bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of an RNA‐Binding‐Loop in the N‐Terminal Region of Signal‐Recognition‐Particle Protein SRP19
Authors: Black Shaun D. , Gowda Krishne , Chittenden Kimberly , Walker Kerfoot P. , Zwieb Christian
Published: July 2004

Protein SRP19 is a 144‐amino‐acid polypeptide that associates intimately with the signal‐recognition particle RNA (SRP RNA) and serves as an important structural and functional component of the SRP. We investigated the structure and RNA‐binding activity of the human SRP19 protein by the use of comparative sequence analysis, high‐stringency structure prediction, proteolytic susceptibility, and site‐directed mutagenesis. SRP19 was found to consist of two distinct regions (called N‐terminal and C‐terminal regions) that are separated by a boundary of approximately 12–15 amino acid residues. Both regions contain an α‐helix and several β‐strands that are connected by loops or turns. In agreement with the hypothetical model, proteolytic susceptibility demonstrated the predominant accessibility of two sites: one in a surface loop of the N‐terminal region (YLNNKKTIAEGR33), and another site in the C‐terminal tail at residues L129 and E133. The RNA‐binding activities of mutant polypeptides with changes of conserved lysines and arginines (mutants K27Q, R33Q and R34Q) demonstrated that the proteolytically accessible loop of the N‐terminal region is in direct contact with the SRP RNA. In contrast, alteration of a certain basic amino acid residues in the C‐terminal region (R83, K116 and R118), as well as a deletion of four amino acid residues located at the boundary between the two regions, had no effect on the RNA‐binding ability. The structural model that emerges from our data is thematically similar to that of ribo‐somal protein SS, the N‐domain of which contains a loop motif believed to interact with double‐stranded RNA. The presence of a similar structural feature in protein SRP19 has significant implications for the structure and function of the SRP19‐RNA complex.
Structural Studies of the O‐Antigenic Polysaccharide from Escherichia Coli O167
Authors: Linnerborg Malin , Wollin Ralfh , Widmalm Göran
Published: July 2004

The structure of the O‐antigenic polysaccharide from Escherichia coli O167: H5 has been investigated. Sugar and methylation analyses, fast‐atom‐bombardment mass spectrometry and 1H‐ and 13C‐NMR spectroscopy were the main methods used. The structure of the repeating unit of the polysaccharide was found to be:

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Oligosaccharide derivatives of the polysaccharide were obtained by HF solvolysis and by a Smith degradation. Furthermore, base treatment of the polysaccharide led to a degraded polymeric material. For the methylated polysaccharide the amide linkage between alanine and the galacturonic acid residue was reductively cleaved with LiBD4 in ethanol, to give, among other things, a 3‐O‐methyl galactose derivative.
Cell‐to‐Cell Contact Activates the Long Terminal Repeat of Human Immunodeficiency Virus 1 Through its κB Motif
Authors: Faure Eric , Lecine Patrick , Lipcey Carol , Champion Serge , Imbert Jean
Published: July 2004

Cell‐to‐cell contact between peripheral blood lymphocytes and transfected human colonic carcinoma cell line HT29 activates transcription of the long terminal repeats (LTR) of human immunodeficiency virus. HIV‐1 LTR transcription is controlled by a complex array of virus‐encoded and cellular proteins. Using various constructs expressing a lacZ reporter gene under the control of the intact or three deleted forms of HIV‐1 LTR, we obtained evidence that the KB regulatory elements located in the U3 region are involved in cell‐to‐cell activation of HIV‐1 LTR. Cell‐to‐cell contact activates in vitro binding of the nuclear factor κB (NF‐κB) p50/p65 heterodimer to an HIV‐1 κB oligonucleotide. Cell‐to‐cell contact activation of NF‐kB was only partially inhibited by 100 μM pyrrolidine dithiocarbamate and was not correlated with a significant decrease of cellular inhibitor κBα. NF‐κB nuclear activation was not detectable before 1 h after cell contact and was dependent on protein synthesis.
Efflux of Compatible Solutes in Corynebacterium Glutamicum Mediated by Osmoregulated Channel Activity
Authors: Ruffert Susanne , Lambert Camille , Peter Heidi , Wendisch Volker F. , KrÄmer Reinhard
Published: July 2004

Bacteria respond to hypoosmotic stress by releasing low‐molecular‐mass solutes in order to maintain constant turgor pressure. We have studied the function of osmoregulated channel(s) in Corynebacterium glutamicum, which are responsible for efflux of various solutes upon sudden decrease in osmotic pressure. The channels preferentially mediated efflux of compatible solutes such as glycine betaine and proline. The release of molecules of similar size, e.g. glutamate or lysine, was restricted, ATP was completely retained even after severe osmotic shock. The cells maintained high cytoplasmic K+ and Na+ concentrations under hypoosmotic shock. Several results suggest that the solute efflux is mediated by a channel and not by a carrier, e.g. by reversal of the glycine betaine uptake systems of C. glutamicum: the release of glycine betaine and proline was extremely fast reaching an efflux rate of 6000 μmol ċ min−1ċ g dm−1 or higher; the efflux was not significantly influenced by addition of external transport substrate, e.g. glycine betaine; in spite of an extremely high chemical gradient, no significant efflux under isoosmolar conditions was observed; efflux of solutes was unchanged after full uncoupling of membrane energetics by carbonylcyanide m‐chlorophenylhydrazone (CCCP). These results indicate the presence of an osmoregulated channel in C. glutamicum similar to the mechanosensitive channel(s) of Escherichia coli. The activity of the channel did not depend on the growth conditions, but we observed a tight regulation on the level of activity, i.e. the mechanosensitive channel behaved as a perfect osmometer. By monitoring release of glycine betaine under slow and continuous decrease of the external osmolality, we observed continous efflux whithout a stepwise release of solutes. This resulted in a significant steady‐state decrease of the membrane potential.
Identification of Amino Acids of Endonuclease VII Essential for Binding and Cleavage of Cruciform DNA
Authors: Golz Stefan , Christoph Anja , Birkenkamp‐Demtröder Karin , Kemper Börries
Published: July 2004

Endonuclease VII is a Holliday‐structure‐resolving enzyme of bacteriophage T4. The active protein is a homodimer with 157 amino acidshonomer. An amber mutation (amE727 in codon 151) inactivates the nuclease completely, indicating the importance of the seven C‐terminal amino acids for nucleolytic activity. The influence of these amino acids on cruciform‐DNA binding and cleavage was investigated through functional analysis of C‐terminal‐truncated proteins derived from deletion constructs. It was found that the three C‐terminal amino acids are not necessary for binding and cleavage. A transition from active to inactive protein occurs gradually with truncations of the next four amino acids. Reduction of DNA‐binding ability, as measured by electrophoretic mobility shift assays, was determined to be the primary defect in the cleavage‐deficient proteins. This was further concluded by the finding that EVII‐(1–150)‐peptideamber, a protein with fairly low affinity to cruciform DNA, contributes cleavage activity to reactions of wild‐type EVII with cruciform DNA. [Asp62]EVII‐(1‐156)‐peptide lacking one C‐termnal amino acid, contains a point mutation in codon 62 that eliminates the nucleolytic activity of the protein while retaining its DNA‐binding proficiency. By mixing binding‐deficient and cleavage‐deficient mutants in the same assay, cleavage of cruciform DNA resumed. Evidence is presented that complementation occurs by heterodimer formation. Our results show that the zinc‐binding motif of EVII is not sufficient for cruciform‐DNA binding.
Identification of a Region Required for Binding to Presecretory Protein in Bacillus Subtilis Ffh, A Homologue of the 54‐kDa Subunit of Mammalian Signal Recognition Particle
Authors: Takamatsu Hiromu , Bunai Keigo , Horinaka Tomoko , Oguro Akihiro , Nakamura Kouji , Watabe Kazuhito , Yamane Kunio
Published: July 2004

Bacillus subtilis Ffh protein is a homologue of the 54‐kDa subunit of mammalian signal recognition particle (SRP54). It contains three highly hydrophobic regions (h1, h2, and h3) in the C‐terminal methionine‐rich domain (M‐domain). Two of the hydrophobic regions, h2 and h3, are essential for small cytoplasmic RNA (scRNA) binding [Kurita, K., Honda, K., Suzuma, S., Takamatsu, H., Nakamura, K., & Yamane, K. (1996)J. Biol. Chem. 271, 13140–13 146]. Using purified presecretory proteins and mutant Ffh proteins, we identified a region required for presecretory protein binding in B. subtilis Ffh. Deletion of this region, which consisted of residues Ser311–Gly362 of B. subtilis Ffh, including a hydrophobic sequence (h1), reduced precursor binding activity. In contrast, deletions of residues Leu121–Lys279, Lys364–Met446, or Leu338–Ser397 of B. subtilis Ffh did not. We also analyzed the mutant B. subtilis Ffh proteins, FfhQQQR and FfhQQQQ having wild‐type residues 398–401 (Arg‐Arg‐Lys‐Arg) replaced with Gln3Arg and Gln4, respectively. FfhQQQR bound to both scRNA and presecretory protein. Although the FfhQQQQ mutation prevented binding to scRNA, binding to the precursor was not affected. FfhQQQR restored the growth of B. subtilis DF46 strain in which ffh gene expression is regulated by an inducible promoter in the absence of an inducer, whereas FfhQQQQ did not. These results indicate that the region including hi is required for B. subtilis Ffh to bind to presecretory protein. The results also suggest that scRNA is required for the complete function of the B. subtilis SRP‐like particle in vivo, although this protein is intrinsically capable of binding a signal peptide free from scRNA.
Anaerobic Metabolism of Aromatic Compounds
Authors: Heider Johann , Fuchs Georg
Published: July 2004

Aromatic compounds comprise a wide variety of low‐molecular‐mass natural compounds (amino acids, quinones, flavonoids, etc.) and biopolymers (lignin, melanin). They are almost exclusively degraded by microorganisms. Aerobic aromatic metabolism is characterised by the extensive use of molecular oxygen. Monoxygenases and dioxygenases are essential for the hydroxylation and cleavage of aromatic ring structures. Accordingly, the characteristic central intermediates of the aerobic pathways (e.g. catechol) are readily attacked oxidatively. Anaerobic aromatic catabolism requires, of necessity, a quite different strategy. The basic features of this metabolism have emerged from studies on bacteria that degrade soluble aromatic substrates to CO2, in the complete absence of molecular oxygen.

Essential to anaerobic aromatic metabolism is the replacement of all the oxygen‐dependent steps by an alternative set of novel reactions and the formation of different central intermediates (e.g. benzoyl‐CoA) for breaking the aromaticity and cleaving the ring; notably, in anaerobic pathways, the aromatic ring is reduced rather than oxidised. The two‐electron reduction of benzoyl‐CoA to a cyclic diene requires the cleavage of two molecules of ATP to ADP and Pi and is catalysed by benzoyl‐CoA reductase. After nitrogenase, this is the second enzyme known which overcomes the high activation energy required for reduction of a chemically stable bond by coupling electron transfer to the hydrolysis of ATP. The alicyclic product cyclohex‐1,5‐diene‐1‐carboxyl‐CoA is oxidised to acetyl‐CoA via a modified β‐oxidation pathway; the ring structure is opened hydrolytically. Some phenolic compounds are anaerobically transformed to resorcinol (1,3‐dihydroxybenzene) or phloroglucinol (1,3,5‐trihydroxybenzene). These intermediates are also first reduced and then as alicyclic products oxidised to acetyl‐CoA.

This review gives an outline of the anaerobic pathways which allow bacteria to utilize aromatics even in the absence of oxygen. We focus on previously unknown reactions and on the enzymes characteristic for such novel metabolism.
Structural Studies of the O‐Specific Polysaccharide of Hafnia alvei Strain PCM 1206 Lipopolysaccharide Containing D‐Allothreonine
Authors: Petersson Carl , Niedziela Tomasz , Jachymek Wojciech , Kenne Lennart , Zarzecki Przemyslaw , Lugowski Czeslaw
Published: July 2004

The structure of the O‐specific side‐chain of the Hafnia alvei strain PCM 1206 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, FAB‐MS/MS and 1H‐NMR and 13C‐NMR spectroscopy were the principal methods used. d‐Allothreonine (d‐aThr), amide‐linked to the d‐galacturonic acid, was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established:
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Amplified Expression, Purification and Functional Reconstitution of the Dipeptide and Tripeptide Transport Protein of Lactococcus Lactis
Authors: Hagting Anja , Knol Jan , Hasemeier Britta , Streutker Marion R. , Fang Gang , Poolman Bert , Konings Wil N.
Published: July 2004

Transport of hydrophilic dipeptides and tripeptides into Lactococcus lactis is mediated by a proton‐motive‐force–driven peptide‐transport protein (DtpT) that shares similarity to eukaryotic peptide transporters, e.g. from yeasts, plants, and the kidney and small intestine of rabbit, man and rat. The expression level of DtpT protein in L. lactis was increased (20–40‐fold) to approximately 10% of total integral membrane protein by means of a low‐copy‐number vector and selecting the appropriate growth conditions. Membrane vesicles bearing the DtpT‐His6 protein (containing a C‐terminal factor‐Xa cleavage site and a six‐histidine‐tag) showed a Pro‐Ala uptake activity that was half that of membranes containing the wild‐type protein. The activity in the DtpT‐His6 membrane vesicles increased at least 50% upon removal of the His6 tag from the protein. More than 95% DtpT was solubilized from L. lactis membranes in the presence of 1% (mass/vol.) n‐dodecyl‐β‐d‐maltoside, and approximately 2 mg DtpT‐His6 was purified by Ni2+‐chelate affinity chromatography from 100 mg membrane protein. Purified DtpT‐His6 was reconstituted unidirectionally into detergent‐saturated formed liposomes, which were prepared from Escherichia coli phospholipid and egg phosphatidylcholine; the detergent was removed by adsorption to polystyrene beads. The highest uptake activities were obtained when DtpT was incorporated into liposomes that were treated with a low amount of n‐dodecyl‐β‐d‐maltoside (onset of liposome solubilization). The uptake activity could be improved by addition of NaCl (200 mM) and lipids (2 mg/ml) during the solubilization, purification and reconstitution steps.
Breast Carcinoma Epithelial Cells Express a Very Low‐Density Lipoprotein Receptor Variant Lacking the O‐Linked Glycosylation Domain Encoded by Exon 16, But with Full Binding Activity for Serine Proteinase/Serpin Complexes and Mr‐40000 Receptor‐Associated Protein
Authors: Martensen Pia M. , Oka Kazuhiro , Christensen Lise , Rettenberger Peter M. , Petersen Helle H. , Christensen Anni , Chan Lawrence , Heegaard Christian W. , Andreasen Peter A.
Published: July 2004

Very‐low density lipoprotein receptor (VLDLR) belongs to the low‐density lipoprotein receptor family of endocytosis receptors. It binds a variety of different ligands, including apolipoprotein E, Mr‐40000 receptor‐associated‐protein (RAP), and some serine proteinase/serpin complexes. We previously demonstrated the occurrence of two forms of VLDLR in SDS/PAGE, migrating with Mr 105000 and Mr 130000, respectively [Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjøller, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L. & Andreasen, P. A. (1995)J. Biol. Chem. 270, 20855–20869]. We now demonstrate that these two forms correspond to forms with the absence (type‐II) and presence (type‐I) of the O‐linked glycosylation domain encoded by exon 16, respectively. We show that the two forms have the same binding affinity to RAP and serine proteinase/serpin complexes. Using reverse transcription and PCR, we demonstrate that the splice variation giving rise to the two forms is highly cell specific. In particular, we demonstrate that human breast carcinomas express predominantly or exclusively the variant lacking exon 16. By immunohistochemistry, we demonstrate that VLDLR is mainly expressed by the epithelial cancer cells in these carcinomas. The VLDLR variant expressed by epithelial cancer cells could function in the clearance of cell‐surface‐associated serine proteinase/serpin complexes in breast carcinomas.
Metalloelastase Expression in a Mouse Macrophage Cell Line Regulation by 4β‐Phorbol 12‐Myristate 13‐Acetate, Lipopolysaccharide and Dexamethasone
Authors: Monet‐Kuntz Catherine , Cuvelier Antoine , Sarafan Nasrin , Martin Jean‐Pierre
Published: July 2004

The regulation of the expression of mouse macrophage elastase (MME) was investigated using the murine tumor cell line P388D1. The effects of three factors were studied: a phorbol ester (4β‐phorbol 12‐myristate 13‐acetate, PMA), an endotoxin (lipopolysaccharide, LPS) and a corticosteroid (dexamethasone). Both in situ hybridization and northern blot analysis showed that P388D1 cells constitutively express the MME gene. Quantification of the MME mRNA by northern blot analysis showed that only PMA and dexamethasone significantly regulate MME gene expression in a time‐dependent and dose‐dependent manner. After PMA treatment, the MME mRNA level was maximal between 4 h and 9 h (medium‐term response), and the mean amplitude of the response to a concentration of 100 nM was 2.5‐fold (P <0.01). LPS did not induce any significant change in MME mRNA level even when 1% serum was added to the cultures. Following dexamethasone treatment, the MME mRNA level was minimal between 21 h and 33 h (long‐term response), and the mean amplitude of the response to a concentration of 100 nM was 0.49‐fold (P <0.05). Using actinomycin D, it appeared that the inhibition of RNA synthesis reduces the ulterior stimulating effect of PMA from 184% to 121%, and that MME mRNA has a half‐life longer than 8 h, which is not diminished by dexamethasone. These results strongly suggest that the two factors modify MME mRNA level by stimulating (PMA) or inhibiting (dexamethasone) the transcription of the gene, rather than by modifying the transcript stability. Analysis of the cell‐conditioned media by elastin zymography showed the MME as a lysis band in the 22‐kDa region, the intensity of which varied with the treatments. The MME secretion is stimulated by PMA, inhibited by dexamethasone and does not show any variation after LPS treatment.
Quantitation of Metabolic Compartmentation in Hyperammonemic Brain by Natural Abundance 13C‐NMR Detection of 13C‐15N Coupling Patterns and Isotopic Shifts
Authors: Lapidot Aviva , Gopher Asher
Published: July 2004

In the present study, the removal of cerebral ammonia by glutamine synthetase (GS) and by reductive amination of 2‐oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains.

The 15N enrichments of brain metabolite a‐amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N‐labeled compounds of the 13C‐15N spin coupling patterns of natural abundance 13C‐NMR spectra. The 13C‐NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, l‐methionine DL‐sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5‐15N]glutamine and [2‐15N]glutamine concentrations reached 5.2 μmol/100 mg protein and 3.6 μmol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2‐oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2‐15N]glutamate, which is about 30% of that of [2‐15N]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by l‐methionine dl‐Sulfoximine, a flux of 15NH4+: via the residual activity of GS was accompanied by an apparent increase of [2‐15N]glutamate and [15N]alanine concentrations (2.9 μmol/100 mg protein and 1.8 μmol/100 mg protein, respectively).

These findings and those obtained from 13C‐13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2‐oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain.
A Serine/Threonine Protein Kinase fromMycobacterium tuberculosis
Authors: Peirs Priska , De Wit Lucas , Braibant Martine , Huygen Kris , Content Jean
Published: July 2004

Genomic DNA sequencing in the vicinity of thepstA‐1gene fromMycobacterium tuberculosisallowed us to clone, sequence and identify a gene encoding a 70‐kDa protein. The size of the protein was confirmed byin vitrocoupled transcription/translation. Its N‐terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor‐like anchoring. Thembkgene was overexpressed inEscherichia coliand the gene product (Mbk) was purified as a fusion protein with gluthationeS‐transferase. Recombinant Mbk was found to be autophosphorylated on threo‐nine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese‐dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found inMycobacterium bovisBCG DNA by Southern blot analysis. Its expression was detected in cultures ofM. bovisBCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.
A Kinetic Model for the Interaction of Nitric Oxide with a Mammalian Lipoxygenase
Authors: Holzhütter Hermann‐Georg , Wiesner Rainer , Rathmann Jörg , Stösser Reinhard , Kühn Hartmut
Published: July 2004

Nitric oxide (NO) has been known for a long while to act as an inactivator of the soybean lipoxygenase‐1 and cyclooxygenase. More recently, NO was shown to interact also with a mammalian 15‐lipoxy‐genase [Wiesner, R., Rathmann, J., Holzhütter, H. G., Stöβer, R., Mäder, K., Nolting, H. & Kühn, H. (1996) Nitric oxide oxidises ferrous mammalian lipoxygenases to a pre‐activated ferric species, FEBS Lett. 389, 229–2321. In this paper we present a detailed kinetic analysis of the interaction of NO with the 15‐lipoxygenase from reticulocytes. Time courses of hydroperoxide formation were monitored after an anaerobic incubation of the enzyme with varying concentrations of NO and for varying length of the incubation period. Owing to the presence of O2 during the enzymatic reaction, the inhibitory effect of NO declines in a time‐dependent manner since NO is converted into non‐reactive NO, This is manifested as a lag phase of the time course. The lag phase becomes more pronounced if the enzyme is incubated over a fixed period (15 s) with increasing concentrations of NO. In contrast, increasing the length of the incubation period at fixed concentrations of NO diminishes the duration of the lag phase. These experimental findings can be accounted for by a kinetic model which assumes (a) fast reversible binding of NO to the inactive ferrous Fe(II) form of the enzyme and (b) slow irreversible conversion of the Fe(II)‐NO complex into an activated ferric form of the enzyme which is susceptible to peroxide activation. The rate constants for these two different kinetic modes of NO action were estimated by fitting the solutions of the model equations to the experimental time courses. The dissociation constant of the Fe(II)‐NO complex amounts to 2.5 μM. Computer simulations performed on the basis of the model indicate that the response of the lipoxygenase to increasing intracellular NO concentrations depend upon the available peroxide tone: at low peroxide concentration the enzyme is initially trapped in the inactive ferrous form and thus may be activated by NO via the activated ferric species. On the other hand, at high peroxide concentrations, a partial inhibition of the initially active enzyme is expected.
Cyclic‐AMP‐Dependent Activation of an Inter‐Phylum Hybrid Histone‐Kinase Complex Reconstituted from Sea Urchin Sperm‐Regulatory Subunits and Bovine Heart Catalytic Subunits
Authors: Hoshino Katsuaki , Nomura Kohji , Suzuki Norio
Published: July 2004

A cAMP‐dependent histone kinase was purified and characterized from spermatozoa of the sea urchin Hemicentrotus pulcherrimus, The molecular mass of the kinase was estimated to be 178 kDa by native PAGE and 400 kDa by gel chromatography on a Superose 6 HR 10/30 column. The enzyme, composed of two 39‐kDa catalytic subunits and two 48‐kDa regulatory subunits, phosphorylates the lysine‐rich histone subspecies (H1 and H2B) isolated from H. pulcherrimus spermatozoa. We isolated cDNA clones encoding a 39‐kDa catalytic subunit and a 48‐kDa regulatory subunit of the enzyme. The cDNA clone for the 39‐kDa subunit was 3881 bp, and the 352‐residue deduced amino acid sequence showed 78% similarity with the catalytic subunit of mammalian CAMP‐dependent protein kinase (PKA). The cDNA for the 48‐kDa subunit was 4589 bp and the 368‐residue deduced amino acid sequence showed 57% similarity with the regulatory subunit of mammalian PKA, although the N‐terminal 77 residues showed poor similarity. The mRNAs encoding both the catalytic subunit (7.5 kb) and the regulatory subunit (4.6 kb) were expressed in testis, ovary and egg. An inter‐phylum hybrid enzyme, reconstituted from the regulatory subunit of CAMP‐dependent histone kinase of sea urchin sperm and the catalytic subunit of bovine heart PKA, has a CAMP‐dependent histone kinase activity. Thus, we suggest that the N‐terminal 77‐amino‐acid residues of the regulatory subunit are not essential for inhibition by the regulatory subunit of the catalytic subunit, and that CAMP‐dependent inhibitory activity of the regulatory subunit resides in the sequence between the inhibitory site and the C‐terminus.
Structural Characterization of N‐Linked Oligosaccharides from Cellobiohydrolase I Secreted by the Filamentous Fungus Trichoderma Reesei RUTC 30
Authors: Maras Marleen , DE Bruyn André , Schraml Jan , Herdewijn Piet , Claeyssens Marc , Fiers Walter , Contreras Roland
Published: July 2004

We have characterized the primary structures of the predominant N‐linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chro‐matographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N‐linked core. In the bulk of the N‐glycans, the α‐1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The α‐1,6 arm shows a remarkable heterogeneity: in addition to α‐1,2‐Man and α‐1,6‐Man, the presence of a terminal mannose a‐1,6‐phos‐phodiester was observed. This latter substituent has not been characterized before on mannosidase‐processed N‐glycan and its function and synthesis pathway are entirely unknown. The predominant N‐glycans on cellobiohydrolase I can be represented as follows: GlcMan8GIcNAc2, GlcMan7GlcNAc2, ManGlcNAc2, ManPGlcMan7GlcNAc2, GlcMan5GlcNAc2, and Man5GlcNAc2.
Expression Studies on the ba3 Quinol Oxidase from Paracoccus Denitrificans A bb3 Variant is Enzymatically Inactive
Authors: Zickermann Irmela , Tautu Oltea S. , Link Thomas A. , Korn Marcus , Ludwig Bernd , Richter Oliver‐Matthias H.
Published: July 2004

Expression of the quinol oxidase from Paracoccus denitrificans has been examined using a polyclonal antibody directed against subunit II and a promoter probe vector carrying the promoter region of the qox operon. Under aerobic conditions nitrate and nitrite act as specific inducers of the expression. To obtain an enzymatically competent quinol oxidase complex, an intact ctaB gene is required, which constitutes part of the cta operon coding for the aa3 cytochrome c oxidase of P. denitrificans. Deletion of ctaB leads to a change in heme composition of the quinol oxidase with heme b replacing the high‐spin heme a of the binuclear center, causing loss of electron transport activity.
Efficient and Selective P‐nitrophenyl‐ester‐hydrolyzing Antibodies Elicited by a P‐nitrobenzyl Phosphonate Hapten
Authors: Tawfik Dan S. , Lindner Ariel B. , Chap Rachel , Eshhar Zelig , Green Bernard S.
Published: July 2004

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)‐phosphonate transition‐state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl‐ester, were also found to catalyze the hydrolysis of the analogous p‐nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np‐ester is higher in terms of rates (kcat as expected from the higher intrinsic reactivity of Np‐esters); however, the rate acceleration (kcat/kuncat) is close to or lower than that observed with the Nbzl‐ester. Unexpectedly, the affinity to the Np‐ester substrate (1/kM) and therefore kcat/KM are significantly higher. The best example is antibody D2.4 having a kcat/KM value of 64 s−1. M−1 with the Nbzl‐ester and 9400 s−1. M−1 with the Np‐ester. Moreover, due to a lower product inhibition by p‐nitrophenol relative to p‐nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np‐ester. The differential affinity of these antibodies to the Nbzl‐phosphonate TSA versus the Nbzl‐ester substrate (KS/KTSA or KM/Ki) correlates well with the observed rate enhancement (kcat/kuncat). For the Np‐ester, however, stabilisation of the transition state (as reflected by Ks/kTSA and by the catalytic proficiencies, kcat/KM/kuncat) does not fully account for the catalytic power (kcat/kuncat), indicating a more complex catalytic mechanism than simply transition‐state stabilization.

A comparison of the kinetic parameters of D2.4 with other Np‐ester‐hydrolyzing antibodies raised against Np‐phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl‐phosphonate hapten. These results appear to be general: anti‐(Nbzl‐phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np‐esterases. They demonstrate the use of an expanded TSA‐hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.
Calponin Inhibits Actin‐Activated MgATPase of Myosin Subfragment 1 (S1) Without Displacing S1 from Its Binding Site on Actin
Authors: Kołakowski Janusz , Karkucińska Agnieszka , Dąbrowska Renata
Published: July 2004

Calponin is a smooth‐muscle thin‐filament protein implicated in the regulation of contraction. Its binding to actin is a prerequisite for inhibition of actin‐activated myosin MgATPase. Investigating the molecular mechanism of this inhibition, it was found that titration of acto‐myosin subfragment 1 with calponin in the presence of either ADP or ATP does not displace weakly or strongly bound myosin subfragment 1 (S1) from actin. S1. ADP, however, is able to release about two‐thirds of the calponin from saturated (equimolar) complexes of actin‐calponin. The remaining calponin is sufficient for almost full inhibition of acto‐Sl MgATPase activity. Bundling of actin filaments by calponin takes place at a higher ratio calponin/actin (above 1:3) and, therefore, is not responsible for inhibition of the ATPase. Bundle formation is inhibited by S1 ADP. These results suggest the existence of two calponin‐binding sites on actin; one, that is insensitive to S1, which is responsible for inhibition of the ATPase, the other, from which calponin is readily displaced by S1.
The two Faces of Pyrrolizidine Alkaloids: the Role of the Tertiary Amine and its N‐Oxide in Chemical Defense of Insects with Acquired Plant Alkaloids
Authors: Lindigkeit Rainer , Biller Andreas , Buch Markus , Schiebel Hans‐Martin , Boppré Michael , Hartmann Thomas
Published: July 2004

Larvae of Creatonotos transiens (Lepidoptera, Arctiidae) and Zonocerus variegatus (Orthoptera, Pyr‐gomorphidae) ingest 14C‐labeled senecionine and its N‐oxide with the same efficiency but sequester the two tracers exclusively as N‐oxide. Larvae of the non‐sequestering Spodoptera littoralis eliminate efficiently the ingested alkaloids. During feeding on the two alkaloidal forms transient levels of senecionine (but not of the N‐oxide) are built up in the haemolymph of S. littoralis larvae. Based on these results, senecionine [18O]N‐oxide was fed to C. transiens larvae and Z. variegatus adults. The senecionine N‐oxide recovered from the haemolymph of the two insects shows an almost complete loss of 18O label, indicating reduction of the orally fed N‐oxide in the guts, uptake of the tertiary alkaloid and its re‐N‐oxidation in the haemolymph. The enzyme responsible for N‐oxidation is a soluble mixed function monooxygenase. It was isolated from the haemolymph of the sequestering arctiid Tyria jacobaeue and purified to electrophoretic homogeneity. The enzyme is a flavoprotein with a native M, of 200000 and a subunit M, of 51 000. It shows a pH optimum at 7.0, has its maximal activity at a temperature of 40–45°C and an isoelectric point at pH 4.9. The reaction is strictly NADPH‐dependent (Km, 1.3 μM). From 20 pyrrolizidine alkaloids so far tested as substrates, the enyzme N‐oxidizes only alkaloids with structural elements which are essential for hepatotoxic and genotoxic pyrrolizidine alkaloids (i.e. 1,2‐double bond, esterification of the allylic hydroxyl group, presence of a second free or esterified hydroxyl group at carbon 7). A great variety of related alkaloids and xenobiotics were tested as substrate, none was accepted. The K, values of senecionine, monocrotaline and heliotrine, representing the three main types of pyrrolizidine alkaloids, are 1.3 μM, 12.5 μM and 290 μM, respectively. The novel enzyme was named senecionine N‐oxygenase (SNO). The enzyme was partially purified from two other arctiids. The three SNOs show the same general substrate specificity but differ in their affinities towards the main structural types of pyrrolizidine alkaloids. The enzymes from the two generalists (Creatonotos transiens and Arctia caja) display a broader substrate affinity than the enzyme from the specialist (Tyria jacobaeae). The two molecular forms of pyrrolizidine alkaloids, the lipophilic protoxic tertiary amine and its hydrophilic nontoxic N‐oxide are discussed in respect to their bioactivation and detoxification in mammals and their role as defensive chemicals in specialized insects. Pyrrolizidine‐alkaloid‐sequestering insects store the alkaloids as nontoxic N‐oxides which are reduced in the guts of any potential insectivore. The lipophilic tertiary alkaloid is absorbed passively and then bioactivated by cytochrome P 450 oxidase.
Characterisation of the Promoter which Regulates Expression of a Phosphoglucomutase‐Related Protein, a Component of the Dystrophidutrophin Cytoskeleton Predominantly Expressed in Smooth Muscle
Authors: Moiseeva Elena P. , Critchley David R.
Published: July 2004

We have recently characterised a 60‐kDa muscle‐specific phosphoglucomutase‐related protein (PGM‐RP) which is expressed predominantly in adult visceral and vascular smooth muscle. Here we show that the adult vascular smooth muscle cell line PAC1, which retains the capacity to synthesise metavinculin (a marker of the contractile phenotype) also expressed PGM‐RP. However, an embryonic smooth muscle cell line A10, which lacks metavinculin, expressed low levels of PGM‐RP. Levels of PGM‐RP increased in quiescent PAC1 and A10 cells, and were elevated in response to angiotensin II. PGM‐RP is therefore a good marker of the contractile/differentiated smooth muscle phenotype. We have sequenced 1.8 kb of the human PGM‐RP promoter and shown that it lacks a conventional TATA box. There are multiple transcription start sites, the most predominant of which are inside an initiator sequence (Inr), which is close to two CT boxes and a GATA element. A minimal promoter‐CAT construct (p57‐CAT) containing the Inr, a CT box and GATA element directed high‐level chloramphenicol acetyltransferase (CAT) expression in the differentiated smooth muscle cell line PACI, and low‐level expression in the embryonic smooth muscle cell line A10. This fits well with the pattern of expression of the endogenous gene. A construct (p146‐CAT) containing all of the mRNA initiation sites directed a reduced level of CAT expression, and constructs containing 1.8 kb and 3.3 kb upstream of the major transcription start site displayed even lower activity. Sequence comparisons suggest that the PGM‐RP promoter evolved from the main phosphoglucomutase promoter which is active in wide range of cell types. The PGM‐RP promoter may have acquired negative regulatory elements as expression of the gene became muscle‐specific.
Alkene Monooxygenase from Nocardia Corallina B‐276 is a Member of the Class of Dinuclear Iron Proteins Capable of Stereospecific Epoxygenation Reactions
Authors: Gallagher Stephen C. , Cammack Richard , Dalton Howard
Published: July 2004

Nocardia coraltina B‐276 possesses a constitutive multi‐component alkene monooxygenase which catalyses the epoxidation of terminal and sub‐terminal alkenes. The epoxygenase component of this system has been purified with an overall yield of 35%. The electron paramagnetic resonance spectrum of the oxidised protein has a weak signal at g= 4.3, which we ascribe to rhombic iron and a free radical signal at gave= 2.01. Upon partial reduction with dithionite using methyl viologen as a mediator, a signal at gave= 1.9 appeared. Upon further reduction with excess dithionite a signal at g= 15 appeared with the concomitant disappearance of the gave= 1.9 signal. These results indicate that the epoxygenase contains a bridged dinuclear iron centre similar to that found in a variety of proteins involved in oxygen transport and activation as well as desaturation of fatty acids. Analysis of the products of the reaction indicates that AM0 is capable of stereospecific epoxidation of alkenes producing the R‐enantiomer in high yield, a reaction catalysed by very few oxygenase enzymes. Whole cells gave lower enantiomeric excess values for the epoxide and a stereospecific epoxidase enzyme has been proposed to account for this difference. Although alkene monooxygenase was not inhibited by ethyne, a potent inhibitor of soluble methane monooxygenase with which alkene monooxygenase shares many common features, it was weakly inhibited by propyne with an apparent K, value of 340 μM. The mechanistic implications of these physico‐chemical features of the enzyme are discussed.
The O‐Specific Polysaccharide Chain of Campylobacter Fetus Serotype A Lipopolysaccharide is a Partially O‐Acetylated 1,3‐Linked α‐D‐Mannan
Authors: Senchenkova Sof'ya N. , Shashkov Alexander S. , Knirel Yuny A. , Mcgovern Joseph J. , Moran Anthony P.
Published: July 2004

A polysaccharide fraction liberated from Cumpylobacter fetus subsp. fetus serotype A lipopolysaccharide by mild acid hydrolysis followed by gel‐permeation chromatography contained a partially O‐acetylated D‐mannan chain, as an O‐specific polysaccharide, with a core oligosaccharide attached. The structure of the polysaccharide was studied by O‐deacetylation, methylation, and 1H‐ and 13C‐NMR spectros‐copy, including computer‐assisted analysis of the 13C‐NMR spectrum. A structure of→3)‐α‐D‐Manp2Ac‐(1→ was established as the structure of the 0‐specific polysaccharide, the degree of O‐acetylation of the mannose residues at position 2 being estimated as 80–907%. As judged by the ratio of mannose to core constituents, the D‐mannan chain consists on average of 10–12 monosaccharide units.
Regulatory Phosphorylation of Banana Fruit Phosphoenolpyruvate Carboxylase by a Copurifying Phosphoedpyruvate Carboxylase‐Kinase
Authors: Law R. David , Plaxton William C.
Published: July 2004

Phosphoenolpyruvate (P‐pyruvate) carboxylase from ripened banana fruit was purified to near homogeneity and a final specific activity of 20–23 U/mg protein; P‐pyruvate carboxylasekinase copurified with P‐pyruvate carboxylase throughout the purification. Gel filtration FPLC indicated that the two proteins form a tightly associated 425‐kDa complex. Both the 103‐kDa and 100‐kDa subunits of the P‐pyruvate carboxylase α2β2 hetetrotetramer were phosphorylated and subsequently dephosphorylated in vitro in a time‐dependent manner when the final preparation was incubated with 0.1 mM [γ‐32P]ATP followed by rabbit muscle protein phosphatase type 2A1. Phosphoamino acid and phosphopeptide map analyses indicated that in vitro phosphorylation of both subunits likely occurs at an identical Ser residue. Maximal stoichiometry of 32P incorporation was 0.2 and 0.4 mol/mol 103‐kDa and 100‐kDa subunit, respectively. The level of 32P incorporation was correlated with the enzyme's activation state when assayed under suboptimal assay conditions (pH 7.3, 75 μM P‐pyruvate, 0.2 mM L‐malate). The main kinetic effect of phosphorylation was to decrease the enzyme's Km(P‐pyruvate), as well as its sensitivity to inhibition by L‐malate and L‐glutamate. Banana P‐pyruvate carboxylase‐kinase: (a) also phosphorylated maize leaf P‐pyruvate carboxylase, histone 111‐S, and dephosphorylated casein; (b) demonstrated Mg2+ dependence and Ca2+ independence, (c) exhibited a broad pH activity optimum of pH 8.0–8.5, and (d) was inhibited by L‐malate and activated by Ba2+ and Co2+. Time‐course kinetic studies suggested that P‐pyruvate carboxylase exists mainly in the dephosphorylated form in preclimacteric, climacteric and postclimacteric fruit, but that its kinase is expressed throughout ripening. In situ32P‐labeling indicated that, while both subunits of ripe banana P‐pyruvate carboxylase are phosphorylated in vivo, it is primarily the 100‐kDa subunit that is radiolabeled. The results suggest that similar to the enzyme from leaves, root nodules and seeds, a fruit P‐pyruvate carboxylase may be subject to regulatory seryl phosphorylation by an endogenous P‐pyruvate carboxylase‐kinase.
Organ‐Specific Activity of the 5′ Regulatory Region of the Glutamine Synthetase Gene in Developing Mice
Authors: Lie‐Venema Heleen , De Boer Piet A. J. , Moorman Antoon F. M. , Lamers Wouter H.
Published: July 2004

Glutamine synthetase (GS) converts ammonia and glutamate into glutamine. We assessed the activity of the 5′ regulatory region of the GS ene in developing transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of 3150 bp of the upstream sequence of the rat GS gene to obtain insight into the spatiotemporal regulation of its pattern of expression. To determine the organ‐specific activity of the 5′ regulatory region CAT and GS mRNA expression were compared by ribo‐nuclease‐protection and semi‐quantitative in situ hybridization analyses. Three patterns were observed: the 5′ region is active and involved in the regulation of GS expression throughout development (peri‐central hepatocytes, intestines and epididymis); the 5′ region shows no activity at any of the ages investigated (periportal hepatocytes and white adipose tissue); and the activity of the 5′ region becomes repressed during development (stomach, muscle, brown adipose tissue, kidney, lung and testis). In the second group, an additional element must be responsible for the activation of GS expression. The last group included organs in which the 5′ regulatory region is active, but not in the cells that express GS. In these organs, the activity of the 5′ regulatory region must be repressed by other regulatory regions of the GS gene that are missing from the transgenic onstruct. These findings indicate that in addition to the 5′ regulatory region, at least two unidentified elements are involved in the spatioteniporal pattern of expression of GS.
Elderberry (Sambucus Nigra) Bark Contains two Structurally Different Neusac(α2,6)Gal/Galnac‐Binding Type 2 Ribosome‐Inactivating Proteins
Authors: VAN Damme Els J. M. , Roy Soma , Barre Annick , Citores Lucia , Mostafapous Kazem , Rougé Pierre , VAN Leuven Fred , Girbés Tomas , Goldstein Irwin J. , Peumans Willy J.
Published: July 2004

A second NeuAc(α2,6)Gal/GalNAc binding type 2 ribosome‐inactivating protein (RIP), called SNAI' has been isolated from elderberry (Sumbucus nigru) bark. SNAI' is a minor bark protein which closely resembles the previously described major Neu5Ac(α2,6)Gal/GalNAc binding type 2 RIP called SNAI with respect to its carbohydrate‐binding specificity and ribosome‐inactivating activity but has a different molecular structure. Molecular cloning revealed that the deduced amino acid sequence of SNAI' is highly similar to that of SNAI and that the difference in molecular structure between both proteins relies on a single cysteine residue present in the B chain of SNAI but absent from SNAI'. The isolation of SNAI' not only identifies a minor bark protein as a type 2 RIP but also further emphasizes the complexity of the type 2 RIP/lectin mixture present in the bark of elderberry.
Specific Modulation of the Fusogenic Properties of the Alzheimer β‐Amyloid Peptide by Apolipoprotein E Isoforms
Authors: Pillot Thierry , Goethals Marc , Vanloo Berlinda , Lins Laurence , Brasseur Robert , Vandekerckhove Joel , Rosseneu Maryvonne
Published: July 2004

C‐terminal fragments of the Alzheimer amyloid peptide (amino acids 29–40 and 29–42) have physico‐chemical properties related to those of the fusion peptides of viral proteins and they are able to induce the fusion of liposomes in vitro. We proposed that these properties could mediate a direct interaction of the amyloid peptide with cell membranes and account for part of the cytotoxicity of the amyloid peptide. In view of the epidemiologic and biochemical linkages between the pathology of Alzheimer's disease and apolipoprotein E (apoE) polymorphism, we examined the potential interaction between the three common apoE isoforms and the C‐terminal fragments of the amyloid peptide. We show that, at low concentration, only apoE2 and apoE3 are potent inhibitors of the amyloid peptide fusogenic and aggregational properties, whereas the apoE4 isoform has no effect. We further show that the protective effect of apoE is mediated by the formation of stable apoE/amyloid peptide complexes, as determined by trypto‐phan emission fluorescence measurements and by gel electrophoresis. The interaction specificity between apoE2 and apoE3 and the amyloid fragments is demonstrated here, since other apolipoproteins (e.g. apolipoprotein A‐I and A‐II), with similar amphipathic structures, do not interact with the amyloid C‐terminal fragments. Finally, we show that, reciprocally, the amyloid peptide can interact directly with the apoE2 and apoE3 isoforms to decrease or perturb their normal association with lipids. These data suggest that the 29‐40 and 29–42 domains of the amyloid peptide could be critical for the amyloid‐apoE interaction, and that apoE2 and apoE3 isoforms, but not apoE4, could play a protective role against the formation of amyloid aggregates and/or against their interaction with cellular membranes.