bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical and genetic characterization of the membrane‐associated malate dehydrogenase (acceptor) from Corynebacterium glutamicum
Authors: Molenaar Douwe , van der Rest Michel E. , Petrović Sonja 
Published: March 2003

In addition to a cytoplasmic, NAD‐dependent malate dehydrogenase ( EC1.1.1.37), Corynebacterium glutamicum possesses a highly active membrane‐associated malate dehydrogenase (acceptor) (
EC1.1.99.16). This enzyme also takes part in the citric acid cycle. It oxidizes
L‐malate to oxaloacetate and donates electrons to ubiquinone‐1 and other artificial acceptors or, via the electron transfer chain, to oxygen. NAD is not an acceptor and the natural direct acceptor for the enzyme is most likely a quinone. The enzyme is therefore called malate :quinone oxidoreductase, abbreviated to Mqo. Mqo is a peripheral membrane protein and can be released from the membrane by addition of chelators. The solubilized form was partially purified and characterized biochemically. FAD is probably a tightly but non‐covalently bound prosthetic group, and the enzyme is activated by lipids.A C. glutamicum mutant completely lacking Mqo activity was isolated. It grows poorly on several substrates tested. The mutant possesses normal levels of cytoplasmic NAD‐dependent malate dehydrogenase. A plasmid containing the gene from C. glutamicum coding for Mqo was isolated by complementation of the Mqo‐negative phenotype. It leads to overexpression of Mqo activity in the mutant. The nucleotide sequence of the mqo gene was determined and is the first sequence known for this enzyme. The derived protein sequence is similar to hypothetical proteins from Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis.
Synthetic peptide‐containing surfactants
Authors: Nilsson Gunhild , Gustafsson Magnus , Vandenbussche Guy , Veldhuizen Edwin , Griffiths William J. , Sjövall Jan , Haagsman Henk P. , Ruysschaert Jean‐Marie , Robertson Bengt , Curstedt Tore , Johansson Jan 
Published: March 2003

Pulmonary surfactant contains two hydrophobic proteins, SP‐B and SP‐C. With the aim of identifying synthetic SP‐B and SP‐C substitutes for replacement therapy of respiratory distress syndromes, we have studied two transmembrane peptides and two amphipathic peptides that are located in the plane of a phospholipid bilayer. One amphipathic peptide was designed by changing the amino acid sequence, but not the composition or size, of the 21‐residue peptide KL4. This peptide, designated KL2.3 from its spacing of nonpolar and polar residues, exhibited similar α‐helical content as KL4 but was oriented along a phospholipid bilayer plane, in contrast to the transmembrane orientation of KL4 in the same environment. The second amphipathic peptide analyzed was succinyl‐LLEKLLEWLK‐amide (WMAP10). KL4 more efficiently accelerated the spreading of a mixture of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (Pam2GroPCho)/phosphatidylglycerol (PtdGro)/palmitic acid (PamOH), 68 : 22 : 9 (by mass), at an air/water interface than did any of the amphipathic peptides. Similarly, KL4, but not KL2.3, when present in an interfacial monolayer composed of Pam2GroPCho/1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoglycerol, 7 : 3 (by mass), increased lipid insertion from subphase vesicles.An SP‐C analogue, SP‐C(Leu), with all helical valyl residues in native SP‐C replaced with Leu and the palmitoylcysteines at positions 5 and 6 replaced with Ser, but otherwise with essentially the same primary structure as the native peptide, was analyzed. SP‐C(Leu) exhibited similar α‐helical content as native SP‐C and a transmembrane orientation and, in contrast to poly‐valyl‐containing synthetic peptides, it folds into a helical conformation after acid‐induced denaturation. SP‐C(Leu) accelerated the spreading of Pam2GroPCho/PtdGro/PamOH, 68 : 22 : 9 (by mass), almost identically to native SP‐C, and lowered the surface tension during rapid cyclic film compressions in a pulsating bubble surfactometer to near zero and 43 mN/m at minimum and maximum bubble size, respectively. Airway instillation of 2 % (by mass) SP‐C(Leu) combined with Pam2GroPCho/PtdGro/PamOH in preterm rabbit fetuses improved dynamic lung compliance by about 30 % compared with untreated controls.
The chemical structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A
Authors: Karlsson Camilla , Jansson Per‐Erik , Sørensen Uffe B. 
Published: March 2003

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3‐position of the 4‐substituted β‐
L‐rhamnose (Rha) residue. Both polysaccharides are substituted with one O‐acetyl group at the 2‐position of the same β‐
L‐rhamnose residue. In addition, the type‐32A polysaccharide is substituted with another O‐acetyl group at the 4‐position of the 2,3‐disubstituted α‐
D‐glucose residue, i.e. the branch‐point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate.R O‐PCho
∣∣
4
3
←4)‐β‐
D‐Glcp‐(1←3)‐α‐
D‐Glcp‐(1←4)‐β‐
L‐Rhap2Ac‐(1←
2
∣α‐
L‐Rhap‐P‐O
32F  R=H
32A
R=Ac
In the type‐32F polysaccharide R=H, and in the type‐32A polysaccharide R=Ac. The structure of C‐polysaccharide found in our preparations of type‐32F and type‐32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C‐polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter‐Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851−857; Kulakowska, M., Brisson, J.‐R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644−648].
Probing of the neuropeptide Y−Y1‐receptors interaction with anti‐receptor antibodies
Authors: Wieland Heike A. , Eckard Christophe P. , Doods Henri N. , Beck‐Sickinger Annette G. 
Published: March 2003

The Y1 receptor, which belongs to the family of rhodopsin‐like GTP‐binding protein‐coupled, seven‐transmembrane helix‐spanning receptors, binds the 36‐mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N‐terminus, extracellular loops and C‐terminus of the Y1 receptor were used to generate 18 anti‐receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C‐terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor.Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand‐receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.
Structure of the O polysaccharide and immunochemical relationships between the lipopolysaccharides of Pseudomonas syringae pathovar tomato and pathovar maculicola
Authors: Knirel Yuriy A. , Ovod Vladimir V. , Zdorovenko Galina M. , Gvozdyak Rostislav I. , Krohn Kai J. 
Published: March 2003

O polysaccharides (OPS) of the lipopolysaccharides (LPS) of Pseudomonas syringae pathovar tomato GSPB 483 and pathovar maculicola IMV 381 were studied by 1H‐NMR and 13C‐NMR spectroscopy, including two‐dimensional COSY, rotating‐frame NOE spectroscopy (ROESY), and H‐detected 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments. The OPS from both strains were shown to be chemically identical. Two structurally different types of repeating units (O repeats 1 and 2) were elucidated in each OPS. The minor O repeat is a pentasaccharide having the structure 2. The same structure has been reported earlier for some other OPS of P. syringae. The major O repeat is a hexasaccharide with the novel structure 1 which is distinguished from 2 by the presence of the second lateral residue of 3‐acetamido‐3,6‐dideoxy‐
D‐galactose (
D‐Fuc3NAc) and by a different position of substitution of one of the
L‐rhamnose (Rha) residues in the backbone.α‐ D‐Fucp3NAc
α‐
D‐Fucp3NAc
1
1
↓↓
3
3
←2)‐α‐
L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←1α‐ D‐Fucp3NAc
1
↓
3
←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←2Structural and serological diversity of OPS of strains belonging to P. syringae pv. tomato and pv. maculicola and phylogenetic relatedness of the strains are discussed.
Oligomerization of the 17‐kDa peptide‐binding domain of the molecular chaperone HSC70
Authors: Fouchaq Benoît , Benaroudj Nadia , Ebel Christine , Ladjimi Moncef M. 
Published: March 2003

Crystallographic and biochemical studies have indicated that the peptide‐binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a β‐sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17‐kDa protein (residues 385–540 of HSC70) consisting mainly of the peptide‐binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60‐kDa fragment of HSC70, which is composed of the 17‐kDa domain and the 44‐kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17‐kDa peptide‐binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide‐binding site may be involved in the oligomerization process.
Site‐directed mutagenesis of proline 204 in the ‘hinge’ region of yeast phosphoglycerate kinase
Authors: McHarg Jane , Kelly Sharon M. , Price Nicholas C. , Cooper Alan , Littlechild Jennifer A. 
Published: March 2003

Site‐specific mutants have been produced in order to investigate the role of proline 204 in the ‘hinge’ region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis‐proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the ‘hinge’ bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild‐type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild‐type. In the denaturation experiments two transitions were observed for both the wild‐type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry29, 8683–8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild‐type protein, suggesting that sequence–distant interactions are more rapidly disrupted in this mutant enzyme than in the wild‐type enzyme, while sequence–local interactions are disrupted in a similar way. The Michaelis constants (Km) for both 3‐phospho‐
d‐glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of ‘hinge’ bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild‐type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the ‘hinge’ region of PGK plays a role in the stability and catalytic mechanism of the enzyme.
Cell wall teichoic acids of Actinomadura viridis VKM Ac‐1315T
Authors: Shashkov Alexander S , Potekhina Natalia V , Naumova Irina B , Evtushenko Ludmila I , Widmalm Goran 
Published: March 2003

The cell walls of Actinomadura viridis contain poly(glycosylglycerol phosphate) chains of complex structure. On the basis of NMR spectroscopy of the polymer and glycosides thereof the following structural units were found: β‐d‐Galp3Me‐(1→4)[β‐D‐Glcp‐(1→6)]‐β‐D‐Galp‐(1→1)‐snGro (G1); β‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→1)‐snGro (G2); β‐d‐Galp3Me‐(1→4)‐β‐d‐Galp‐(1→1)‐snGro (G2a); β‐d‐Galp‐(1→1)‐snGro (G3);β‐d‐Galp‐(1→1)[β‐d‐Galp‐(1→2)]‐snGro (G4); β‐d‐Glcp‐(1→2)‐snGro (G5). Glycosides G1, G2 and G3 were the predominant components of the teichoic acid: they formed the polymer chain via phosphodiester bonds involving C‐3 of the glycerol residue and C‐3 of the galactosyl residue which in turn glycosylates C‐1 of the glycerol residue. Whether the different glycosides make up the one chain or whether there are several poly(glycosylglycerol phosphate) chains in the cell wall remains to be determined. It was suggested that the minor component G5 is located at the nonterminal end of the chains. Compound G4 which contains disubstituted glycerol resudues (unusual for the teichoic acid) was also found as a minor component; this may be the glycoside of a new type of teichoic acid, or a glycoside on the terminal end of the above mentioned chains. In addition, small amounts of 1,3‐poly(glycerol phosphate) chains were found in the cell wall.
Evidence for a dynamic role for proline376 in the purine–cytosine permease of Saccharomyces cerevisiae
Authors: Ferreira Thierry , Napias Christian , Chevallier Jean , Brèthes Daniel 
Published: March 2003

The purine–cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem.272, 9697–9702] suggest that the hydrophilic segment 371–377 (‐I‐A‐N‐N‐I‐P‐N‐) of the polypeptide chain may play a key role in the correct three‐dimensional structure of the active carrier. This paper describes the effects of mutations in this particular segment: a four‐residue deletion, Δ374–377, and two substitutions, P376G and P376R. The Δ374–377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild‐type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base‐binding sites [1000–1200 pmol·(mg protein)−1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5–6.0), showing that the conformational change induced by ligand binding was no longer present. Our results show that the 374–377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a β‐turn motif, which must play a crucial dynamic role in the translocation process.
Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp.
Authors: Corsaro M. Michela , Grant William D. , Grant Susan , Marciano Carmela E. , Parrilli Michelangelo 
Published: March 2003

An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp. was found to contain d‐galactopyranuronic acid (GalpA), 2,4‐diacetamido‐2,4,6‐trideoxy‐d‐glucopyranose (QuipNAc4NAc), 2‐acetamido‐2‐deoxy d‐mannopyranuronic acid (ManpNAcA) and one uncommon unit of d‐galactopyranuronic acid with the carboxyl group amide‐linked to glycine [GalpA(Gly)]. The polysaccharide was studied by one‐dimensional and two‐dimensional 1H‐NMR and 13C‐NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis. The following linear structure of the repeating unit was established:→3)‐α‐d‐GalpA(Gly)‐(1→4)‐β‐d‐ManpNAcA‐(1→4)‐α‐d‐GalpA‐(1→3)‐α‐d‐QuipNAc4NAc‐(1→.A preliminary phylogenetic assignment for the bacterium is also reported.
Structural elucidation of the O‐antigenic polysaccharides from Escherichia coli O21 and the enteroaggregative Escherichia coli strain 105
Authors: Staaf Mikael , Urbina Felipe , Weintraub Andrej , Widmalm Göran 
Published: March 2003

The structure of the O‐antigen polysaccharide of the lipopolysaccharide from an enteroaggregative Escherichia coli (strain 105) has been elucidated, using primarily one‐dimensional and two‐dimensional NMR experiments. The sequence of residues was deduced with heteronuclear multiple‐bond correlation and NOESY experiments. The structure of the repeating unit of the polysaccharide from the enteroaggregative E. coli is as follows:The structure of the O‐antigen from enteroaggregative E. coli strain 105 was shown to be identical with that of E. coli O21 by sugar and methylation analyses as well as by 1H‐NMR and 13C‐NMR spectroscopy.
Biochemical properties of a minimal functional domain with ATP‐binding activity of the NTPase/helicase of hepatitis C virus
Authors: Borowski Peter , Kuehl Reinhard , Mueller Oliver , Hwang Lih‐Hwa , Schulze zur Wiesch Julian , Schmitz Herbert 
Published: March 2003

The RNA‐stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP‐binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N‐ and C‐terminal sequencing and by its capability to bind 5′‐fluorosulfonylbenzoyladenosine (FSBA).The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 µm. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP‐binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides.Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 µm), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 µm). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP‐binding domain.
The structure of the carbohydrate backbone of core‐lipid A region ofthe lipopolysaccharides from Proteus mirabilis wild‐type strain S1959 (serotype O3) and its Ra mutant R110/1959
Authors: Vinogradov Evgeny , Radziejewska‐Lebrecht Joanna , Kaca Wieslaw 
Published: March 2003

The following structure of core‐lipid A region of the lipopolysaccharide (LPS) from Proteus mirabilis strain 1959 (serotype O3) and its rough mutant R110/1959 (Proteus type II core) was determined using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS:Incomplete substitutions are indicated by italics. All sugars are in pyranose form, α‐Hep is the residue lglycero‐α‐dmanno‐Hep, α‐dd‐Hep is the residue dglycero‐α‐dmanno‐Hep. The differences with the previously reported structures are discussed.
Production of recombinant human β2‐microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis
Authors: Linke Reinhold P. , Schäeffer Jürgen , Gielow Peter , Lindner Peter , Lottspeich Friedrich , Plückthun Andreas , Weiss Elisabeth H. 
Published: March 2003

Amyloid of β2‐microglobulin (β2m) origin can be diagnosed using 131I‐radiolabelled‐β2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer β2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products.In order to exclude these risks we have produced recombinant human β2m (rhβ2m) in Escherichia coli. The expression vector pASK40ΔLβ2m(His)5 contains a C‐terminal (His)5‐tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification.The isolated rhβ2mH5 reacted in an immunochemically identical manner to native human β2m, and showed a single band of ≈ 11.8 kDa in Western blot analysis and revealed a single spot in two‐dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N‐terminal amino‐acid sequence analysis.The rhβ2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium‐111‐labelled rhβ2mH5 was monitored in hemodialysed patients with and without known β2m‐amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid.Thus, this rhβ2mH5 preparation is suitable for detecting amyloid‐containing organs of the β2m‐class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium‐111 instead of iodine‐131 has reduced the radioactive load and resulted in higher resolution.