscopy (absorption, fluorescence emission and anisot‐ropy decay). The investigated proteins were as follows: yellow fluorescence protein (YFP) with bound riboflavin, FMN or 6,7‐dimethyl‐8‐ribityllumazine; a blue fluorescence protein (BFP) with bound 6,7‐dimethyl‐8‐ribityllumazine, riboflavin, or 6‐methyl‐7‐oxo‐8‐ribityllumazine; thioredoxin reductase with FAD as ligand; and two c‐type diheme cytochromes, c551 and c554. We present evidence that the riboflavin‐bound YFP has an N‐terminal sequence corresponding to that published for the dimeric YFP. We show that an equilibrium replacement of the riboflavin can be made with excess lumazine derivative and that lumazine‐bound YFP has different bioluminescence properties to those of the lumazine protein from Photobacterium leiognathi. BFP is a different protein again, and in the bacterial lysate it occurs in multiple forms, ligated to either riboflavin, lumazine, or the 7‐oxolumazine derivative. The N‐terminal sequence for BFP shows similarities to those of the YFP proteins and to lumazine protein and riboflavin synthase from Photobacterium BFP in any form has no bioluminescence or riboflavin‐synthase activity. A 70‐kDa fluorescent flavoprotein with FAD as ligand has an N‐terminal sequence highly similar to those of thioredoxin reductases from Haemophilus influenzae and Escherichia coli. Cytochrome contaminations in previous preparations of YFP have been removed and are identified as the two c‐type cytochromes c551 and c554. Both inhibit the NADH‐induced bioluminescence in the reductase/luciferase system with the luciferases from P. leiognathi and V. fischeri. The N‐terminal amino acid sequence of the cytochrome (c551) corresponds to a diheme cytochrome c4. The spectral properties of c554 are similar to those of other c5 cytochromes, and both c554 and c551 have absorption spectra similar to those of the respective cytochromes from the gram‐negative bacteria Pseudomonas and Azotobacter.
Analysis of the Large Aqueous Pores Produced by a Bacillus Turingiensis Protein Insecticide in Manduca Sexta Midgut‐Brush‐Border‐Membrane Vesicles
Authors: Carroll Joe , Ellar David J.
Published: July 2004

An osmotic swelling assay utilising carboxyfluorescein self‐quenching to measure intravesicular volume changes was adapted to investigate permeability changes induced by the Bacillus thuringiensis Cry1Ac ‐endotoxin in Manduca sexta midgut‐brush‐border‐membrane vesicles (BBMV). This assay provides a more quantitative analysis of Cry‐toxin‐induced BBMV permeability changes, extending our previously published protocol which employed a light‐scattering signal to monitor ‐endotoxin activity [Carroll, J. & Ellar, D. J. (1993)Eur. J. Biochem. 214, 771–7781, The fluorescence signal changes, supported by electron microscopy of the BBMV, demonstrated that CrylAc altered the membrane permeability for large non‐electrolyte solutes. With this approach CrylAc was observed to induce or form pores freely permeant for raffinose (1.14 nm diameter) and using non‐electrolytes of increasing size the pores were estimated to have a limiting diameter of approximately 2.4–2.6 nm under alkaline pH conditions.
Antigenic Mapping of Human Thyroglobulin
Authors: Erregragui Khadija , Prato Sabine , Miquelis Raymond , Barrande Christophe , Daniel Cecile , Fert Vincent
Published: July 2004

We characterized 26 mAb to human thyroglobulin to obtain a topographic map of the thyroglobulin antigenic surface. Among these mAb, three bind thyroglobulin peptides that are located in the primary sequence of thyroglobulin at either the N terminus or in the middle part of the molecule, three bind thyroglobulin via epitopes comprising the thyroid‐hormone moiety, and three bind thyroglobulin through epitopes involved in the recognition of the molecule by its receptor. The 18 remaining mAb bind thyroglobulin through undetermined epitopes; most of these epitopes are resistant to trypsinization. We used two methods to map the antigenic regions of thyroglobulin: all 26 mAb were grouped, by means of cross‐inhibition experiments, in 11 clusters corresponding to 11 antigenic regions of the thyroglobulin surface; by means of thyroglobulin peptides of decreasing size, obtained by time‐controlled tryptic digestion, we analyzed the relative distance between pairs of epitopes in sandwich immunoassays. By combining these two methods, we organized most of the 11 antigenic regions on a topographic representation of the thyroglobulin surface. This new topographic map of thyroglobulin led us to some unexpected features of the thyroglobulin structure. First, antigenic region 8 located far from the N‐terminal region is in close contact with two remote N‐terminal antigenic regions (1 and 4), both involved in hormone formation. This antigenic region is likely to play a role in the correct positioning of hormonogenic tyrosines so as to optimize iodination‐coupling reactions. Secondly, the domain involved in the binding of thyroglobulin to its receptor, probed by three mAb, is shared by two distinct mid‐molecule antigenic regions, one being the main autoantigenic region of thyroglobulin.
Electron Transfer Between Spinach Plastocyanin Mutants and Photosystem 1
Authors: Sigfridsson Kalle , Young Simon , Hansson örjan
Published: July 2004

Two distinct regions of plastocyanin, one hydrophobic and one acidic, are generally thought to be involved in the electron‐transfer reactions with its physiological partners, cytochrome f and photosystem 1. To probe the importance of the hydrophobic patch in the reaction with photosystem 1, seven mutant plastocyanin proteins have been constructed with the following mutations: Gly7A1a, Gly8Asp, Ser11Asp, Ser11Gly, Pro36Gly, Ser85Thr and Gln88Asn. The electron‐transfer reaction was investigated by transient flash‐photolysis absorption spectroscopy. All proteins remained active in photosystem 1 reduction, showing a biphasic reaction. However, the substitution in position 36 resulted in a drastic decrease in efficiency, suggesting that this residue is involved in a specific contact with photosystem 1. Measurements over a wide range of plastocyanin concentration, ionic strength and pH, showed different properties for the two kinetic phases. A mechanism involving a rate‐limiting conformational change accounts well for the observations. Electron transfer from plastocyanin to photosystem 1 would thus require a conversion from an inactive to an active conformation of the complex. Both hydrophobic and electrostatic interactions are important in the dynamics. The structural integrity of a few critical residues, including Pro36, is essential for efficient photosystem 1 reduction.
Purification of the Cardiac Sarcoplasmic Reticulum Membrane Protein Phospholamban from Recombinant Escherichia Coli
Authors: KrÖMer Wolfgang J. , Carafoli Ernesto , Bailey James E.
Published: July 2004

Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S‐transferase (GST). GST–PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein. Attempts to suppress inclusion body formation or to use GST as an affinity‐purification tag failed. A successful purification method is based on preparative SDS/PAGE and electrodialysis. From 1g cells we typically purified 13.5 nig fusion protein with a PLN content of 2.8 mg. We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein. The approach described represents a substantial advancement in PLN expression and purification.
Structures of Cobalt(III)‐Pepleomycin and Cobalt(III)‐Deglycopepleomycin (green forms) Determined by NMR Studies
Authors: Caceres‐Cortes Janet , Sugiyama Hiroshi , Ikudome Kenji , Saito Isao , Wang Andrew H.‐J.
Published: July 2004

Pepleomycin (PEP) is a metalloglycopeptide that has stronger anticancer activity and less pulmonary toxicity than bleomycin (BLM). PEP, like BLM, exerts its action by binding to and degrading DNA in the presence of oxygen and certain metals. Obtaining detailed structural information of PEP and PEP‐DNA complexes is crucial to understanding its anticancer activity. The structures of two green forms of cobalt‐PEP species, HO2−‐Co(III)‐PEP (denoted CoPEP) and deglycosylated HO2−‐Co(III)‐PEP (denoted CodPEP) have been obtained by NOE restrained refinements. Earlier studies of the related HO2−‐Co(III)‐BLM A2 proposed that two chiral conformers (form A or B) could exist with either the β‐aminoalanine primary amine (A,NH2) or the mannose carbamoyl nitrogen (M,NH2) as the axial ligand. Analysis of our NOESY data shows convincingly that form A is the most probable conformer with the mannose carbamoyl M,NH2 and the β‐aminoalanine primary amine A,NH2 as the axial ligands in CoPEP and CodPEP, respectively. The NOE cross‐peaks resulting from the interactions between the N‐terminus (i.e., the metal‐binding domain) and the C‐terminus of CoPEP and CodPEP have similar patterns, suggesting that they both adopt compact structures with the bithiazole group folded back over the N‐terminus.
Molecular Characterization of Fdx1, a Putidaredoxin‐Type [2Fe‐2S] Ferredoxin Able to Transfer Electrons to the Dioxin Dioxygenase of Sphingomonas sp. RW1
Authors: Armengaud Jean , Timmis Kenneth N.
Published: July 2004

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo‐p‐dioxin. The first step of the pathway is performed by a ring‐dihydroxylating enzyme. Bünz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bünz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467–64751. The gene encoding this [2Fe‐2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested‐PCR amplification. Primers for the amplification were designed based on the N‐terminus sequence of the purified ferredoxin and on sequence comparisons with related proteins, Several cosmids were obtained and the ferredoxin gene, fdxl, was subcloned from one of them. The nucleotide sequence of a 4.6‐kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi‐component dioxygenases, genes encoding the a and β subunits are found to be contiguous with the gene of the specific electron carrier, the fdxl gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylaseslisomerases and a glutathione S‐transferase.

The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin‐type [2Fe‐2S] cluster in this protein. Its redox potential was determined to be ‐245 (±5) mV versus the normal hydrogen electrode at 25°C, pH 8.0. Therefore, the protein is closely related to [2Fe‐2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin‐type [2Fe‐2S] ferredoxin, namely Fdxl, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.
Cloning of Two Putative Ecdysteroid Receptor Isoforms from Tenebrio Molitor and their Developmental Expression in the Epidermis during Metamorphosis
Authors: Mouillet Jean‐François , Delbecque Jean‐Paul , Quennedey Brigitte , Delachambre Jean
Published: July 2004

Using the Drosophila EcR‐B1 cDNA as a probe, we have cloned the putative ecdysteroid receptor from the mealworm Tenebrio molitor. We have isolated two cDNAs with different 5′ termini that contain a complete open reading frame. These two cDNAs encode two proteins with distinct N‐terminal regions corresponding to two isoforms. The coleopteran receptor is obviously related to the ecdysteroid receptor of other insects, but shares only 89% and 61% amino acid identities with the DNA‐binding and ligand‐binding domains of the Drosophila receptor, respectively. Its expression pattern has been examined in the epidermis during the last larval instar and pupal stage of T. molitor, in correlation with the hemolymph ecdysteroid titer. Hybridizations revealed two transcripts of 7 kb and 6.5 kb detected in most stages during metamorphosis and corresponding to the A and B1 isoforms. These two mRNAs are highly evident just before the rise of each ecdysteroid peak both in prepupae and in pupae. They show almost the same expression pattern in epidermis except for the second part of the pupal stage, during which only the A isoform is detected.
Determination of Functional Domains of the Human Transcription Factor PAX8 Responsible for its Nuclear Localization and Transactivating Potential
Authors: Poleev Andrej , Okladnova Olga , Musti Anna‐Maria , Schneider Susanne , Royer‐Pokora Brigitta , Plachov Dimitrij
Published: July 2004

The conserved structure of the transcription factors of the Pax gene family may reflect functional conservation. We have demonstrated that the human Pax8 transcription factor is organized in several functional domains and contains two regions responsible for its nuclear localization, in addition to an activating region at the carboxy terminus of the protein and an inhibitory region encoded by the exon 9 present only in a splice variant PAX8a. Regions of PAX8 determining the nuclear localization of the PAX8flacZ fusions contain short amino acid sequences similar to several described nuclear localization sites (NLS). These NLS were identified in the paired domain and between the octapeptide and the residual homeodomain, respectively. The activating domain is encoded by the exons 10 and 11 and its function is modulated by the adjacent domains encoded by the exons 9 and 12. The domain encoded by exon 9 significantly inhibits the function of the activating domain. Pax8 is expressed in thyroid cells and its product binds promoters of the thyroglobulin and thyroperoxidase genes through its paired domain. Thyroid cell growth and differentiation depend on thyrotropin which, by stimulating cAMP synthesis, activates the CAMP‐dependent protein kinase A (PKA). We have investigated a link between thyrotropin stimulation and gene activation by Pax8. Stimulation of cAMP synthesis augments Pax8‐specific transcription in thyroid cells, indicating that PKA is involved in Pax8 activation. Cotransfection of GAL4/PAX8 fusions and the catalytic subunit of PKA in A126, a PKA‐deficient derivative of the PC12 pheo‐chromocytoma cell line, synergistically activates the GAL4‐specific reporter, suggesting the activating domain of PAX8 is dependent upon the catalytic subunit of the PKA. We propose that this dependence is due to a hypothetical adaptor which forms a target for PKA and interacts with the activating domain of PAX% We show that PAX8 isolated from the thyroid cell line FTRL5 is a phosphoprotein in which phosphorylation is not dependant on cAMP pathway activation. Our results suggest that Pax8 is part of the cAMP signaling pathway and mediates thyrotropin‐dependent gene activation in thyroid cells. Investigation of the PAX8 expression in a panel of Wilms' tumors shows a striking correlation between the expression of PAX8 and another transcription factor, WT1, indicating that these two genes may interact in vivo.
Characterization of the Embryonic Globin Chains of the Marsupial Tammar Wallaby, Macropus Eugenii
Authors: Holland Robert A. B. , Gooley Andrew A.
Published: July 2004

The embryonic hemoglobins of the marsupial Tammar wallaby (Macropus eugenii) are known to aggregate, which was shown by the finding that the Hill coefficient, h, was greater than 4.0 in the upper part of the oxygen equilibrium curve. Here, we have undertaken a detailed primary structure analysis of the Tammar wallaby pouch young hemoglobin complement, which we hoped might provide clues into the residues that cause aggregation and a high embryonic h.

The Tammar wallaby embryonic hemoglobin complement is principally four major hemoglobins each with a different isoelectric point. Two early expressed hemoglobins contain the same embryonic β‐like chain, epsilon (ɛ), but two separate α‐like chains, termed zeta and zeta prime (ζ and ζ′)both of which are N‐terminally blocked. The later two expressed hemoglobins contain the same adult α‐chain, but different β‐like chains. The latest expressed hemoglobin contains the same β‐like chain, ɛ, as the two early expressed forms, but the third expressed hemoglobin contains a unique β‐like chain which we have termed omega (ω). A protein database similarity search using the first 54 N‐terminal amino acids of the ω‐chain showed a range of sequence identities of 57–72% to all known mammalian β‐like chains, including the other marsupial ɛ‐chains. The closest identity, reflected by both the highest percentage identity and Smith‐Waterman score, was with the embryonic β‐chains of the aves.

While the primary structures of the hemoglobins reported here do not explain the low hemoglobin‐oxygen affinity in embryonic marsupial blood, the finding of the similarity with the bird globin‐like sequence with one of the marsupial chains has implications on mammalian globin evolution. How many other marsupials and placental mammals are harboring a bird‐like globin in their embryos?
Phosphoprotein PII from Cyanobacteria
Authors: Forchhammer Karl , Hedler Andrea
Published: July 2004

The signal transduction protein PII from Escherichia coli is modified by uridylylation, whereas its counterpart from the cyanobacterium Synechococcus PCC 7942 is phosphorylated at a seryl residue. To elucidate functional conservations between these proteins, we compared the Synechococcus PII protein with the known properties of the E. coli PII protein. Similar to the E. coli protein, Synechococcus PII binds the metabolites 2‐oxoglutarate and ATP in a mutually dependent manner. The synergism of ligand binding was analyzed in detail. The ATP‐binding site of Synechococcus PII could be labelled with 5′‐p‐fluorosulfonylbenzoyladenosine. By heterologous expression of the cyanobacterial glnB gene in E. coli we showed that Synechococcus PII can be modified by the E. coli PII uridylyltransferase. The presence of Synechococcus PII prevents signal transduction of E. coli PII to NtrB, presumably by non‐functional competition. We therefore propose that the primary function of Synechococcus PII is to sense 2‐oxoglutarate, the carbon skeleton required for nitrogen assimilation.
Identification in a Fish Species of Two Id (Inhibitor of DNA Binding/Differentiation)‐related helix‐loop‐helix Factors Expressed in the Slow Oxidative Muscle Fibers
Authors: Rescan Pierre‐Yves
Published: July 2004

Helix‐loop‐helix (HLH) proteins related to the inhibitor of DNA binding/differentiation (Id) serve as general antagonists of cell differentiation. They lack a basic DNA‐binding domain and are thought to function in a dominant negative manner by sequestering basic HLH (bHLH) transcription factors that are involved in cell determination and differentiation. Four Id‐encoding genes have been shown in mammals, they have a distinct pattern of expression suggesting different functions for each member in different cell lineage. In this study we describe the identification and cloning of two trout cDNAs which encode helix‐loop‐helix proteins showing a high degree of similarity with mammalian Id family members. One cDNA encodes a trout putative Id1 protein (TId1) that is 63% identical to the human Id1 protein over the entire length and 78% identical within the HLH region. The other cDNA encodes a trout putative Id2 protein (TId2) that shows 82% identity to the human Id2 protein and only one change that is conservative over the HLH region. In the 3′ untranslated region, TId2 mRNA exhibits 16 nucleotides upstream from the AATAAA site, a palindromic sequence similar to the cytoplasmic polyadenylation element (CPE) which is also present in Id2 and Id3 mRNAs from mammals and in XIdx/XIdI mRNA from Xenopus. In the fish, TIdl and TId2 are expressed in a tissue‐specific manner, with slightly different patterns. During myogenesis, TId1 and TId2 are highly expressed in the myotomal musculature of fish embryos and of early alevins but are down‐regulated in that of late alevins. In muscle from juveniles and adults, TId1 and TId2 transcripts are abundant in the slow oxidative fibers while they are absent in the fast glycolytic fibers. This expression pattern suggests that Id genes play a role in the regulation of muscle fiber pheno‐type in addition to controlling early myogenesis. On the whole. the identification of two HLH‐Id encoding genes in a major taxonomic group like teleosts, suggests an early divergence of Id genes in vertebrate evolution. The observation that Id transcripts are present selectively in the slow muscle reveals that their expression is more complicated than previously appreciated.
Two‐Dimensional1H‐NMR and CD Structural Analysis in a Micellar Medium of a Bovine αs1‐Casein Fragment having Benzodiazepine‐Like Properties
Authors: Lecouvey Marc , Frochot Céline , Miclo Laurent , Orlewski Piotr , Driou Alain , Linden Guy , Gaillard Jean‐Luc , Marraud Michel , Cung Manh Thong , Vanderesse Régis
Published: July 2004

The conformation of the benzodiazepine‐like decapeptide, YLGYLEQLLR, corresponding to residues 91–100 of bovine αs1‐casein, has been examined in SDS micelles using CD, two‐dimensional1H‐NMR and restrained molecular‐dynamics simulation. Evidence is presented that the decapeptide adopts a rigid structure in water/SDS micellar medium, but not in water or dimethylsulfoxide. The three‐dimensional structure, consistent with the proton‐proton distances obtained from the quantitative analysis of the two‐dimensional NOES, was generated by restrained energy minimization and molecular‐dynamics simulation. In water/SDS micellar medium, YLCYLEQLLR adopts an amphipathic helicoid structure with distinct hydrophobic and hydrophilic faces. The relative disposition of the tyrosine aromatic rings was compared with that of the aromatic rings in the benzodiazepines.
Cloning and Characterization of the Oxytricha Granulifera Chaperonin Containing Tailless Complex Polypeptide 1 γ Gene
Authors: Palmedo Gabriele , Ammermann Dieter
Published: July 2004

Here we report the cloning and characterization of the Oxytricha granulifera CCTγ (chaperonin containing tailless complex polypeptide 1) gene encoding a protein that consists of 559 amino acids. The derived amino acid sequence shares 68% identity with Tetrahymena pyriformis CCTγ and 58% identity with Mus musculus CCTγ. The O. granulifera CCTγ gene is located on a 2.07‐kbp macronuclear gene‐sized piece. Its transcription initiation site was determined by primer extension analysis. The expression of the CCTγ gene was investigated by northern blot hybridization of the coding region with exponentially growing cells, heat shocked cells, and cells treated with 50 μM CdCl2. After heat shock, a decrease in the amount of the transcript compared to that of exponentially growing cells could be shown. In contrast, higher amounts of the transcript could be detected after treatment with CdCl2.
A 20‐Amino‐Acid Autonomous RNA‐Binding Domain Contained in an Enoyl‐CoA Hydratase
Authors: Nakagawa Junichi , Moroni Christoph
Published: July 2004

A + U‐rich elements in the 3′ untranslated region of mRNA species coding for lymphokines and early response genes play a pivotal role in the control of their rapid turnover. In a search for corresponding trans‐acting factors, we have previously affinity‐purified and cloned a human 32‐kDa A+U‐binding protein, termed AUH. AUH exhibited dual activities, namely A+U‐specific RNA‐binding and catalytic activity as enoyl‐CoA hydratase. In this report we map the RNA‐binding site by analysis of a series of deletion and substitution recombinant proteins. Ultraviolet cross‐linking experiments demonstrated that the deletion of a 20‐amino‐acid segment, Lys109‐Ile128, abolished more than 80% of the relative RNA‐binding activity. This segment conferred RNA‐binding activity when fused to maltose binding protein. Binding of this fusion protein to A+U‐rich RNA was significantly competed by an AUUUA cluster and poly(U), followed by poly(G), but not by poly(A) nor poly(C). Furthermore, RNA binding of the fusion protein was competed by a synthetic peptide corresponding to Lys109‐Ile128. Circular dichroic measurement indicated formation of a specific complex between this peptide and poly(U) but not with poly(A). The identified 20 amino acids therefore constitute an automonous RNA‐binding domain, distinct from the RNA‐recognition motifs of the family of ribonucleoproteins or NAD/RNA‐binding sites in dehydroge‐nases found in hitherto reported A+U‐binding proteins. Replacement of Arg125 in this motif with Glu reduced binding twofold, indicating this residue is integral to the binding function. Deletion of other parts of the protein did not impair RNA binding to any significant extent. By contrast, the hydratase activity of AUH required an intact three‐dimensional conformation, as most mutations downstream of Ser68 impaired enzymatic activity.
The FLG Motif in the N‐Terminal Region of Glucoprotein 41 of Human Immunodeficiency Virus Type 1 Adopts a Type‐I β Turn in Aqueous Solution and Serves as the Initiaion Site for Helix Formation
Authors: Chang Ding‐Kwo , Chien Wei‐Jyun , Cheng Shu‐Fang
Published: July 2004

NMR and CD studies were carried out on a peptide representing the hydrophobic N‐terminal domain of envelope glycoprotein of human immunodeficiency virus type‐1 in solutions of varying polarity. It was found that in aquaeous solution the amide proton of glycine in the FLG motif resonated at a considerably high field and its chemical shift, within the limit of experimental precision, had a temperature coefficient of zero in the range studied. The upfield shift of NH of the glycine could be largely attributed to the ring‐current effect of phenylalanine in the FLG motif that participated in a type‐1 β turn with a short Cβ(i)‐NH(i+2) distance. The slower proton‐deuterion exchange for the glycine amide proton relative to that of other glycines was consistent with a folded structure for the motif in aquaeous solution. Results of the molecular simulation showed that this proton was shielded from the solvent by non‐polar side chains of the amino acid residues surrounding the turn stabilized by hydrophobic interactions, thus explaining the zero temperature coefficient of the proton chemical shift. The structural stabilizing effect of the hydrophobic interaction was supported by the behavior of the proton in less polar Me2SO solution, in which the anomaly in the chemical shift and its temperature coefficient was less prominent. Detailed secondary‐structure analysis suggested that the β turn of the FLG motif may act as an initiation core for helix formation, probably because the turn readily transforms into helical form.
Small‐Angle X‐Ray Scattering Analysis of Pneumococcal Lipoteichoic Acid Phase Structure
Authors: Fischer Werner , Markwitz Sabine , Labischinski Harald
Published: July 2004

X‐Ray‐scattering analysis was performed on micelles of lipoteichoic acid from Streptococcus pneumoniae. This lipoteichoic acid differs from poly(glycerophosphate) and poly(glycosylglycerophosphate) lipoteichoic acids by a unique positively charged diacylglyceroglycolipid anchor and a complex structure of the hydrophilic chain which is composed of zwitterionic tetrasaccharide ribitol phosphate repeats each carrying two zwitterionic phosphocholine substituents. The size distribution of the lipoteichoic acid micelles was sufficiently homogenous to determine their size and some related molecular parameters by small‐angle scattering analysis. Nearly independent on the ionic strength of the aqueous dispersion, an average micelle contained about 150 lipoteichoic acid molecules arranged in a spherical assembly with a diameter of about 23 nm, whereby the hydrophilic region occupied an outer shell of about 8.5 nm thickness. Taken this as the average chain length of LTA in the micelle and 7.2 repeats per chain, each repeat contributed about 1.2nm to the thickness of the hydrophilic shell as compared to 2.4 nm for a fully extended chain conformation and 1.1 nm estimated for a more helical arrangement [Klein, R. A., Hartmann, R., Egge, H., Behr, T. & Fischer, W. (1994) Carbohydr. Res. 256, 189‐222].

A comparison with the micelle of poly(glycerophosphate) lipoteichoic acid of Staphylococcus aureus suggests that the supramolecular structure is largely independent of the structure of the hydrophilic chain and solely dictated by the small cross‐sectional area of the diacylglycerol moiety common to all lipoteichoic acids and lipoglycans of gram‐positive bacteria.
Influence of Osmotic Stress on Protein Methylation in Resealed Erythrocytes
Authors: Ingrosso Diego , Cotticelli Maria Grazia , D'angelo Stefania , Buro Maria Domenica , Zappia Vincenzoa , Galletti Patrizia
Published: July 2004

Resealed erythrocytes are a useful means of targeting exogenous proteins or extending their activity. We tested human resealed erythrocytes as a model system for studying protein methyl esterification, a reaction involved in the processing of spontaneously dearnidated/isomerized polypeptides. Our results show that resealed erythrocytes are still active in the metabolic processes that lead to the formation of methyl‐esterified proteins. The methylation pattern of endogenous membrane proteins appeared to be similar to that of normal erythrocytes, with bands 2.1, 3, 4.1 and 4.2 as the major methyl acceptors. We detected methyl esterification of ovalbumin, as an exogenous substrate trapped within resealed erythrocytes. Methyl incorporation was almost completely inhibited by simultaneously loading red cells with adenosine and homocysteine thiolactone, in vivo precursors of the transmethylation inhibitor S‐adenosyl‐homocysteine. We investigated the effects of repeated resealing procedures on methyl acceptability of endogenous membrane proteins. We found that methyl‐incorporation levels increased, despite an apparent conserved protein composition of the membrane. This result suggests that osmotic stress to the membrane may be responsible for increased protein methylation due to the appearance of new sites or an increased accessibility of existing sites.
The Active Species of ‘CO2’ Utilized by Formylmethanofuran Dehydrogenase from Methanogenic Archaea
Authors: Vorholt Julia A. , Thauer Rudolf K.
Published: July 2004

Formylmethanofuran dehydrogenase from methanogenic Archaea catalyzes the reversible conversion of CO2 and methanofuran to formylmethanofuran, which is an intermediate in methanogenesis from CO2 a biological process yielding approximately 0.3 billion tons of CH4 per year. With the enzyme from Methanosarcina barkeri, it is shown that CO2 rather than HCO3 is the active species of ‘CO2’ utilized by the dehydrogenase. Evidence is also presented that the enzyme catalyzes a methanofuran‐dependent exchange between CO2 and the formyl group of formylmethanofuran. The results are consistent with N‐carboxymethanofuran being an intermediate in CO2 reduction to formylmethanofuran.
Identification of the Regions on the M110 Subunit of Protein Phosphatase 1M That Interact with the M21 Subunit and with Myosin
Authors: Johnson Deborah , Cohen Philip , Chen Mao Xiang , Chen Yu Hua , Cohen Patricia T. W.
Published: July 2004

We have previously isolated a form of protein phosphatase‐1 (PP1M) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PP1C) bound to an M‐complex consisting of 110‐kDa (M110) and 21‐kDa (M21) subunits. The interaction of PP1C with an N‐terminal region of the M110 subunit enhances the dephosphorylation of myosin and suppresses the dephosphorylation of other substrates [Alessi, D. R., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023–1035; Chen, Y. H., Chen, M. X., Alessi, D. R., Campbell, D. G., Shanahan, C, Cohen, P. & Cohen, P. T. W. (1994) FEES Lett. 356, 51–6; Johnson, D. R, Moorhead, G., Caudwell, F. B., Cohen, P., Chen, Y. H., Chen, M. X. & Cohen, P. T. W. (1996) Eur. J. Biochem. 239, 317–325]. In this paper, we establish that PP1M accounts for nearly all the myosin phosphatase activity in myofibrils, that the M110 and M21 subunits are present at similar concentrations in the myofibrillar fraction, and that these subunits are entirely bound to PP1. We demonstrate that the M21 subunit does not interact with PP1C, but with the C‐terminal 72 residues of the M110 subunit, a region which is 43% identical to residues 87–161 of the M21 subunit. A fragment of the M21 subunit, M21‐(M1‐L146), which lacks the C‐terminal leucine zipper, also bound to the M110 subunit, but two other fragments M21‐(M1‐E110) and M21‐(E110–K186) did not. The M110 and M21 subunits were both found to be myosin‐binding proteins. The C‐terminal 291 residues of the M110 subunit, but not the C‐terminal 72 residues, bound to myosin, but the N‐terminal fragments M110‐(M1‐E309) and M110‐(M1‐S477) did not. Thus, the region of the M110 subunit that stimulates the dephosphorylation of myosin by PP1C is distinct from the region that targets PP1M to myosin. Remarkably, each myosin dimer was capable of binding about 20 mol M21 subunit and many of the M21‐binding sites were located in the myosin rod domain. The potential significance of this observation is discussed.
C‐Cytosolic and Transmembrane Domains of the N‐benzoyl‐L‐tyrosyl‐p‐aminobenzoic Acid Hydrolase α Subunit (Human Meprin α)are Essential for its Retention in the Endoplasmic Reticulum and C‐Terminal Processing
Authors: Hahn Dagmar , Lottaz Daniel , Sterchi Erwin E.
Published: July 2004

N‐benzoyl‐L‐tyrosyl‐p‐aminobenzoic acid hydrolase (PPH, human meprin) is a member of the astacin family of Zn‐metalloendopeptidases and is highly expressed in the microvillus membrane of human small intestinal epithelial cells. It is a type I transmembrane protein consisting of differentially processed glyco‐sylated α and β subunits. Biosynthesis experiments using transfected, metabolically labelled simian virus 40 (SV40) transformed african green monkey kidney cells (COS‐1) and Madin Darby canine kidney (MDCK) cells, have previously shown that PPHα was retained in the endoplasmic reticulum (ER) and that for subsequent secretion removal of the α‐tail was necessary [Grünberg, J., Dumermuth, E., Eldering, J. A. & Sterchi, E. E. (1993) FEBS Lett. 335, 376–379]. We proposed an involvement of the α‐tail in ER retention. To investigate the possible role of the transmembrane and/or the C‐terminal domain of the α‐subunit, tailswitch mutants were constructed in which these domains were exchanged between the a and β subunits. Biosynthesis and post‐translational processing of these mutants were investigated in transiently transfected COS‐1 cells. The β/α tailswitch mutant, in which the transmembrane and C‐cytosolic parts of PPHP were substituted by the corresponding parts of the PPHα subunit, was transported much slower compared with the wild‐type PPHP subunit, In addition, fusion of the α‐tail to a C‐terminally truncated secretory form of PPHα leads to its retention in the ER. This mutant, but not the secretory form, coimmu‐noprecipitated with calnexin, indicating an involvement of this molecular chaperone in retaining PPHα in the ER. The α/β tailswitch mutant, in which the transmembrane domain and the C‐cytosolic part of PPHα were substituted by the corresponding parts of PPHβ, was processed less efficiently in comparison with PPHα, resulting in a lower secretion rate. Taken together these data suggest a role of the α‐tail in mediating association with ER‐resident machinery, facilitating C‐terminal processing.
Structural Aspects of the Interaction of peptidyl‐glycylleucine‐carboxyamide, a Highly Potent Antimicrobial Peptide from Frog Skin, with Lipids
Authors: Latal Angelika , Degovics Gabor , Epand Raquel F. , Epand Richard M. , Lohner Karl
Published: July 2004

The interaction of PGLa (peptidyl‐glycylleucine‐carboxyamide), a 21‐amino‐acid residue cationic peptide, isolated from the skin of the South African clawed frog, Xenopus laevis, with model membrane systems was investigated. Our studies focussed on the importance of the difference in the phospholipid composition of bacterial and erythrocyte membranes. This is of particular interest to gain information on the specificity of membranolysis exhibited by this peptide against bacteria but not against erythrocytes.

In phosphate buffer at physiological pH, as well as in the presence of the zwitterionic phosphatidylcholine and sphingomyelin, the peptide had a random structure but it adopted an α‐helical conformation in the presence of negatively charged lipids. Furthermore, calorimetric experiments showed that PGLa had no effects on the thermotropic phase behavior of liposomes composed of the choline phosphatides, while separation of a distinct peptide‐rich domain was observed for phosphatidylglycerol liposomes. In addition to the main transition of pure 1,2‐dipalmitoylglycerophosphoglycerol at 40°C a second transition owing to the peptide‐perturbed lipid domains was found at 41°C. This conclusion is supported by X‐ray diffraction experiments which indicated that PGLa penetrates into the hydrophobic core of the bilayer inducing an untilting of the hydrocarbon chains as observed in the gel phase of the pure lipid. These results demonstrate that this antibacterial peptide specifically interacts with negatively charged lipid membranes, which are characteristic of bacterial membranes. This can be explained based on the structural features of PGLa.
Structure of the O‐Specific Polysaccharide of Proteus Vulgaris O25 Containing 3‐O‐[(R)‐1‐carboxyethyl]‐d‐glucose
Authors: Knirel Yuriy A. , Kaca Wieslaw , Paramonov Nikolay A. , Cedzynski Maciej , Vinogradov Evgeny V. , Ziolkowski Andrzej , Shashkov Alexander S. , Rozalski Antoni
Published: July 2004

The O‐specific polysaccharide of Proteus vulgaris 025 was studied by acid hydrolysis and by 1H‐NMR and 13C‐NMR spectroscopies, including one‐dimensional NOE and two‐dimensional COSY and heteronuclear 13C,1H correlation (HETCOR) spectroscopy. The polysaccharide was found to contain 3‐O‐[(R)‐1‐carboxyethyl]‐D‐gIucose (D‐RGLC), and the following structure of the pentasaccharide repeating unit was established:
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Characterization of Recombinant Yeast Dolichyl Mannosyl Phosphate Synthase and Site‐Directed Mutagenesis of Its Cysteine Residues
Authors: Forsee W. Thomas , Mcpherson David , Schutzbach John S.
Published: July 2004

Dolichyl mannosyl phosphate synthase is associated with membranes of the rough endoplasmic reticulum and catalyzes mannosyl transfer from GDP‐mannose to the hydrophobic long‐chain acceptor dolichyl‐phosphate. The gene for the yeast enzyme encodes a protein with a molecular mass of 30.36 kDa containing three cysteine residues, at positions 93, 172 and 259 [Orlean, P., Albright, C. & Robbins, P. W. (1988) J. Biol. Chem. 263, 17499–17507]. Inhibition of the synthase by thiol‐specific reagents, including N‐ethylmaleimide, p‐hydroxymercuribenzoate, 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2), and lucifer yellow iodoacetamide (LYI), suggests that sulfhydryl groups might play a role in the catalytic mechanism of the enzyme. Titration of the synthase with Nbs2 or LYI indicated that 1 mol sulfhydryl/mol protein was accessible to these reagents, and that saturation of this site completely inhibited enzyme activity. To ascertain the reactive group and its possible function in enzyme catalysis, each of the cysteine residues was replaced individually by site‐directed mutagenesis. The mutant enzymes had specific activities comparable to that of the wild‐type enzyme, demonstrating that none of the cysteine residues were essential for catalytic activity. All of the mutant proteins except those containing a substitution at Cys93 were inhibited by thiol‐blocking reagents, indicating that Cys93 might be physically located near the catalytic site of the enzyme. GDP‐mannose, dolichyl phosphate and substrate analogs were found to protect against Nbs2 inactivation, further suggesting that Cys93 was physically near, or within, the substrate‐binding site of the enzyme.
Involvement of Lysine Residues in the Gating of the Ryanodine Receptor/Ca2+‐Release Channel of Skeletal Muscle Sarcoplasmic Reticulum
Authors: Feng Wei , Shoshan‐Barmatz Varda
Published: July 2004

In this study, the modification of skeletal muscle ryanodine receptor (RyR)/Ca2+‐release channel with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3,‐diazole (Nbd‐Cl) demonstrates that lysyl residues are involved in the channel gating. Nbd‐Cl was found to have a dual effect: stimulation and inhibition of ryanodine binding and single channel activities. Nbd‐CI, in a time‐dependent manner, first stimulated and subsequently inhibited ryanodine binding to both membrane‐bound and purified RyR. Incubation of sacroplasmic reticulum membranes with Nbd‐CI for 5–20 s resulted in enhanced ryanodine‐binding activity by 2–4‐fold due, to an increased binding affinity by about tenfold, with no effect on the total binding sites (Bmax). However, under prolonged incubation (5–20 min), Nbd‐CI strongly inhibited ryanodine binding by decreasing the Bmax with no effect on the binding affinity. Similar effects of stimulation and inhibition by Nbd‐CI were obtained with single channel activity of RyR reconstituted into planar lipid bilayer. Nbd‐CI initially (within a few seconds) activated the channel to a highly open state, then (within a few minutes) inactivated it to the completely closed state. Nbd‐CI‐modified protein, as assayed by ryanodine binding or single channel activities, was stable against thiolysis by dithiothreitol, suggesting Nbd‐CI modification of lysyl residues. Evidence from absorption and fluorescence excitation and emission spectra also demonstrated that lysyl residues in RyR were modified by Nbd‐CI. Spectrophotometric data were used to estimate a ratio of up to 1 mol Nbd boundmol RyR (tetramer) and up to 4 mol Nbd bound per mol RyR (tetramer) for Nbd‐CI stimulated and inhibited RyR activities, respectively. The results clearly indicate the involvement of two classes of lysyl residues in RyR activity. Modification by Nbd‐CI of the fast‐reacting group led to stimulation of ryanodine binding and single channel activities, while modification of the slow‐reacting group resulted in inhibition of these activities. Thus, the involvement of lysine residues in the gating of the RyR channel is proposed.