bluuebunny/crossref_metadata_2025 huggingface dataset:
Flexibility of myosin‐subfragment‐1 in its complex with actin as revealed by fluorescence resonance energy transfer
Authors: Nyitrai Miklós , Hild Gábor , Bódis Emõke , Lukács András , Somogyi Béla
Published: March 2003

The flexibility of the acto–myosin complex in rigor conditions was characterized by measuring the temperature profile of normalized fluorescence resonance energy transfer efficiency, f′[Somogyi, B., Matkó, J., Papp, S., Hevessy, J., Welch, G.R. & Damjanovich, S. (1984) Biochemistry23, 3403–3411]. Fluorescence acceptors were introduced to the Cys374 residues of actin and the donors were covalently attached either to Cys707 in the catalytic domain or to Cys177 in the essential light‐chain of myosin S1. Fluorescence resonance energy transfer measurements revealed that the protein matrix between Cys374 of actin and Cys707 of S1 is rigid. In contrast, the link between the catalytic and light‐chain‐binding domains in myosin S1 is flexible. We have recently shown that the positional distribution of Cys707 was narrow relative to the actin filament, while that of the Cys177 was broad. Accordingly, the broad positional distribution of Cys177 is likely to be due to the large flexibility of the link between the catalytic and light‐chain‐binding domains. This flexibility is probably essential for the interdomain reorganization of the myosin head during the force generation process and for accommodating the symmetry difference between actin and myosin filaments to allow the formation of cross‐bridges.
Homo‐oligomer formation by basigin, an immunoglobulin superfamily member, via its N‐terminal immunoglobulin domain
Authors: Yoshida Seiya , Shibata Maki , Yamamoto Satoshi , Hagihara Masako , Asai Naoya , Takahashi Masahide , Mizutani Shigehiko , Muramatsu Takashi , Kadomatsu Kenji
Published: March 2003

Basigin (Bsg) is a highly glycosylated transmembrane protein with two immunoglobulin (Ig)‐like domains. A number of studies, including gene targeting, have demonstrated that Bsg plays pivotal roles in spermatogenesis, implantation, neural network formation and tumor progression. In the present study, to understand the mechanism of action of Bsg, we determined its expression status on the plasma membrane. Cotransfection of Bsg expression vectors with two different tags clarified that Bsg forms homo‐oligomers in a cis‐dependent manner on the plasma membrane. If the disulfide bond of the more N‐terminally located Ig‐like domain was destroyed by mutations, Bsg could not form oligomers. In contrast, the mutations of the C‐terminal Ig‐like domain or N‐glycosylation sites did not affect the association. The association of mouse and human Bsgs, which exhibit high homology in the transmembrane and intracellular domains but low homology in the extracellular domain, was very weak as compared with that within the same species, suggesting the importance of the extracellular domain in the association. If the extracellular domain of the human Ret protein was replaced with the N‐terminal Ig‐like domain of Bsg, the resulting chimera protein was associated with intact wild‐type Bsg, but not if the C‐terminal Ig‐like domain, instead of the N‐terminal one, of Bsg was used. No oligomer formation took place between the intact wild‐type Ret and Bsg proteins. In conclusion, these data indicate that the N‐terminal Ig‐like domain is necessary and sufficient for oligomer formation by Bsg on the plasma membrane.
Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN
Authors: Geoghegan Susan M. , Mayhew Stephen G. , Yalloway Gary N. , Butler Geraldine
Published: March 2003

The gene for the electron‐transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N‐terminus, the physico‐chemical properties of the protein, including its optical spectrum and oxidation‐reduction properties, are very similar to those of native flavodoxin. A site‐directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation‐reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from −114 to −242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from −373 mV to −271 mV. A redox‐linked pKa associated with the hydroquinone is decreased from 5.8 to ≤ 4.3. The spectra of the hydroquinones of wild‐type and mutant proteins depend on pH (apparent pKa values of 5.8 and ≤ 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side‐chain is associated with the redox‐linked pKa of 5.8 in the hydroquinone.
Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa
Authors: Hill Justine M. , Alewood Paul F. , Craik David J.
Published: March 2003

The three‐dimensional solution structure of conotoxin TVIIA, a 30‐residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D 1H NMR spectroscopy. TVIIA contains six cysteine residues which form a ‘four‐loop’ structural framework common to many peptides from Conus venoms including the ω‐, δ‐, κ‐, and µO‐conotoxins. However, TVIIA does not belong to these well‐characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side‐chain torsion angle restraints derived from spin‐spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2–27 of 0.18 ± 0.05 Å for the backbone atoms and 1.39 ± 0.33 Å for all heavy atoms. The structure consists of a triple‐stranded, antiparallel β sheet with +2x, −1 topology (residues 7–9, 16–20 and 23–27) and several β turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino‐acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four‐loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure‐activity relationship for these molecules and their interaction with target receptors.
Role of redox‐active iron ions in the decomposition of S‐nitrosocysteine in subcellular fractions of porcine aorta
Authors: Sorenson Elizabeth , Skiles Emily H. , Xu Bin , Aleryani Samir , Kostka Peter
Published: March 2003

We recently reported that degradation of S‐nitrosocysteine in homogenates of porcine aorta increased severalfold in the presence of Mg2+ ions [Kostka, P., Xu, B. & Skiles, E.H. (1999) J. Cardiovasc. Pharmacol.33, 665–670]. The objective of the present study was to examine this in greater detail. The rate of S‐nitrosocysteine degradation by aortic homogenates in the presence of Mg2+ ions exhibited differential sensitivity to chelators of iron ions. Terpyridine and diethylenetriamine penta‐acetic acid (5–500 µm) caused a concentration‐dependent inhibition of S‐nitrosocysteine decay, whereas deferoxamine (100 µm) was ineffective. o‐Phenanthroline (250 µm), a selective chelator of Fe2+ ions, potentiated the reaction at low initial concentrations of S‐nitrosocysteine (≤ 15 µm) and inhibited the reaction at higher concentrations. The inhibitory effects of o‐phenanthroline were related to suppression of S‐nitrosocysteine decay by cysteine‐mediated reduction of Fe3+. In the presence of o‐phenanthroline, S‐nitrosocysteine decomposition followed saturable kinetics with K0.5 = 3.8 ± 0.3 µm and h = 1.8 ± 0.1 (mean ± SE, n = 4). Comparison of the rates of S‐nitrosocysteine decay in different subcellular fractions showed selective association with the cytosolic fraction, as documented by copurification with lactate dehydrogenase activity. At non‐limiting concentrations of S‐nitrosocysteine, the rate of degradation in the cytosolic fraction was 4.1 ± 0.3 nmol·min−1·(mg protein)−1 (n = 4). It is concluded that the cytosolic fraction of porcine aorta contains a protein factor, presumably an enzyme, capable of catalyzing heterolytic decomposition of the S–NO bond of S‐nitrosocysteine in a process involving redox cycling of iron ions.
Identification of foetal brain proteins by two‐dimensional gel electrophoresis and mass spectrometry
Authors: Oppermann Madalina , Cols Neus , Nyman Tuula , Helin Jari , Saarinen Juhani , Byman Irene , Toran Núria , Alaiya Ayodele A. , Bergman Tomas , Kalkkinen Nisse , Gonzàlez‐Duarte Roser , Jörnvall Hans
Published: March 2003

Protein expression in foetal brain with or without chromosome 21 trisomy (Down's syndrome) was analyzed by two‐dimensional gel electrophoresis and mass spectrometry. Data generated by in‐gel digestion and matrix‐assisted laser desorption/ionization mass spectrometry allowed identification of 40 proteins. Most of these are common to syndrome and healthy subjects and represent different types of protein. However, a few proteins, identified as truncated structural proteins (tubulin, actin), were present in part of the trisomy samples but absent from the controls. This is interpreted to indicate increased proteolysis in the syndrome samples but could also reflect some altered expression or processing. Independent of the apparently increased proteolysis in the syndrome samples, and in spite of the use of total brain tissues, the results show that two‐dimensional protein separation patterns are largely similar between the syndrome and control samples upon silver‐staining, but that differences associated with structural components can be detected and identified.
In vitro hyperprocessing of Drosophila tRNAs by the catalytic RNA of RNase P
Authors: Hori Yoshiaki , Baba Hideo , Ueda Ryu , Tanaka Terumichi , Kikuchi Yo
Published: March 2003

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNAMeti) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNAMeti. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed.
Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels
Authors: Fajloun Ziad , Carlier Edmond , Lecomte Catherine , Geib Sandrine , di Luccio Eric , Bichet Delphine , Mabrouk Kamel , Rochat Hervé , De Waard Michel , Sabatier Jean‐Marc
Published: March 2003

Pi1 is a 35‐residue toxin cross‐linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme‐based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half‐cystine pairings between Cys4–Cys25, Cys10–Cys30, Cys14–Cys32 and Cys20–Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 µg per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nm) and rat Kv1.2 channels (IC50 of 0.44 nm) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I‐labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pm. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK‐ and Kv1‐types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide‐bridged scorpion toxin that exhibits a different half‐cystine pairing pattern.
Purification and characterization of the single‐strand‐specific and guanylic‐acid‐preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus
Authors: Desai Neelam A. , Shankar Vepatu
Published: March 2003

An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE‐cellulose, and chromatography on phenyl‐Sepharose followed by FPLC on phenyl‐Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8.5 and 60 °C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2‐mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA ≈ 3′AMP ≫ RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5′dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5′dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5′dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3′ or the 5′ end, indicates that C‐linkages are resistant to cleavage by nuclease Bh1.
Characterization and kinetic mechanism of mono‐ and bifunctional ornithine acetyltransferases from thermophilic microorganisms
Authors: Marc Frédéric , Weigel Pierre , Legrain Christianne , Almeras Yann , Santrot Marie , Glansdorff Nicolas , Sakanyan Vehary
Published: March 2003

The argJ gene coding for N2‐acetyl‐l‐ornithine: l‐glutamate N‐acetyltransferase, the key enzyme involved in the acetyl cycle of l‐arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N‐acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His‐tagged ArgJ enzyme of M. jannaschii only catalyzes N2‐acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N‐acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ‐mediated catalysis follows ping‐pong bi–bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2‐acetyl‐l‐glutamate as acetyl donors. In this catalysis l‐ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of l‐arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate α and β subunits which appear to assemble to α2β2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM‐ATML motif detected in all available ArgJ sequences.
Regulation of protein function by S‐glutathiolation in response to oxidative and nitrosative stress
Authors: Klatt Peter , Lamas Santiago
Published: March 2003

Protein S‐glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol‐group modifications. Special attention will be paid to recently published work supporting a role for S‐glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S‐glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post‐translational protein modification will be discussed.
Biosynthesis of the cyanobacterial reserve polymer multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin)
Authors: Berg Holger , Ziegler Karl , Piotukh Kirill , Baier Kerstin , Lockau Wolfgang , Volkmer‐Engert Rudolf
Published: March 2003

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi‐l‐arginyl‐poly‐l‐aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP‐binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin‐like polymers of 25–30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C‐terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino‐acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.
Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS‐modified cytochrome c
Authors: Kotlyar Alexander , Borovok Natalia , Hazani Miron , Szundi Istvan , Einarsdóttir Ólöf
Published: March 2003

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene‐3,6,8‐trisulfonate in complex with cytochrome oxidase. The thiouredopyrene‐3,6,8‐trisulfonate‐labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso‐1‐cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene‐3,6,8‐trisulfonate‐cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene‐3,6,8‐trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of ≈ 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.
KATP channels gated by intracellular nucleotides and phospholipids
Authors: Baukrowitz Thomas , Fakler Bernd
Published: March 2003

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir6.1 or Kir6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir6.x subunits, whereas Mg‐ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4,5‐bisphosphate and phosphatidylinositiol 4‐monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications.
Spectroscopic probing of the influence of calcium and the Gla domain on the interaction between the first EGF domain in factor VIIa and tissue factor
Authors: Österlund Maria , Owenius Rikard , Persson Egon , Lindgren Mikael , Carlsson Uno , Freskgård Per‐Ola , Svensson Magdalena
Published: March 2003

The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor‐like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4‐carboxyglutamic acid (Gla)‐domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla‐domainless and full‐length FVIIa.
Thioredoxin reductase
Authors: Williams Charles H. , Arscott L. David , Müller Sylke , Lennon Brett W. , Ludwig Martha L. , Wang Pan‐Fen , Veine Donna M. , Becker Katja , Schirmer R. Heiner
Published: March 2003

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH‐dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.
The myxochelin iron transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15
Authors: Silakowski Barbara , Kunze Brigitte , Nordsiek Gabriele , Blöcker Helmut , Höfle Gerhard , Müller Rolf
Published: March 2003

The biosynthetic gene cluster of the myxochelin‐type iron chelator was cloned from Stigmatella aurantiaca Sg a15 and characterized. This catecholate siderophore was only known from two other myxobacteria. The biosynthetic genes of 2,3‐dihydroxybenzoic acid are located in the cluster (mxcC–mxcF). Two molecules of 2,3‐dihydroxybenzoic acid are activated and condensed with lysine in a unique way by a protein homologous to nonribosomal peptide synthetases (MxcG). Inactivation of mxcG, which encodes an adenylation domain for lysine, results in a myxochelin negative mutant unable to grow under iron‐limiting conditions. Growth could be restored by adding Fe3+, myxochelin A or B to the medium. Inactivation of mxcD leads to the same phenotype. A new type of reductive release from nonribosomal peptide synthetases of the 2,3‐dihydroxybenzoic acid bis‐amide of lysine from MxcG, catalyzed by a protein domain with homology to NAD(P) binding sites, is discussed. The product of a gene, encoding a protein similar to glutamate‐1‐semialdehyde 2,1‐aminomutases (mxcL), is assumed to transaminate the aldehyde that is proposed as an intermediate. Further genes encoding proteins homologous to typical iron utilization and iron uptake polypeptides are reported.
Effect of cadmium on the relationship between replicative and repair DNA synthesis in synchronized CHO cells
Authors: Banfalvi Gaspar , Littlefield Neil , Hass Bruce , Mikhailova Marina , Csuka Ildiko , Szepessy Edit , Chou Ming Wu
Published: March 2003

Repair and replicative DNA synthesis were measured at different stages of the cell cycle in control and cadmium‐treated Chinese hamster ovary (CHO‐K1) cells. Cells were synchronized by counterflow centrifugal elutriation. Elutriation resulted in five repair and four replication subphases. On Cd treatment, repair synthesis was elevated in certain subphases. Replicative subphases were suppressed by Cd treatment, with some of the peaks almost invisible. The number of spontaneous strand breaks measured by random oligonucleotide primed synthesis assay showed a cell‐cycle‐dependent fluctuation in control cells and was greatly increased after Cd treatment throughout the S phase. Elevated levels of the oxidative DNA damage product, 8‐oxodeoxyguanosine, were observed after Cd treatment, with the highest level in early S phase, which gradually declined as damaged cells progressed through the cell cycle.
Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site‐directed mutagenesis
Authors: Labrou Nikolas E. , Rigden Danyel J. , Clonis Yannis D.
Published: March 2003

The 2′,3′‐dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first‐order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min−1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme–oADP product, a process that was reversed after dialysis or after treatment with 2‐mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2‐mercaptoethanol was prevented if the enzyme–oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP‐modified enzyme with trypsin and subsequent separation of peptides by RP‐HPLC gave only one radioactive peak. Amino‐acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+‐binding site. Site‐directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360→Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360→Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.
Chaperone function of mutant versions of αA‐ and αB‐crystallin prepared to pinpoint chaperone binding sites
Authors: Derham Barry K. , van Boekel Martinus A. M. , Muchowski Paul J. , Clark John I. , Horwitz Joseph , Hepburne‐Scott Henry W. , de Jong W. W. , Crabbe M. James C. , Harding John J.
Published: March 2003

A major stress protein, α‐crystallin, functions as a chaperone. Site‐directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat‐induced aggregation method at elevated temperature and using enzyme methods at 37 °C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of α‐crystallin. A mutant corresponding to a minor component of rodent αA‐crystallin, αAins‐crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139→ Arg, Thr144→Arg, Ser59→Ala mutants of αB‐crystallin and double mutants Ser45→Ala/Ser59→Ala, Lys103→ Leu/His104→Ile, and Glu110→His/His111→Glu. A mutant Phe27→Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.
Active site characterization of the single endo‐polygalacturonase produced by Fusarium moniliforme NCIM 1276
Authors: Niture Suryakant K. , Pant Aditi , Kumar Ameeta R.
Published: March 2003

Fusarium moniliforme NCIM 1276 isolated from a tropical mangrove ecosystem produces a single extracellular endo‐polygalacturonase with an Mr of 38 kDa and a carbohydrate content of 4%. It has an alkaline pI of 8.1. The Km is 0.12 mg·mL−1, Vmax is 111.1 µmol·min−1·mg−1 and the kcat is 4200 min−1. It has a pH optimum of 4.8. Kinetic and fluorescence data show that tryptophan is involved in binding. An arginine residue at or near the active site may be involved in extended binding of the substrate. A carboxylate and a histidine residue are involved in catalysis. These data are discussed with reference to current literature.
Structural study of N‐linked oligosaccharides of human intercellular adhesion molecule‐3 (CD50)
Authors: Funatsu Osamu , Sato Takeshi , Kotovuori Pekka , Gahmberg Carl G. , Ikekita Masahiko , Furukawa Kiyoshi
Published: March 2003

The N‐linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)‐3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM‐3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase‐treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio‐Gel P‐4 column chromatography. Structural studies of each oligosaccharide by sequential exo‐ and endo‐glycosidase digestion and by methylation analysis revealed that N‐linked oligosaccharides of ICAM‐3 are mainly of tri‐ and tetra‐antennary complex‐type, about 60% of which contain two to three poly N‐acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose‐type oligosaccharide with six α‐mannose residues, which could act as a ligand for the dendritic cell‐specific ICAM‐3 grabbing nonintegrin, was detected.
Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore
Authors: Pontoppidan Bo , Ekbom Barbara , Eriksson Susanna , Meijer Johan
Published: March 2003

Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion‐exchange and phenyl‐Sepharose chromatography. The protein has an apparent subunit molecular mass of 57–58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 °C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases. Using sinigrin as the substrate Km was determined as 0.41 mm, and the kcat as 36 s−1. With glucotropaeolin the Km and kcat values were determined as 0.52 mm and 22.8 s−1, respectively. The enzyme was stable upon storage at 4 °C for many months, but lost some activity upon freezing. The insect myrosinase did not cross‐react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to β‐glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.
Novel methylated triterpenoids of the gammacerane series from the nitrogen‐fixing bacterium Bradyrhizobium japonicum USDA 110
Authors: Bravo Jean‐Michel , Perzl Michael , Härtner Thomas , Kannenberg Elmar L. , Rohmer Michel
Published: March 2003

The nitrogen‐fixing, symbiotic root‐nodule forming bacterium Bradyrhizobium japonicum USDA 110 contained gammacerane derivatives next to triterpenoids of the hopane series. Diploptene, diplopterol, 2β‐methyldiplopterol, aminobacteriohopanetriol and adenosylhopane were accompanied by tetrahymanol and the corresponding novel methylated homologues 2β‐methyltetrahymanol, 20α‐methyltetrahymanol, and 2β,20α‐dimethyltetrahymanol. Incorporation of [2H3]methyl–l‐methionine indicated that the additional methyl groups originated from methionine, probably with S‐adenosylmethionine acting as methyl donor, with retention of the three deuterium atoms. The simultaneous presence of hopane and gammacerane derivatives seems a characteristic feature of the genus Bradyrhizobium and the phylogenetically closely related Rhodopseudomonas palustris.
Resistance to induced apoptosis in the human neuroblastoma cell line SK‐N‐SH in relation to neuronal differentiation
Authors: Lombet Alain , Zujovic Violetta , Kandouz Mustapha , Billardon Claude , Carvajal‐Gonzalez Santos , Gompel Anne , Rostène William
Published: March 2003

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK‐N‐SH to undergo apoptosis induced by thapsigargin was examined. SK‐N‐SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4β‐phorbol 12‐myristate 13‐acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by γ‐enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin‐induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro‐(Bcl‐2, Bcl‐xL) and anti‐apoptotic (Bax, Bak) protein expression of the Bcl‐2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl‐2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl‐xL expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl‐2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl‐2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G‐protein RhoA and PtdIns3 kinase in the control of Bcl‐2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl‐2 protein family and the resistance of differentiated SK‐N‐SH to apoptosis.
Effects of methylcyclodextrin on lysosomes
Authors: Jadot Michel , Andrianaivo Fanjambolatiana , Dubois Franz , Wattiaux Robert
Published: March 2003

The cholesterol complexing agent methyl‐cyclodextrin (MCD) provides an efficient mean for the removal of cholesterol from biological membranes. In order to study the effects of this agent on the lysosomal membrane in situ, we treated HepG2 cells with MCD and studied the effects of this treatment on lysosomes in isolated fractions. We found that lysosomes prepared from treated cells are more sensitive to various membrane perturbing treatments such as: incubation of lysosomes in isotonic glucose, in hypotonic sucrose or in the presence of the lytic agent glycyl‐l‐phenylalanine 2‐naphthylamide. The lysosomal membrane is also less resistant to increased hydrostatic pressure. Centrifugation methods were used to analyse the effect of MCD on lysosomes. Isopycnic centrifugation in sucrose density gradients demonstrates that the drug induces a reversible density increase of the lysosomes. Our study indicates that extracellularly added MCD can modify the properties of the lysosomal membrane in living cells. It suggests that MCD could be an effective tool to modulate the physical properties of lysosomes within intact cells and to monitor the cellular responses to such modifications.
The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3
Authors: Vinogradov Evgeny , Cedzynski Maciej , Ziolkowski Andrzej , Swierzko Anna
Published: March 2003

The structure of lipid A–core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS:where P is H or α‐Hep; J is H or β‐GalA; R is H or P (in the deacylated oligosaccharides).Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain α‐Hep‐(1→4)‐α‐Kdo‐(2→6)‐GlcN and α‐Kdo‐(2→6)‐GlcN fragments.
Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius
Authors: Uhrigshardt Helge , Walden Michael , John Harald , Anemüller Stefan
Published: March 2003

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27‐fold to a specific activity of ≈ 55 µmol·min−1·mg−1, based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 °C, slightly under the growth optimum of S. acidocaldarius around 80 °C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 °C. Even at 95 °C, a half‐life of ≈ 14 min was determined, clearly defining it as a thermostable protein. The apparent Km values for the three substrates cis‐aconitate, citrate and isocitrate were found as 108 µm, 2.9 mm and 370 µm, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans‐aconitate and tricarballylate. Amino‐acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino‐acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR‐spectroscopy, the enzyme hosts an interconvertible [3Fe−4S] cluster.
The role of exon 5 in fibroblast collagenase (MMP‐1) substrate specificity and inhibitor selectivity
Authors: Knäuper Vera , Patterson Margaret L. , Gomis‐Rüth Franz X. , Smith Bryan , Lyons Alan , Docherty Andrew J. P. , Murphy Gillian
Published: March 2003

Interstitial collagen is degraded by members of the matrix metalloproteinase (MMP) family, including MMP‐1. Previous work has shown that the region of MMP‐1 coded for by exon 5 is implicated both in substrate specificity and inhibitor selectivity. We have constructed a chimeric enzyme, the exon 5 chimera, consisting primarily of MMP‐1, with the region coded for by exon 5 replaced with the equivalent region of MMP‐3, a noncollagenolytic MMP. Unlike MMP‐3, the exon 5 chimera is capable of cleaving type I collagen, but the activity is only 2.2% of trypsin‐activated MMP‐1. ‘Superactivation’ of the chimera has no discernible effect, suggesting that the salt bridge formed in ‘superactive’ MMP‐1 is not present. The kinetics for exon 5 chimera cleavage of two synthetic substrates display an MMP‐3 phenotype, however, cleavage of gelatin is slightly impaired as compared to the parent enzymes. The Kiapp values for the exon 5 chimera complexed with synthetic inhibitors and N‐terminal TIMP‐2 also show a more MMP‐3‐like behaviour. However, the kon values for N‐terminal TIMP‐1 and N‐terminal TIMP‐2 are more comparable to those for MMP‐1. These data show that the region of MMP‐1 coded for by exon 5 is involved in both substrate specificity and inhibitor selectivity and the structural basis for our findings is discussed.
Exonucleolytic proofreading by p53 protein
Authors: Bakhanashvili Mary
Published: March 2003

The tumour suppressor p53 protein plays an important role in maintaining genetic integrity. Recently, p53 was shown to have an intrinsic 3′→5′ exonuclease activity. The current study has extended the characterization of purified wild‐type recombinant p53‐associated 3′→5′ exonuclease function to demonstrate proofreading activity. p53‐associated 3′→5′ exonuclease shows clear preference for degradation of ssDNA over dsDNA substrate. On partial duplex structures, this exonucleolytic activity displays a marked preference for excision of a mismatched vs. a correctly paired 3′ terminus which enables the p53 protein to act as a proofreader. However, p53 displays variation in excision of mismatched base pairs. The results demonstrate that p53 exhibits mispair excision with a specificity of A:A > A:G > A:C opposite the template adenine residue and with a specificity of G:A > G:G > G:T opposite the template guanine residue. Hence, the observed specificity of mismatch excision shows that p53 exonucleolytic proofreading preferentially repairs transversion mutations. As part of an investigation of the functional interaction between p53 and DNA polymerase, the influence of p53 on the accuracy of DNA synthesis was determined with exonuclease‐deficient murine leukemia virus (MLV) reverse transcriptase (RT), representing a relatively low fidelity enzyme. Using an in vitro biochemical assay with 3′‐terminal mismatch‐containing DNA template primers, it was shown that wild‐type recombinant p53 protein enhanced the DNA replication fidelity of MLV RT. A functional interaction between the exonuclease (p53) and polymerase (MLV RT) activities was observed; excision of mispairs by p53 was followed by further elongation onto correctly base‐paired 3′‐termini by MLV RT. Furthermore, the formation of 3′‐mispair and subsequent mispair extension by the enzyme were decreased substantially in the presence of p53. The fact that the exonuclease‐deficient MLV RT is more accurate in the presence of p53, suggests that p53 protein may function as an external proofreading exonuclease for viral enzyme. The observed decrease in initial nucleotide misincorporation and 3′‐terminal mispair extension by MLV RT in the presence of p53, indicates the mechanism by which p53 affects the DNA replication fidelity of exonuclease‐deficient DNA polymerase.