bluuebunny/crossref_metadata_2025 huggingface dataset:
The Energy Conserving N5‐Methyltetrahydromethanopterin:Coenzyme M Methyltransferase Complex from Methanobacterium thermoautotrophicum is Composed of Eight Different Subunits
Authors: Harms Ulrike , Weiss David S. , Gärtner Peter , Linder Dietmar , Thauer Rudolf K.
Published: March 2005

N
5‐Methyltetrahydromethanopterinxoenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum strain Marburg is a membrane‐associated enzyme complex which catalyzes an energy‐conserving, sodium‐ion‐translocating step in methanogenesis from H2 and CO2. We report here that the complex is composed of eight different subunits for which evidence was obtained at the protein, DNA and RNA levels: (a) SDS/PAGE of the purified complex revealed the presence of eight different polypeptides of apparent molecular masses of 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 (MtrD), 13 (MtrG), 12.5 (MtrB) and 12 kDa (MtrF). The N‐terminal amino acid sequences of the 12‐, 12.5‐ and 13‐kDa polypeptides, which had previously not been accessible, were determined; (b) cloning and sequencing of the corresponding genes revealed the presence of the eight mtr genes organized in a 4.9‐kbp gene cluster in the order mtrEDCBAFGH; (c) Northern‐blot analysis revealed the presence of a 5‐kbp transcript. DNA probes derived from the mtrE and mtrH genes hybridized to the transcript, indicating that the eight mtr genes are organized in a transcription unit. By primer extension, the 5′ end of the mtrEDCBAFGH mRNA was analyzed. The mtr operon was found to be located between the methyl‐coenzyme M reductase I operon (mcr) and a downstream open reading frame predicted to encode a Na+/Ca2+, K+ exchanger.
Insertion of Glycophorin A, A Transmembraneous Protein, in Lipid Bilayers can be Mediated by Electropermeabilization
Authors: Raffy Sophie , Teissie Justin
Published: March 2005

Transmembraneous back‐insertion of a solubilized membrane protein, glycophorin A, has been obtained in 1,2 dipalmitoyl‐sn ‐glycero‐3‐phosphocholine (Pam2GroP Cho) cell‐size‐like liposomes by submitting the lipid/protein mixture to calibrated electric field pulses. Field conditions which are prone to trigger glycophorin insertion are similar to those which mediate lipid layer electropermeabilization. The efflux of calcein, trapped in the liposomes during their preparation, was observed only when field strength is higher than 1.3 kV/cm. Electroinsertion was detected only above the same critical field intensity. Calcein efflux as well as glycophorin insertion were increased by increasing field intensity, pulse duration and/or number of pulses. Experimental evidence of protein insertion was provided by physico‐chemical as well as biochemical methods. Direct observation of the pulsed vesicles under the microscope revealed the insertion by means of immunofluorescence and fluorescence. Electroinsertion of fluorescent glycophorin A revealed that the inner bilayers were also labeled. The gel‐to‐liquid phase transition temperature of Pam2GroP Cho decreased after insertion but its cooperativity was not affected. A narrow 31P‐NMR peak was observed after electroinsertion showing that the polar headgroups of phospholipids had been altered. Analysis of trypsin‐digested peptides revealed that the two trans ‐orientations of the protein across the external lipid layer were present after electroinsertion. Localized perturbation of the polar headgroup region of phospholipids, which supports the transient permeabilization of lipid layers, allows spontaneous transinsertion of glycophorin across the lipid bilayers.
Limitations to in vivo Import of Hydrophobic Proteins into Yeast Mitochondria
Authors: Claros Manuel G. , Perea Javier , Shu Youmin , Samatey Fadel A. , Popot Jean‐Luc , Jacq Claude
Published: March 2005

The apocytochrome b gene, exclusively encoded by the mitochondrial genome, was engineered so that it could be expressed in the yeast cytoplasm. Different combinations of the apocytochrome b trans‐membrane domains were produced in the form of hybrid proteins fused to both the N‐terminal mito‐chondrial targeting sequence of the ATPase subunit 9 from Neurospora crassa and to a cytoplasmic version of the bI4 RNA maturase, localised on the N‐terminal and C‐terminal sides, respectively, of the hydrophobic stretches. The bI4 RNA maturase, which can complement mitochondrial mutations, was used as an in vivo reporter to assess the mitochondrial import of the different groups of transmembrane helices. This new, reliable and sensitive reporter activity allowed us to experimentally determine the limitations to the mitochondrial import of hydrophobic proteins. All eight transmembrane helices of apocytochrome b could be imported into mitochondria, either alone or in combination, but no more than three to four transmembrane helices could be imported together at one time. This limit is close to that observed in the population of nuclear‐encoded mitochondrial proteins. The hydrophobic characteristics of engineered and natural proteins targeted to the mitochondrial inner membrane revealed two factors important in the import process. These were (a) the local hydrophobicity of a transmembrane segment, and (b) the average regional hydrophobicity of the protein over an extended length of 60–80 residues. Such features may have played a major role in the evolution of mitochondrial genomes.
Functional Identification of Three Major Phosphoproteins in Endocytic Fractions from Rat Liver
Authors: Enrich Carlos , Vergés Marcel , Evans W. Howard
Published: March 2005

Liver plasma membranes originating from the sinusoidal, lateral and canalicular domains and ‘early’ and ‘late’ endosomes were prepared from rats injected with [32P]orthophosphate. The phosphorylated polypeptides in these subcellular fractions, resolved by gel electrophoresis, were analysed and compared with those obtained by in vitro phosphorylation of the fractions by endogenous protein kinases. The polypeptides phosphorylated in vitro were different in plasma membranes, endosomes and lysosomes. Three of the major phosphoproteins in the endocytic membranes were shown to be the polymeric immunoglobulin receptor, the β subunit of the insulin receptor and the 550‐kDa low‐density‐lipoprotein‐receptor‐related protein (LRP). An additional 35‐kDa polypeptide of unknown function was a major phosphorylated component and thus emerges as a candidate marker protein of hepatic endosomes. Phosphoserine was shown to be the major amino acid phosphorylated in vitro in the phosphoproteins of endocytic membranes. The subcellular distribution in liver tissue of protein kinase activity was also investigated and activity shown to be recovered mainly in blood‐sinusoidal and lateral plasma membranes; bile canalicular plasma membranes and endosomes contained low protein kinase activities. The results show that receptor phosphorylation is an ‘early’ event in endocytosis and the trafficking of ligands that is sustained especially in early endosomes in liver, and emphasizes the biochemical and thus functional distinctiveness of the plasma membrane and the endosomal and lysosomal compartments with regard to their population of phosphorylated proteins.
Isolation and Structural Characterization of Trimeric Cyanobacterial Photosystem I Complex with the Help of Recombinant Antibody Fragments
Authors: Tsiotis Georgios , Haase Winfried , Engel Andreas , Michel Hartmut
Published: March 2005

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb.

A one‐step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N‐terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139±9 chlorophyll a/primary electron donor. Flash‐induced absorption‐change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe‐S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high‐resolution electron microscopy and digital image processing.
Isolation, Characterization and Expression of Cationic Peroxidase Isozymes Released into the Medium of Cultured Tobacco Cells
Authors: Narita Hiroshi , Asaka Yukiko , Ikura Koji , Matsumoto Shinya , Sasaki Ryuzo
Published: March 2005

Three glycoproteins of 34, 38 and 40 kDa were isolated from the spent medium of suspension‐cultured tobacco cells. The 38‐kDa and 40‐kDa proteins were highly cationic peroxidases with indistinguishable enzymic properties but their structural difference was confirmed by sequence analysis of the amino‐terminal regions and the recognition specificity of monoclonal antibodies. The 34‐kDa protein was a moderately cationic peroxidase with enzymic properties quite different from those of the 38‐kDa and 40‐kDa enzymes. They were undetectable in the spent medium during the cell‐proliferation phase but became abundant in the medium during the cell‐expansion phase. This was confirmed quantitatively with the 40‐kDa protein using the 40‐kDa‐specific monoclonal antibody. The mRNA expression for 40‐kDa protein was at a constant basal level in the cell‐proliferation phase but increased in the cell‐expansion phase.
The (2′‐5′)Oligoadenylate Synthetase is Present in the Lowest Multicellular Organisms, the Marine Sponges
Authors: Kuusksalu Anne , Pihlak Arno , Müller Werner E. G. , Kelve Merike
Published: March 2005

We have proved the presence of (2′‐5′)oligoadenylates [(2′‐5′)An] and oligoadenylate synthetase [(2′‐5′)An synthetase] in the marine sponge Geodia cydonium. (2′‐5′)An isolated from sponge crude extract competed with authentic (2′‐5′)An for binding to polyclonal antiserum against (2′‐5′)An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2′‐5′)A oligomers. The biological activity of sponge (2′‐5′)An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2′‐5′)An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon‐treated cell extract. The (2′‐5′)An synthetase from sponge extract binds to poly(I) · poly(C) as does the mammalian enzyme. Western blot analysis with antibodies to recombinant rat 43‐kDa (2′‐5′)An synthetase revealed in sponge immunologically related proteins with molecular masses of approximately 110, 65, 61 and 34 kDa. We conclude, that the (2′‐5′)An system has evolved from receptors and enzymes involved in cell adhesion and/or growth control.
Localization of the Palmitoylation Site in the Transmembrane Protein p12E of Friend Murine Leukaemia Virus
Authors: Hensel Jutta , Hintz Martin , Karas Michael , Linder Dietmar , Stahl Bernd , Geyer Rudolf
Published: March 2005

Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10‐3H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size‐exclusion chromatography and purified by narrowbore reverse‐phase HPLC. Analysis of the purified product by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI‐TOF‐MS or fractionated by microbore reverse‐phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C‐terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.
Structure of the Capsular Polysaccharide of Vibrio cholerae O139 Synonym Bengal Containing D‐galactose 4,6‐Cyclophosphate
Authors: Knirel Yuriy A. , Paredes Liliana , Jansson Per‐Erik , Weintraub Andrej , Widmalm Göran , Albert M. John
Published: March 2005

The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O‐specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel‐permeation chromatography. The CPS contained d‐galactose, 3,6‐dideoxy‐l‐xylo‐hexose (colitose, Col), 2‐acetamido‐2‐deoxy‐d‐glucose, 2‐acetamido‐2,6‐dideoxy‐d‐glucose (N‐acetyl‐d‐quinovosamine, d‐QuiNAc),d‐galacturonic acid (d‐GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter aliad‐galactose 4,6‐cyclophosphate and having the following structure

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These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835–838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate.
A Nuclear Protein, Synthesized in Growth‐Arrested Human Hepatoblastoma Cells, is a Novel Member of the Short‐Chain Alcohol Dehydrogenase Family
Authors: Gabrielli Franco , Donadel Giulia , Bensi Giuliano , Heguy Adriana , Melli Marialuisa
Published: March 2005

We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723–729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short‐chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear‐targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post‐transcriptional level. The possible nuclear role of the Hep27 protein is discussed.
Structures of the O‐specific Polysaccharide Chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis Lipopolysaccharides
Authors: Senchenkova Sof'ya N. , Shashkov Alexander S. , Moran Antony P. , Helander Ilkka M. , Knirel Yuriy A.
Published: March 2005

Mild acid hydrolysis of the smooth‐type lipopolysaccharide (LPS) of Pectinatus frisingensis afforded no polysaccharide but monomeric 6‐deoxy‐l‐altrose (l‐6dAlt) which was identified by anion‐exchange chromatography in borate buffer, GLC/MS, 1H‐NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely O‐deacylated polysaccharide, which was studied by methylation analysis, 1H‐NMR and 13C‐NMR spectroscopy, including sequential, selective spin‐decoupling, two‐dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two‐dimensional heteronuclear 13C,1H‐COSY., one‐dimensional NOE and two‐dimensional rotating‐frame NOE spectroscopy. It was found that the O‐specific polysaccharide chain of P. frisingensis LPS is a homopolymer of 6‐deoxy‐l‐altrofuranose built up of tetrasaccharide‐repeating units having the following structure:
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Similarly, mild acid degradation of smooth‐type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of d‐glucose and d‐fucose. Study of the disaccharide by methylation analysis and alkali‐degraded LPS by one‐dimensional and two‐dimensional 1H‐NMR and 13C‐NMR spectroscopy showed that the O‐specific polysaccharide of P. cerevisiiphilus has the following structure:
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A Plant Acyltransferase Involved in Triacylglycerol Biosynthesis Complements an Escherichia Coli sn‐1‐acylglycerol‐3‐phosphate Acyltransferase Mutant
Authors: Hanke Christiane , Wolter Frank P. , Coleman Jack , Peterek Gabriele , Frentzen Margrit
Published: March 2005

The second acylation reaction in glycerolipid biosynthesis is catalyzed by an sn‐1‐acylglycerol‐3‐phosphate acyltransferase. The enzyme of Limnanthes douglusii involved in triacylglycerol synthesis has an unusual specificity for very long chain acyl groups in both of its substrates, namely acyl‐CoA and sn‐1‐acylglycerol‐3‐phosphate, and causes the enrichment of erucoyl groups in the sn‐2 position of the seed oil of this plant species. We have isolated a cDNA clone encoding this embryo‐specific, microsomal acyltransferase via heterologous complementation of an Escherichia coli mutant deficient in sn ‐1‐acyl‐glycerol‐3‐phosphate acyltransferase activity. The open reading frame of the cDNA insert encodes a protein with a length of 281 amino acids, with three predicted membrane‐spanning domains and of about 31.7 kDa. The sequence exhibits substantial sequence similaritiy to the sn‐1‐acylglycerol‐3‐phosphate acyltransferase of E. coli. The corresponding transcript was detectable in developing embryos but not in leaves of L. douglasii, and expression of the open reading frame in E. coli caused sn‐1‐acylglycerol‐3‐phosphate acyltransferase activity which showed properties different from those of the bacterial acyltransferase but typical of the L. douglusii enzyme involved in triacylglycerol biosynthesis.
Import and Insertion of Proteins into the Mitochondrial Outer Membrane
Authors: Shore Gordon C. , McBride Heidi M. , Millar Douglas G. , Steenaart Nancy A. E. , Nguyen Mai
Published: March 2005

Nuclear‐encoded proteins destined for insertion into the mitochondrial outer membrane, follow the same general pathway for import as proteins that are translocated to interior compartments within the organelle. This observation is true both for β‐barrel‐type proteins and for proteins that contain hydrophobic α‐helical transmembrane segments. In this review, we describe what is known about the various steps leading to protein insertion into the outer membrane, and discuss the energetics that favor vectorial translocation into and across this membrane. The selection of the outer membrane during import may involve a lateral release of the translocating polypeptide from the import machinery so that the appropriate domains of the protein become embedded in the lipid bilayer. One type of topogenic domain that can guarantee such selection of the outer membrane is a signal‐anchor sequence of the type characterized for the bitopic protein Mas70p. It is suggested that a signal‐anchor sequence selective for the mitochondrial outer membrane causes abrogation of polypeptide translocation and triggers the release of the transmembrane segment into the surrounding lipid bilayer, prior to any possibility for the commitment of translocation to the interior of the organelle. Specific structural features of the signal‐anchor sequence specify its orientation in the membrane, and can confer on this sequence the ability to form homo‐oligomers and hetero‐oligomers. Strategies other than a signal‐anchor sequence may be employed by other classes of proteins for selection of the outer‐membrane. Of note is the ability of the outer‐membrane import machinery to catalyze integration of the correct set of proteins into the outer‐membrane bilayer, while allowing proteins that are destined for integration into the bilayer of the inner membrane to pass through unimpeded. Again, however, different proteins may employ different strategies. One model proposes that this can be accomplished by a combination of a matrix‐targeting signal and a distal stop‐transfer sequence. In this model, the formation of contact sites, which is triggered when the matrix‐targeting signal engages the import machinery of the inner membrane, may prevent the outer‐membrane translocon from recognizing and responding to the downstream stop‐transfer domain. This allows the transmembrane segment to pass across the outer‐membrane, and subsequently integrate into the inner membrane.
Molecular Structure and Mechanism of Action of the Crotoxin Inhibitor from Crotalus durissus terrificus Serum
Authors: Perales Jonas , Villela Christina , Domont Gilberto B. , Choumet Valérie , Saliou Bernard , Moussatché Haity , Bon Cassian , Faure Grazyna
Published: March 2005

An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action.

CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G‐200, ion‐exchange chromatography on DEAE‐Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non‐covalent association of 23–25‐kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N‐terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues.

The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex.

The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.
Probing Protein Structure by Solvent Perturbation of NMR Spectra
Authors: Improta Sabina , Molinari Henriette , Pastore Annalisa , Consonni Roberto , Zetta Lucia
Published: March 2005

We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et al., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine α‐lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces.
Activin A and Retinoic Acid Synergize in Cyclooxygenase‐1 and Thromboxane Synthase Induction During Differentiation of J774.1 Macrophages
Authors: Nüsing Rolf Michael , Mohr Susanne , Ullrich Volker
Published: March 2005

The murine macrophage cell line J774.1 was used to study the development of prostanoid biosynthesis under the influence of activin A and retinoic acid. Treatment of cells with 3 nM activin A for 48 h increased the biosynthesis of the prostaglandins E2, D2, F2α and thromboxane A2 more than fourfold due to an induction of cyclooxygenase‐1 while cyclooxygenase‐2 was unaffected. Transforming growth factor‐β acted in a similar way. Retinoic acid, when present alone, was without effect on the total cyclooxygenase products and only slightly changed the pattern of prostanoids. However, when coincubated with activin A, retinoic acid specifically induced the synthesis of thromboxane‐A‐synthase‐specific mRNA and induced an increase in enzyme activity with a synergistic effect on cyclooxygenase‐1 protein and mRNA. JunB, but not c‐jun, mRNA expression was found under these conditions in addition to a transient c‐fos mRNA increase. The combination of activin A and retinoid acid may be regarded as a differentiation model to study the development of cell‐specific prostanoid patterns in macrophages and possibly other differentiating cells.
Identification and Partial Purification of (Ca2+ or Mg2+)‐ATPase in Renal Brush‐Border Membranes
Authors: Van Erum Monique , Lemmens Raf , Berden Jan , Teuchy Henri , Vanduffel Luc
Published: March 2005

The protein responsible for the (Ca2+ or Mg2+)‐ATPase activity in brush‐border membranes from pig kidney tubular cells was characterized to distinguish this enzyme from the N‐ethylmaleimide‐sensitive Mg2+‐ATPase, also present in renal brush borders. Both enzymes are clearly different in their pH optimum and their sensitivity to divalent cations, nucleoside 5′‐triphosphates and inhibitors.

Solubilization of the (Ca2+ or Mg2+)‐ATPase from brush‐border membrane vesicles was accomplished with Nonidet P‐40 or dodecylmaltoside. However, simultaneous inactivation of the enzyme was inevitable.

A tenfold enrichment of the ATPase activity was obtained by chromatofocusing of Nonidet‐P‐40‐solubilized brush borders. A similar degree of purification was achieved by ion‐exchange chromatography of dodecylmaltoside‐solubilized preparations. From the SDS/polyacrylamide gels of partially purified (Ca2+ or Mg2+)‐ATPase, a few protein bands could still be tentatively identified as responsible for the enzyme activity.

Labeling of solubilized brush‐border preparations with several radioactive ATP analogues also revealed that a protein band of molecular mass 90 kDa is the most probable candidate for the catalytic peptide of the (Ca2+ or Mg2+)‐ATPase.

Finally, immunoprecipitation as well as semi‐dry blotting with antibodies generated against partially purified enzyme preparations, confirmed that a 90‐kDa component is a reasonable candidate for the (Ca2+ or Mg2+)‐ATPase in renal brush‐border membranes.
Benzoyl‐Coenzyme‐A 3‐Monooxygenase, a Flavin‐Dependent Hydroxylase
Authors: Niemetz Ruth , Altenschmidt Uwe , Brucker Susanne , Fuchs Georg
Published: March 2005

A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl‐CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic benzoate oxidation pathway via benzoyl‐coenzyme A and 3‐hydroxybenzoyl‐coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851–4858)]. The initial reaction is catalyzed by benzoate‐CoA ligase (AMP‐forming), converting benzoate into benzoyl‐CoA. The next step is 3‐hydroxylation of benzoyl‐CoA to 3‐hydroxybenzoyl‐CoA catalyzed by a flavin‐nucleotide‐dependent monooxygenase, benzoyl‐CoA 3‐monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl‐CoA, but not benzoate, is hydroxylated. The protein is a monomer of Mr 65 000 and is induced when cells are grown aerobically with benzoate. 3‐Hydroxybenzoyl‐CoA is further hydroxylated para to the hydroxyl group affording 2,5‐dihydroxybenzoate (gentisate). This reaction requires another monooxygenase, 3‐hydroxybenzoyl‐CoA 6‐monooxygenase, which is unspecific with respect to the pyridine nucleotide. Cells contain a second 6‐monooxygenase activity which acts on free 3‐hydroxybenzoate. Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced. The proposed intermediates are benzoyl‐CoA, 3‐hydroxybenzoyl‐CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate.
Cloning and Expression in Insect Cells of two Pancreatic Lipases and a Procolipase from Myocastor coypus
Authors: Thirstrup Kenneth , Carrière Frédéric , Hjorth Siv A. , Rasmussen Poul B. , Nielsen Per F. , Ladefoged Claus , Thim Lars , Boel Esper
Published: March 2005

The physiological role of pancreatic lipases has traditionally been assigned solely to triacylglyceride metabolism, while the digestion of phospholipids requires the presence of the pancreatic phospholipase A2, a 14‐kDa enzyme unrelated to pancreatic lipases. However, in the guinea pig, it was observed that the pancreatic phospholipase A2 was absent and that a guinea pig pancreatic‐lipase‐related protein 2 (GPL‐RP2) was responsible for phospholipase activity, in contrast to the situation observed in other mammalian species. As the guinea pig is a member of the hystricomorph rodents, it was of interest to investigate if other species within this evolutionary suborder display similar characteristics. The coypu (Myocastor coypus) also a member of the hystricomorph rodents, was chosen for further investigations. The cDNAs encoding two pancreatic lipases and a procolipase from the coypu were cloned, expressed and characterized. One lipase, CoPL‐RP2, was identified as belonging to the RP2 subfamily, while the second, CoPL, was found to belong to the classical pancreatic lipase subfamily. Enzymic characterization and sequence data suggest a role for coypu colipase as a specific cofactor for CoPL, while this coypu colipase cannot be an important cofactor for CoPL‐RP2 in vivo. Also, the new lipase cDNA sequences were used in a phylogentic analysis to reinvestigate the taxonomical position of the hystricomorph rodents (e.g. cloypu and guinea pig) with respect to the myomorph rodents (e.g. rat and mouse)
Platelet‐derived Growth Factor Stimulates de‐Novo Synthesis of Mitogen‐activated Protein Kinase in Renal Mesangial Cells
Authors: Huwiler Andrea , Fabbro Doriano , Pfeilschifter Josef
Published: March 2005

Platelet‐derived growth factor (PDGF) BB is a potent mitogen for renal mesangial cells and stimulates a biphasic mitogen‐activated protein kinase (MAP kinase) activation. A rapid increase in activity (maximal at 10 min) is followed by a lower persistent level of activity which is maximal at 4–6 h. The second peak of MAP kinase activity is markedly attenuated by the protein synthesis inhibitor cycloheximide and, consequently, is paralleled by a marked de‐novo synthesis of p42 and p44 MAP kinases, as measured by immunoprecipitation of [35S]methionine‐labeled mesangial cells and by a 700 % increase in total MAP kinase protein, as detected by Western‐blot analysis. A 30‐min treatment with PDGF‐BB is sufficient to induce pronounced de‐novo synthesis of MAP kinase. However, for maximal induction of MAP kinase synthesis, PDGF is required to be present for at least 4 h. In addition, an increased de‐novo synthesis of MAP kinase kinase, the upstream activator of MAP kinase, is observed in response to PDGF stimulation. We propose that PDGF‐induced de‐novo synthesis of MAP kinase and MAP kinase kinase is important for the potent mitogenic activity of this growth factor.
Disulfide Bonds and Glycosylation in Fungal Peroxidases
Authors: Limongi Paola , Kjalke Marianne , Vind Jesper , Tams Jeppe W. , Johansson Tomas , Welinder Karen G.
Published: March 2005

Four conserved disulfide bonds and N‐linked and O‐linked glycans of extracellular fungal peroxidases have been identified from studies of a lignin and a manganese peroxidase from Trametes versicolor, and from Coprinus cinereus peroxidase (CIP) and recombinant C. cinereus peroxidase (rCIP) expressed in Aspergillus oryzae. The eight cysteine residues are linked 1–3, 2–7, 4–5 and 6–8, and are located differently from the four conserved disulfide bridges present in the homologous plant peroxidases. CIP and rCIP were identical in their glycosylation pattern, although the extent of glycan chain heterogeneity depended on the fermentation batch. CIP and rCIP have one N‐linked glycan composed only of GlcNAc and Man at residue Asn142, and two O‐linked glycans near the C‐terminus. The major glycoform consists of single Man residues at Thr331 and at Ser338. T. versicolor lignin isoperoxidase TvLP10 contains a single N‐linked glycan composed of (GlcNAc)2Man5 bound to Asn103, whereas (GlcNAc)2Man5 was found in T. versicolor manganese isoperoxidase TvMP2 at the same position. In addition, mass spectrometry of the C‐terminal peptide of TvMP2 indicated the presence of five Man residues in O‐linked glycans. No phosphate was found in these fungal peroxidases.
Inhibition of 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase and Citrate Synthase by Sulfoxides and Sulfones of Substrate‐analogue CoA‐thioether Derivatives
Authors: Karl Rosa Maria , Hupperich Martin , Thomer Anna , Eggerer Hermann
Published: March 2005

Sulfoxides and sulfones were prepared by specific oxidation of 3‐hydroxy‐3‐methylglutaryl‐CoA‐analogue CoA‐thioether derivatives and their kinetic properties were determined with 3‐hydroxy‐3‐methylglutaryl‐CoA reductase. The oxidized CoA‐thioether derivatives with a hydroxyl group at C3 were powerful competitive inhibitors, their Ki values being much smaller than the Km for 3‐hydroxy‐3‐methyl‐glutaryl‐CoA.

Sulfoxides and sulfones of substrate analogues of citrate synthase were also prepared. When tested in the appropriate reaction with citrate synthase, the sulfoxide and sulfone derivatives were competitive inhibitors, but their Ki values were greater than the Km values of the corresponding unmodified substrates.
Significant Amounts of Glycogen are Synthesized from 3‐Carbon Compounds in Astroglial Primary Cultures from Mice with Participation of the Mitochondrial Phosphoenolpyruvate Carboxykinase Isoenzyme
Authors: Schmoll Dieter , Führmann Elgin , Gebhardt Rolf , Hamprecht Bernd
Published: March 2005

The incorporation was studied of the gluconeogenic substrates lactate, alanine, aspartate and glutamate into glycogen of astroglial primary cultures derived from mouse brain. The incorporation was inhibited by 3‐mercaptopicolinate, an inhibitor of one of the characteristic gluconeogenic enzymes, phosphoenolpyruvate carboxykinase. Only the mitochondrial isoenzyme of phosphoenolpyruvate carboxykinase was detectable in the astroglial primary cultures. After the incubation of glucose‐starved cells with medium containing a mixture of [6‐3H]glucose and [U‐14C]glucose, the newly synthesized glycogen showed a 3H/14C ratio which was approximately 15% less than the isotope ratio for the medium. The decrease of the isotope ratio was not significantly inhibited by 3‐mercaptopicolinate, indicating a cycling of approximately 15% of the glucose to the level of the triose phosphates before its incorporation into astroglial glycogen. During the initial phase of glycogen resynthesis, the contribution of the gluconeogenic substrates appeared to be higher. This was in agreement with the accumulation of fructose 2,6‐bisphosphate during refeeding. A participation of gluconeogenic substrates in glycogen metabolism was also detectable when the glycogen content was not changing significantly.
Stereoselectivity of Microbial Lipases
Authors: Stadler Peter , Kovac Andrea , Haalck Lutz , Spener Fritz , Paltauf Fritz
Published: March 2005

In the present study, the stereoselectivity of purified lipases from Candida rugosa, Chromobacterium viscosum, Pseudomonas species and Rhizopus arrhizus towards triacylglycerols in comparison to various structural analogs were investigated. Different triacylglycerol analogs with distinct polarities at position sn‐2 of the glycerol backbone (1,3‐diacy1‐2‐X‐glycerol, where 2‐X = 2‐acyloxy, 2‐alkyloxy, 2‐deoxy‐2‐alkyl, or 2‐deoxy‐2‐phenyl) were synthesized. Substrate hydrophobicity and steric requirement was modified by variation of the alkyl and acyl chain length. Hydrolysis of these substrates demonstrated that minor structural variations at C2 of triacylglycerol strongly affect the stereoselectivity of the lipases tested. It was noteworthy that the variation of substrate structure did not only affect the quantity of stereoselectivity expressed as percentage enantiomeric excess, but also resulted in a reversal of stereo‐preference in some cases. Replacement of the acylester in position 2 of glycerol by a non‐ester‐linked aliphatic moiety shifted the preference of Chromobacterium viscosum lipase from sn‐3 to sn‐1. Lipases from Chromobacterium viscosum, Pseudomonas species and Rhizopus arrhizus exhibited sn‐3 preference with 2‐deoxy‐2‐phenyl analogs, while towards substrates with a 2‐deoxy‐2‐alkyl moiety sn‐1 stereobias was recorded. Candida rugosa lipase was rather insensitive to substrate variations concerning the polarity at position 2 of the glycerol backbone. However, variation of the acyl chain length significantly influenced stereoselectivity of this lipase.
Preferred DNA‐binding‐Sites of Polyomavirus Large T‐antigen
Authors: Bondeson Kåre , Rönn Ola , Magnusson Göran
Published: March 2005

Polyomavirus large T‐antigen is a multifunctional protein. Its essential function in virus infection is to control the synthesis of viral RNA and DNA. For this activity specific DNA binding is necessary. Large T‐antigen can bind to several sites in the regulatory region of viral DNA, consisting of clustered GRGGC nucleotide motifs. Since large T‐antigen also regulates the activity of cellular genes and cellular DNA synthesis, it seemed possible that there are alternative, as yet unrecognized, binding sites. To identify sites preferred by large T‐antigen, double‐stranded polynucleotides with random sequence were used. These polymers had a 31‐bp central segment synthesized from a mixture of all four nucleotides and flanking segments of defined sequence. They were subjected to several cycles of binding to large T‐antigen with intervening PCR amplification. Individual polynucleotides with affinity for large T‐antigen were then isolated by cloning and their nucleotide sequences were determined. The majority of the polynucleotides contained two or three GRGGC motifs separated by between five and eight variable nucleotides. This result suggests that there are not any alternative high‐affinity binding sites of large T‐antigen. By comparing all the individual binding motifs an extended consensus sequence was observed. The dinucleotide TG was predominant immediately 5′ to the binding pentanucleotide. On the 3′‐side, at position +2, C residues were very rare. Although the pentanucleotide motif is the same as in polyomavirus DNA, the extended consensus sequence is not observed in viral DNA. In semi‐quantitative experiments, binding of purified large T‐antigen to a few of the selected DNA molecules was tested. Stable complexes were formed with DNA substrates containing two or three binding motifs in tandem. Binding to DNA with only one complete motif was weaker, but significantly stronger than non‐specific association. This result has implications for the number of large T‐antigen binding sites in cellular DNA. When mutant bc1081 large T‐antigen, that is defective in specific DNA binding, was used in selection of polynucleotides, a different result was obtained. Neither bc1081 nor wild‐type large T‐antigen bound strongly to these polynucleotides. However, the presence of the motif TTGCCGTT, or part of it, in five of the six isolated polynucleotides suggested that the T‐antigen selection was specific.
Stathmin is a Major Substrate for Mitogen‐activated Protein Kinase During Heat Shock and Chemical Stress in HeLa Cells
Authors: Beretta Laura , Dubois Marie‐Fraçoise , Sobel André , Bensaude Olivier
Published: March 2005

Stathmin is a ubiquitous, highly conserved 19‐kDa cytoplasmic protein whose expression and phosphorylation are regulated in relation to cell proliferation, differentiation or activation, in many biological systems. In this report, we show that stathmin undergoes major phosphorylation in HeLa cells submitted to heat or chemical stress. Heat‐shock‐induced stathmin phosphorylation was very rapid, as maximal incorporation of phosphate was observed at 5 min. Phosphorylation of stathmin might, therefore, occur as a very early step in the intracellular response to heat shock. The sites of phosphorylation of stathmin involved during the stress response were identified as mostly Ser25 and, to a lesser extent, Ser38. These sites are both followed by a proline residue, and known to be good substrates in vitro for mitogen‐activated protein kinase (MAP‐kinase) and p34cdc2 kinase, respectively. In lysates from heat‐shocked cells, an increased stathmin‐kinase activity, distinct from the histone‐H1‐kinase activity, was found to phosphorylate stathmin mostly on Ser25, the main site for MAP‐kinase in vitro. This stathmin‐kinase coeluted quantitatively with the stress‐activated MAP‐kinase from an FPLC MonoQ column. Furthermore, a stathmin kinase activity was precipitated from lysates of heat‐shocked HeLa cells by an anti‐(MAP‐kinase) serum. Together, these results indicate that the phosphorylation of stathmin by MAP‐kinase is likely to be a significant component of the signalling array controlling the cellular response to stress, and they further underline the general involvement of stathmin in intracellular signalling.
Functional Consequences of Proline Mutations in the Putative Transmembrane Segments 6 and 10 of the Glucose Transporter GLUT1
Authors: Wellner Maren , Monden Ingrid , Mueckler Mike M. , Keller Konrad
Published: March 2005

Proline residues are thought to play a characteristic structural and/or dynamic role in various membrane proteins [Williams, K. A. & Deber, C. M. (1991) Biochemistry 30, 8919–8923]. By use of site‐directed mutagenesis and functional expression of mutant glucose transporters in Xenopus oocytes, we investigated the effects of single proline substitutions in the putative helices 6 and 10 on GLUT1‐mediated glucose transport. Proline residues of helix 6, that are conserved in all human glucose‐transporter isoforms except for the human GLUT2, were mutated either to alanine or to the corresponding residues of GLUT2, i.e. to histidine (P187H), arginine (P196R) or phenylalanine (P205F). In addition, the three proline amino acids within the domain G382‐P‐G‐P‐I‐P of helix 10 were individually replaced with either alanine or glutamine residues. In all cases, transport function was retained when each individual proline residue was replaced with alanine. Substitution of proline 196 by arginine (P196R), however, resulted in reduction of 2‐deoxy‐d‐glucose uptake rates by approximately 70 %. Since the amount of this mutant transporter protein in plasma membrane and total membrane preparations was found to be decreased, as detected by immunoblotting, no single proline residue seemed to play a critical role in maintaining the catalytic activity of GLUT1. However, structural changes introduced by incorporation of the neutral polar amino acid glutamine at each single proline position of helix 10 almost completely abolished 2‐deoxy‐d‐glucose uptake. Thus, the specific chemical structure of the side chain of the substituted amino acid rather than the unique property of proline residues for cis‐trans isomerization seemed to determine the consequences on glucose transport.
A Reactive, Surface Cysteine Residue of the Class‐II Fructose‐1,6‐Bisphosphate Aldolase of Escherichia coli Revealed by Electrospray Ionisation Mass Spectrometry
Authors: Packman Leonard C. , Berry Alan
Published: March 2005

The state of post‐translational modification of the class‐II fructose‐1,6‐bisphosphate aldolase (FBP‐aldolase) purified from Escherichia coli was examined by electrospray ionisation mass spectrometry (ESI‐MS). The mass was larger than that expected from the known DNA sequence by approximately 80±6 Da, suggesting the presence of a covalent modification on the protein. Phosphorylation (+80 Da), a known modification in an FBP‐aldolase from Bacillus subtilis and a suspected modification in this E. coli aldolase, was ruled out as the extra mass was readily removed by treatment with dithiothreitol. Purification of aldolase by a protocol which omitted 2‐mercaptoethanol from all buffers resulted in the purified protein having the expected mass (39 016 Da). The extra mass was therefore established as a covalent adduct of the protein with 2‐mercaptoethanol (+76 Da). Reduction and alkylation studies, followed by isolation of tryptic peptides, established that the site of attachment was Cys36. Although no significant effect of the modification on the activity of the protein was observed, the study underlines the ease with which a protein can be modified covalently by a simple and mild purification procedure; such labelling, which may not always be benign, would be undetectable without the routine use of mass spectrometric analysis.
Site‐specific N‐terminal Auto‐degradation of Human Serum Albumin
Authors: Chan Bernard , Dodsworth Neil , Woodrow John , Tucker Alan , Harris Roy
Published: March 2005

Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30°C. Mass spectrometry and N‐terminal sequencing of the protein identified degradation corresponding to the loss of the first two residues, aspartic acid and alanine. The reaction was shown to be dependent upon temperature and the N‐terminal α‐amino group. In addition, comparison with serum albumins derived from other species showed that the instability of the N‐terminus was specific to the human albumin sequence. An intact aspartyl‐alanyl dipeptide, purified from degraded albumin solutions, differed substantially from a synthetic dipeptide on amino acid analysis, N‐terminal sequencing and NIMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.
Characterisation of a Protease from Escherichia coli Involved in Hydrogenase Maturation
Authors: Rossmann Reinhild , Maier Thomas , Lottspeich Friedrich , Böck August
Published: March 2005

The large subunits of nickel‐containing hydrogenases are synthesised in a precursor form which, after nickel incorporation, is processed by proteolytic cleavage at the C‐terminal end. The protease involved in processing of HycE, the large subunit of hydrogenase 3 from Escherichia coli, was purified by three chromatographic steps to apparent homogeneity. Its gene was identified by using a hybridisation probe generated by PCR with oligonucleotide primers the sequence of which was derived from the N‐terminal and internal amino acid sequences. Determination of the nucleotide sequence showed that the gene is located distally and as a hitherto uncharacterised gene within the hyc operon, coding for hydrogenase 3 components. It was designated hycI. The HycI protease has a molecular mass of 17 k Da and is a monomer. Its cleavage reaction is not inhibited by conventional inhibitors of serine and metalloproteases, which correlates with the fact that the sequence does not contain signature motifs characteristic of serine‐, metallo‐, cysteine‐ or acid proteases. Homologous genes are present in other transcriptional units coding for hydrogenases.