bluuebunny/crossref_metadata_2025 huggingface dataset:
Production and purification of active snowdrop lectin in Escherichia coli
Authors: Longstaff Marian , Powell Kevin S. , Gatehouse John A. , Raemaekers Romaan , Newell Christine A. , Hamilton William D. O.
Published: March 2003

Recombinant snowdrop lectin was produced in Escherichia coli from a cDNA clone encoding mature Galanthus nivalis agglutinin. After induction with isopropylthio‐β‐
D‐galactoside, inclusion bodies from E. coli were solubilised and the G. nivalis agglutinin purified by metal‐affinity chromatography using a carboxy‐terminal hexahistidine tag. The protein was refolded on the metal‐affinity column prior to elution. After purification, the recombinant G. nivalis agglutinin agglutinated rabbit erythrocytes to a dilution similar to that determined for ’native' lectin purified from snowdrop, and showed similar specific binding to mannose. The toxicity of the recombinant G. nivalis agglutinin towards rice brown planthopper (Nilaparvata lugens) was shown to be similar to that of ’native' G. nivalis agglutinin when incorporated into an artificial diet. The recombinant G. nivalis agglutinin is thus functionally similar to ’native' snowdrop lectin.
Characterization of wheat thioredoxin h cDNA and production of an active Triticum aestivum protein in Escherichia coli
Authors: Gautier Marie‐Françoise , Lullien‐Pellerin Valérie , de Lamotte‐Guéry Frédéric , Guirao Anne , Joudrier Philippe
Published: March 2003

Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h, respectively, were isolated from mid‐maturation seed cDNA libraries. The T. aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T. durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5 % similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three‐step procedure including heat treatment, anion‐exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13 391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N‐terminal end sequence AASAAT. Using the 5,5′‐dithiobis(2‐nitrobenzoic acid)‐reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP : thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed α‐amylase inhibitors (chloroform/methanol‐soluble proteins). The presence of a putative transmembrane domain at the N‐terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.
Non‐detergent sulphobetaines enhance the recovery of membrane and/or cytoskeleton‐associated proteins and active proteases from erythrocytes infected by Plasmodium falciparum
Authors: Blisnick Thierry , Morales‐Betoulle Maria Eugenia , Vuillard Laurent , Rabilloud Thierry , Braun Breton Catherine
Published: March 2003

A better understanding of the causative agent's biology and the definition of new targets for the development of drugs and/or specific immune responses is necessary to face the spred of drug‐resistant malaria in developing countries and the absence of an efficient vaccine against this most important infectious disease. Non‐detergent sulphobetaines enhance the recovery and isoelectric focussing of active Plasmodium falciparum proteases, cytoskeleton‐associated proteins and Maurer's cleft‐associated proteins. This is a significant advantage for the purification of such proteins and might help pinpoint their role for red blood cell rupture and merozoite release.
Molecular cloning of cDNAs and expression of mRNAs encoding α and β subunits of soluble guanylyl cyclase from medaka fish Oryzias latipes
Authors: Mikami Toshiro , Kusakabe Takehiro , Suzuki Norio
Published: March 2003

Two cDNA clones encoding α and β subunits of soluble guanylyl cyclase (GC) were isolated from medaka fish (Oryzias latipes) cDNA libraries and designated as OlGCS‐α1 and OlGCS‐β1, respectively. The ORF of OlGCS‐α1 encodes 678 amino acids and that of OlGCS‐β1 encodes 614 amino acids. Both subunits have a putative catalytic domain conserved with membrane GC and adenylyl cyclases. The amino acid sequences of OlGCS‐β1 and mammalian soluble GC subunits are highly conserved throughout the entire protein, suggesting that amino acid sequences outside the catalytic domain are important for the functioning of the β subunit. On the contrary, amino acid sequences of α subunits outside the catalytic domain are fairly divergent between fish and mammals. Reverse transcription/PCR (RT/PCR) analysis showed that OlGCS‐α1 and OlGCS‐β1 transcripts were abundant in the brain, eye, spleen and testis. RT/PCR analysis demonstrated that both transcripts were present in unfertilized eggs, were reduced immediately after fertilization, then increased again. In addition, our results suggest that expression patterns of OlGCS‐α1 and OlGCS‐β1 genes are different from each other both during development and in adults.
Delayed production of biologically active O‐glycosylated forms of human eotaxin by tumor‐necrosis‐factor‐α‐stimulated dermal fibroblasts
Authors: Noso Norio , Bartels Joachim , Mallet Antony I. , Mochizuki Mitsuru , Christophers Enno , Schröder Jens‐M.
Published: March 2003

Since a number of inflammatory skin diseases are characterized by selective eosinophil infiltration preferentially in the dermis, we speculated that dermal fibroblasts might represent a potential cellular source of eosinophil‐selective attractants. Cultivated dermal fibroblasts treated with tumor necrosis factor α secreted, not before day 3 of stimulation, eosinophil‐specific chemotactic activity. Purification of this activity revealed a heparin‐binding protein with an apparent molecular mass of 13 kDa in SDS/polyacrylamide gel electrophoresis. Peptide mapping with subsequent amino acid sequence analyses revealed it to be human eotaxin. Natural eotaxin preparations contain 50 % N‐terminally truncated forms missing two or three amino acids. It is O‐glycosylated at Thr71, resulting in at least two sialylated O‐glycosylated variants. Electrospray ionization mass spectrometric analyses revealed the natural eotaxin preparation to be heterogeneous with principal masses of 9033 Da and 9317 Da.

Natural eotaxin stimulated eosinophil chemotaxis with identical potency and efficacy as recombinant human eotaxin. Neither neutrophils, monocytes or lymphocytes responded towards natural eotaxin preparations indicating that N‐terminal truncation and O‐glycosylation did not affect the cell‐specificity of chemotactic activity.

Treatment of eosinophils with natural eotaxin desensitizes chemotactic responses towards eotaxin, regulated an normal T‐lymphocyte expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP‐3), whereas RANTES and MCP‐3 were unable to desensitize natural eotaxin‐dependent responses.
The C‐terminal helix of human apolipoprotein AII promotes the fusion of unilamellar liposomes and displaces apolipoprotein AI from high‐density lipoproteins
Authors: Lambert Gilles , Decout Anne , Vanloo Berlinda , Rouy Didier , Duverger Nicolas , Kalopissis Athina , Vandekerckhove Joel , Chambaz Jean , Brasseur Robert , Rosseneu Maryvonne
Published: March 2003

To assess the functional properties of apolipoprotein (apo) AII and to investigate the mechanism leading to the displacement of apo AI from native and reconstituted high‐density lipoproteins (HDL and r‐HDL) by apo AII, wild‐type and variant apo AII peptides were synthesized. The wild‐type peptides, residues 53−70 and 58−70, correspond to the C‐terminal helix of apo AII and are predicted to insert at a tilted angle into a lipid bilayer. We demonstrate that both the apo AII‐(53−70) peptide, and to a lesser extent the apo AII‐(58−70) peptide are able to induce fusion of unilamellar lipid vesicles together with membrane leakage, and to displace apo AI from HDL and r‐HDL. Two variants of the apo AII‐(53−70)‐wild‐type (WT) peptide, designed either to be parallel to the water/lipid interface [apo AII‐(53−70)‐0+] or to retain an oblique orientation [apo AII‐(53−70)‐30+], were synthesized in order to test the influence of the obliquity on their fusogenic properties and ability to displace apo AI from HDL. The parallel variant did not bind lipids, due to its self‐association properties. However, the apo AII‐(53−70)‐30+ variant was fusogenic and promoted the displacement of apo AI from HDL. Moreover, the extent of fusion of the apo AII‐(53−70)‐WT, apo AII‐(58−70)‐WT and apo AII‐(53−70)‐30+ peptides was related to the α‐helical content of the lipid‐bound peptides measured by infrared spectroscopy. Infrared measurements using polarized light also confirmed the oblique orientation of the helical component of the three peptides. In native and r‐HDL, the tilted insertion of the C‐terminal helix of apo AII resulting in a partial destabilization of the HDL external lipid layer might contribute to the displacement of apo AI by apo AII.
Expression of fatty‐acid‐binding proteins in cells involved in lung‐specific lipid metabolism
Authors: Guthmann Florian , Hohoff Carsten , Fechner Henry , Humbert Peter , Börchers Torsten , Spener Friedrich , Rüstow Bernd
Published: March 2003

Members of the fatty‐acid‐binding protein (FABP) family are thought to play an important role in fatty acid transport within the cytosol and thus to be involved in lipid metabolism. As previous data on the occurrence of distinct FABP types in total lung are contradictory, we determined the expression of FABP types in three isolated cell types of rat lung, which are characterised by active lipid metabolism. Alveolar type‐II cells synthesise, store and secrete pulmonary surfactant, a phospholipid‐rich surface‐tension‐lowering agent, whereas lung fibroblasts, localised adjacent to the alveolar type‐II cells, are assumed to provide neutral lipid substrate to alveolar type‐II cells around birth, and alveolar macrophages are known to degrade complex lipids. Initial screening by reverse transcriptase PCR revealed the occurrence of heart (H‐), epidermal (E‐) and liver FABP in rat lung, the latter being not detectable in the three cell types studied. Cells were analysed by northern and western blotting, then quantitatively by sandwich ELISA, for which recombinant rat E‐FABP was prepared. E‐FABP mRNA was found in all three cell types, and E‐FABP was detected in the following amounts : 240.9 ± 19.0 ng/mg cytosolic protein in alveolar type‐II cells; 172.3 ± 0.7 ng/mg protein for lung fibroblasts; and 36.9 ± 3.5 ng/mg protein for alveolar macrophages. This indicates a basic function of E‐FABP in cellular lipid metabolism. In contrast, H‐FABP probably is involved in the metabolism of neutral lipids because H‐FABP mRNA was found only in lung fibroblasts with a corresponding protein level of 315.5 ± 6.9 ng/mg. Small amounts of H‐FABP protein were present in alveolar type‐II cells and alveolar macrophages.
A plant surface protein sharing structural properties with animal integrins
Authors: Faik Ahmed , Labouré Anne Marie , Gulino Danielle , Mandaron Paul , Falconet Denis
Published: March 2003

Using a polyclonal antibody (P23) generated against the human platelet integrin αIIbβ3 and a FITC‐conjugate secondary antibody, fluorescence is observed at the surface of protoplasts isolated from Arabidopsis thaliana and Rubus fruticosus. Arabidopsis thaliana cells grown in suspension culture containing P23 and glycylarginylglycylaspartylserine (GRGDS), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins demonstrated aberrant cell wall/plasma membrane interactions and organization. When glycoproteins from these plants, purified on a concanavalin A Sepharose 4B, were subjected to SDS/PAGE and Western blotting, under reduced and non‐reduced conditions, immunoblots probed with P23 revealed bands in both species. A shift in electrophoretic mobility is observed to different apparent molecular mass when no reducing agent is present. When purified by immunoaffinity chromatography on anti‐αIIbβ3 Sepharose or Sepharose linked to the synthetic peptide
D‐Arg‐Gly‐Asp‐Trp, the major antigenic components detected migrate at 30 kDa and 60 kDa in the first experiment and 60 kDa in the second one. Only the 60‐kDa component is immunodetected with antibodies specific for either the β3 platelet chain or the αIIb polypeptide, suggesting the presence of two polypeptides co‐migrating. To address more precisely the structure of this complex in plants, competition assays were performed. A significant inhibition is observed with CS3 a monoclonal antibody that interacts with the complexed form αIIbβ3 but not the dissociated subunits. Further structural similarities with the animal αIIbβ3 complex is demonstrated with Western blotting detection after plant glycoproteins immunoprecipitation with CS3 in absence or presence of 5 mM EDTA to dissociate the complex. We also present data on the characterization of a polyclonal antibody, named AcAt2, raised against Arabidopsis glycocoproteins purified by affinity chromatography on a
D‐RGDW column and eluted with the same peptide, that specifically interacts with the animal αIIbβ3 receptor.
Dicyclohexylcarbodiimide interaction with the voltage‐dependent anion channel from sarcoplasmic reticulum
Authors: Shafir Iris , Feng Wei , Shoshan‐Barmatz Varda
Published: March 2003

In a recent study [Shoshan‐Barmatz, V., Orr, I., Weil, S., Meyer, H., Varsanyi, M. & Heilmeyer, L. M. G. (1996) FEBS Lett. 386, 205−210] we have demonstrated the presence of the voltage‐dependent anion channel (VDAC) in skeletal muscle sarcoplasmic reticulum (SR) as supported here by co‐localization of VDAC and (Ca2++Mg2+)ATPase in the SR using double‐immunogold labeling. The interaction of the carboxyl‐modifying reagent dicyclohexylcarbodiimide with the SR‐VDAC is characterized by labeling with [14C]dicyclohexylcarbodiimide and by dicyclohexylcarbodiimide modification of the reconstituted‐purified VDAC channel activity. In both SR and mitochondrial membranes, [14C]dicyclohexylcarbodiimide most specifically labeled a 35‐kDa protein, identified as VDAC by specific anti‐VDAC Ig. Labeling of the SR‐VDAC was about twofold higher than that of the mitochondrial VDAC, which could result either form higher labeling of the SR protein or from relatively higher amounts of VDAC/mg total protein in the SR membranes. [14C]Dicyclohexylcarbodiimide labeling of the SR, but not the mitochondrial VDAC, was biphasic with respect to time and concentration of [14C]dicyclohexylcarbodiimide. Partial digestion of [14C]dicyclohexylcarbodiimide‐labeled SR‐VDAC with chymotrypsin yielded five proteolytic fragments which were recognized by the anti‐VDAC Ig, and the dicyclohexylcarbodiimide‐binding site was localized in the 19‐kDa fragment. VDAC was purified from SR and mitochondrial membranes by spermine‐agarose column.

The interaction of dicyclohexylcarbodiimide with functional carboxyl residue(s) in the purified VDAC is demonstrated by recording its channel activity, following its reconstitution into planar lipid bilayer (PLB). Dicyclohexylcarbodiimide inhibited the channel activity in a voltage‐dependent manner, requiring incubation with dicyclohexylcarbodiimide at high (negative or positive) potentials. Dicyclohexylcarbodiimide slowed down the transition from the high‐conducting to a long‐lived low‐conducting states of the channel (approximately 20 % of its maximal conductance), by stabilizing the intermediate states. Similar results were also obtained with purified‐reconstituted mitochondrial VDAC. Hydrophilic carboxyl reagents {[1‐ethyl‐3‐(3‐dimethylamino)propyl] carbodiimide, N‐ethyl‐phenylisoxazolium‐3′‐sulfonate} neither modified the channel activity nor prevented [14C]dicyclohexylcarbodiimide labeling. These results indicate that dicyclohexylcarbodiimide interacts with a carboxyl group located in a hydrophobic region of the protein which is involved in the channel gating.
Kinetic analysis of integrin‐dependent cell adhesion on vitronectin
Authors: Germer Matthias , Kanse Sandip M. , Kirkegaard Tove , Kjøller Lars , Felding‐Habermann Brunhilde , Goodman Simon , Preissner Klaus T.
Published: March 2003

Distinct binding interactions between cell‐surface receptors and extracellular matrix components are characteristic of multifunctional adhesion proteins such as vitronectin. The close proximity of binding sites for αv‐integrins and plasminogen activator inhibitor‐1 (PAI‐1) on vitronectin may have consequences for cell adhesion and migration, or for the localized inhibition of plasminogen activators. In this study, the kinetics and reversibility of vitronectin‐dependent cell adhesion via αv‐integrins was investigated using RGD peptides and PAI‐1 as competitors. Active, but not latent or cleaved PAI‐1, and RGD peptides were effective in preventing cell adhesion to vitronectin provided the inhibitor was present at the time of cell seeding. In a concentration‐dependent manner urokinase or thrombin abrogated the inhibitory effect of PAI‐1. Following cell seeding onto a vitronectin substratum, delayed addition of RGD peptides or active PAI‐1 (10−20 min post‐seeding) resulted in the loss of their inhibitory potential. These data were supported by experiments in a purified system where delayed addition of active PAI‐1 could no longer prevent vitronectin binding to immobilized αvβ3, while a cyclic RGD peptide gave some moderate inhibition. The apparent stabilization of vitronectin−integrin contacts was observed with immobilized native or multimeric vitronectin but not with the more rigid form of denatured, aggregated multimers. These results demonstrate that the cell adhesive properties of vitronectin depend on its conformational flexibility and can be tightly regulated in a spatio‐temporal manner through direct competition of cellular integrins by soluble or matrix‐bound factors such as PAI‐1.
O‐Demethylase from Acetobacterium dehalogenans
Authors: Kaufmann Franz , Wohlfarth Gert , Diekert Gabriele
Published: March 2003

The ether‐cleaving O‐demethylase isolated from syringate‐grown cells of Acetobacterium dehalogenans (formerly named strain MC) consists of four proteins, components A, B, C and D. The enzyme system converts only phenyl methyl ethers with a hydroxyl group in the ortho position to the methoxyl moiety. The presence of a carboxyl group in the aromatic compound was not required for O‐demethylase reaction. Component B mediated the conversion of vanillate to 3,4‐dihydroxybenzoate in the presence of the Ti(III)‐reduced corrinoid‐containing component A. After addition of component D and tetrahydrofolate, methyl tetrahydrofolate was formed from vanillate in stoichiometric amounts. Titanium(III) citrate as a reductant could be replaced by H2, methyl viologen or ferredoxin, partially purified hydrogenase, purified component C obtained from A. dehalogenans, and ATP. From these findings, it was deduced that component B serves as vanillate : corrinoid protein methyltransferase (methyltransferase I) mediating the methyl transfer from vanillate to the reduced corrinoid protein component A. Component D functions as methylcorrinoid protein : tetrahydrofolate transferase (methyltransferase II). The role of component C is probably that of an activating protein reversing accidental oxidation of the protein‐bound cob(I)alamin to cob(II)alamin in the presence of ATP and reducing equivalents supplied by the enzymatic oxidation of hydrogen.
Engineering, expression and biochemical characterization of the hemoglobin domain of a Erwinia chrysanthemi flavohemoprotein
Authors: Labesse Gilles , Craescu Constantin T. , Mispelter Joël , Chottard Geneviève , Marden Michael C. , Pin Serge , Forest Eric , Mornon Jean‐Paul , Boccara Martine
Published: March 2003

An artificial hemoglobin‐like domain has been constructed by engineering the gene coding for the multi‐domain flavohemoprotein from the bacterium Erwinia chrysanthemi. This domain was designed by molecular modelling, cloned and over‐expressed in Escherichia coli. The holo‐protein was obtained in large quantities after extraction from inclusion bodies and refolding in presence of alkaline hemin. The purified 140‐residue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein.

The structural and functional features of this domain in solution were studied using far‐ultraviolet circular dichroism, resonance Raman, proton‐NMR spectroscopy, flash laser photolysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin‐like domain is able to bind oxygen and carbon monoxide with very high affinity. It exhibits a rapid auto‐oxidation which may explain its tight association with a flavin containing reductase domain.

A functional model of this hemoglobin is discussed and compared with the X‐ray structures of other hemoproteins.
Amino acid sequence determination and biological activity of therin, a naturally occuring specific trypsin inhibitor from the leech Theromyzon tessulatum
Authors: Chopin Vincent , Matias Isabel , Stefano George B. , Salzet Michel
Published: March 2003

We purified a trypsin inhibitor, designated therin, from the rhynchobdellid leech Theromyzon tessulatum. Therin was purified to apparent homogeneity by gel‐permeation and anion‐exchange chromatography followed by reverse‐phase HPLC. By a combination of reduction and S‐β‐pyridylethylation, Edman degradation and electrospray mass spectrometry measurement, the complete sequence of therin (48 amino acid residues; m/z, 5376.35 ± 0.22 Da) was determined. Therin exhibits an approximately 30 % sequence similarity with peptides of the antistasin‐type inhibitors family, i.e. the first and second domains of antistasin, hirustasin, ghilanthen and guamerins (I, II). Therin is a tight‐binding inhibitor of trypsin (Ki, 45 ± 12 pM) and has no action towards elastase or cathepsin G. Furthermore, therin (10−6 M) in conjunction with theromin, a Theromyzon thrombin inhibitor (10−6 M) significantly diminish the level of human leucocytes activation induced by lipopolysaccharide (10 μg) in a manner similar to that of aprotinin. These data suggest a leech trypsin inhibitor with possible biomedical signifance.
The porin RafY encoded by the raffinose plasmid pRSD2 of Escherichia coli forms a general diffusion pore and not a carbohydrate‐specific porin
Authors: Andersen Christian , Krones Daniela , Ulmke Christine , Schmid Kurt , Benz Roland
Published: March 2003

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin‐deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion‐permeable channel with a single‐channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high‐molecular‐mass carbohydrates through the outer membrane.
The cotton‐top tamarin carries an extended semenogelin I gene but no semenogelin II gene
Authors: Lundwall Åke
Published: March 2003

Previous studies have shown that the predominant proteins secreted by the seminal vesicles are transglutaminase substrates which have undergone major structural alterations during evolution. In man, they are known as semenogelin I and II; recently it was shown that, similar to man, several new world and old world monkeys carry two semenogelin genes as well, the exception being the cotton‐top tamarin (Saguinus oedipus) with a single gene. This gene has now been cloned and identified as a semenogelin I gene, because of a higher number of conserved nucleotides in the human semenogelin I gene (89 %) than in the human and the rhesus monkey semenogelin II genes (82 %). Furthermore, the difference in sequence similarity indicates that the semenogelin II gene was deleted from the genome of a progenitor to the cotton‐top tamarin after the duplication that yielded the two semenogelin genes seen in man. Like several other genes expressing seminal‐vesicle‐secreted transglutaminase substrates, the cotton‐top tamarin semenogelin I gene consists of three exons of 97, 1816 and 146 bp. It codes for a signal peptide of 23 amino acid residues and the secreted protein of 592 amino acid residues. The molecular mass of 66 kDa is 32 % larger than that of the human counterpart and, contrary to human semenogelin I, the cotton‐top tamarin protein has the potential to be highly glycosylated as there are 14 sites with the consensus sequence for N‐linked glycosylation. Approximately half of the primary structure consists of five nearly identical tandem repeats of 58 amino acid residues, that probably evolved relatively late.
Specific cleavage of amino side chains of serine and threonine in peptides and proteins with S‐ethyltrifluorothioacetate vapor
Authors: Kamo Masaharu , Tsugita Akira
Published: March 2003

A vapor of S‐ethyltrifluorothioacetate was found to specifically cleave the amino side of serine and threonine peptide bonds. The cleavage reactions were carried out at 50 +C for 6 h−24 h or at 30 +C for 24 h. When vapors were generated in a solution containing several conventional organic solvents, the cleavage reactions were reduced or stopped, or modification took place. When the reagent vapor was made in an aqueous solution, the cleavage reaction at glycine residues was enhanced. This reagent did not oxidize any amino acid residues, such as methionine, cysteine and tryptophan. The cleavage was also effective on proteins on membranes blotted or electroblotted from polyacrylamide gels. This method therefore may be used for the peptide mass fingerprinting [Patterson, S. D. (1995) Electrophoresis 16, 1104−1114] after two‐dimensional electrophoresis.
GTP‐binding‐protein‐coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin
Authors: Haga Kazuko , Ogawa Haruo , Haga Tatsuya , Murofushi Hiromu
Published: March 2003

Tubulin was found to bind to a glutathione S‐transferase fusion protein containing the carboxy‐terminal domain of GTP‐binding‐protein‐coupled receptor kinase 2 (GRK2) (residues 467−689), which is known to contain a pleckstrin homology site and to bind GTP‐binding protein βγ subunits. The binding of tubulin to the fusion protein was not affected by GTP‐binding protein βγ subunits, indicating that tubulin and βγ subunits bind GRK2 independently. Western‐blotting analysis with anti‐GRK2 Ig indicated that GRK2 was copurified with tubulin through the polymerization‐depolymerization procedure. Tubulin was phosphorylated by GRK2, in contrast with the facts that the known substrates of GRK2 are restricted to activated forms of GTP‐binding‐protein‐coupled receptors and that tubulin is a poor substrate for most kinases. GRK2 did not phosphorylate microtubule‐associated proteins (MAPs), under conditions where MAPs were well phosphorylated by endogenous kinases copurified with tubulin. The Km for tubulin was estimated to be 3 μM, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by βγ subunits and agonist‐bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2.
Cross‐linked DNA duplexes
Authors: Antsypovich Sergei , Kubareva Elena , Tashlitsky Vadim , Oretskaya Tatiana , Shabarova Zoe
Published: March 2003

Investigation of substrate properties of DNA duplexes with covalently connected strands has been performed. The kinetic measurements of the hydrolysis processes of these compounds by snake venom phosphodiesterase has been carried out. It has been shown that the exonuclease stability of cross‐linked DNA duplexes is greatly increased as compared with analogs without linkages between strands. The interaction of cross‐linked DNA duplexes with the p50 subunit of human nucleic transcription factor of the κ light‐chain enhancer (NF‐κB) has been studied. The presence of linkages between strands did not prevent the binding of protein with DNA duplexes and, in specific cases, even promoted this process. The speculation of using cross‐linked DNA duplexes as efficient decoys for DNA‐binding proteins such as NF‐κB from the point of view of sense technology are discussed.
Stability of Escherichia coli phosphoenolpyruvate carboxykinase against urea‐induced unfolding and ligand effects
Authors: Encinas María Victoria , Evangelio Juan A. , Andreu José Manuel , Goldie Hughes , Cardemil Emilio
Published: March 2003

The urea‐induced unfolding at pH 7.5 of Escherichia coli phosphoenolpyruvate (P‐pyruvate) carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism spectra, and 1‐anilino‐8‐naphthalenesulfonate binding. These studies were performed in the absence and presence of substrates and ligands. ATP or P‐pyruvate plus MnCl2, or of the combined presence of ATP plus MnCl2 and oxalate, conferred great protection against urea‐induced denaturation. The unfolding process showed the presence of at least one stable intermediate which is notably shifted to higher urea concentrations in the presence of substrates. This intermediate protein structure was inactive, contained less tertiary structure than the native protein and retained most of the original secondary structure. Hydrophobic surfaces were more exposed in the intermediate than in the native or unfolded species. Refolding experiments indicated that the secondary structure was completely recovered. Total recovery of tertiary structure and activity was obtained only from samples denatured at urea concentrations lower than those where the intermediate accumulates.
Analysis of protein self‐association at constant concentration by fluorescence‐energy transfer
Authors: Hassiepen Ulrich , Federwisch Matthias , Mülders Thomas , Lenz Volker J. , Gattner Hans‐Gregor , Krüger Peter , Wollmer Axel
Published: March 2003

Fluorescence‐resonance‐energy transfer from subunits labelled with a fluorescence donor group to subunits labelled with a fluorescence acceptor group can be used for quantitative analysis of protein self‐association. The present approach evaluates fluorescence measurements on mixtures of equimolar solutions of donor‐labelled and acceptor‐labelled protein composed by systematic variation of the volume ratio. Its attractive feature is that it allows the determination of equilibrium constants at fixed total concentration. Problems encountered by most other methods, which require the equilibria to be followed to high dilution, are avoided. Conditions to be fulfilled are that a reactive site is available on the protein for specific introduction of the labels and that labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Förster distance of the donor/acceptor pair complies with its separation. While dimerisation constants can be determined exclusively by fluorescence measurements, the analysis of more complex cases of self‐association depends on additional independent information. This communication reports on an application of the approach to the association/dissociation equilibrium between insulin monomers and dimers. Labelling of insulin at the ε‐amino group of LysB29 does not disturb the conformation nor does it affect dimerisation. 2‐Aminobenzoyl and 3‐nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most other fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Förster distance of 29 Å matches their 32‐Å separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor‐insulin and acceptor‐insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K12, of 0.72×105 M−1. Its agreement with values obtained with other methods demonstrates that the present approach is a reliable alternative.
Two metal‐binding sites in a lead ribozyme bound to competitively by Pb2+ and Mg2+
Authors: Katahira Masato , Kim Mi Hee , Sugiyama Takashi , Nishimura Yoshifumi , Uesugi Seiichi
Published: March 2003

We reported recently that a lead ribozyme with modified bases cleaved at an additional site at high Pb2+ concentrations (> 0.1 mM), and that the cleavage at a canonical site was enhanced nearly fourfold at the optimum combination of Pb2+ and Mg2+ concentrations [Kim, M. H., Katahira, M., Sugiyama, T. & Uesugi, S. (1997) J. Biochem. (Tokyo) 122, 1062−1067]. Here we have identified two metal‐binding sites (sites 1 and 2) of the lead ribozyme at the residue level by NMR. Both sites are located in an asymmetric internal loop of the lead ribozyme. Site 1 is composed of residues of an enzyme strand and site 2 of residues of a substrate strand. The two sites are bound to competitively by Pb2+ and Mg2+. It was revealed that at certain Pb2+ and Mg2+ concentrations, site 1 is occupied by Pb2+ and site 2 is occupied by Mg2+. The dependency of the cleavage at the canonical and other sites on the Pb2+ and Mg2+ concentrations is interpreted by considering the species of metal ions bound to the two sites. It is suggested that the addition of the two metal ions produces similar and different effects on the structure of the lead ribozyme, and the two metal ions have a synergistic effect on the structure.
Monitoring the conformational flexibility of cytochrome c at low ionic strength by 1H‐NMR spectroscopy
Authors: Banci Lucia , Bertini Ivano , Reddig Tim , Turano Paola
Published: March 2003

Horse heart cytochrome c at pH 7 and low ionic strength is present as two conformers, as evidenced by 1H‐NMR spectroscopy. The two structures have been calculated using NOE and pseudocontact shift constraints. They have the same folding patterns and are essentially equal, within the rmsd of the families. The two average structures have rmsd values of 0.049 nm and 0.093 nm for the backbone and the heavy atoms, respectively. Such a difference has been analyzed through a detailed analysis of the NOEs. It appears that the species at low ionic strength differs from the species present at high ionic strength by the displacement of some external residues, such as Gln16, Ile81 and Glu90. Other changes are monitored by the chemical shifts but they cannot be quantified at the present level of resolution. Ionic‐strength‐dependent structural rearrangements may be relevant with respect to the problem of molecular recognition.
Analysis of the ternary complex formation of human urokinase with the separated two domains of its receptor
Authors: Oda Masayuki , Shiraishi Akihiro , Hasegawa Mamoru
Published: March 2003

Human urokinase‐type‐plasminogen‐activator receptor (uPAR) is a glycolipid‐anchored membrane glycoprotein comprising three structurally similar domains. We have succeeded in direct observation of the ternary complex formation of single‐chain urokinase (scuPA) or its N‐terminal fragment (ATF) with the separated domain‐1 (N‐terminal domain) and domain‐(2+3) (internal and C‐terminal domain) of human uPAR, by means of gel‐filtration HPLC analysis. This complex was found to consist of the three components in an equimolar ratio (thus referred to as the three‐part complex). To determine the nature of the interaction between these components, cross‐linking experiments involving various kinds of cross‐linkers and competitive binding assay on ELISA were performed. These experiments have shown that each uPAR domain can bind directly to scuPA at low affinity, and that both these domains contribute to the high‐affinity binding between scuPA and uPAR in a synergistic manner. It can be considered that the synergistic effect of domain‐1 and domain‐(2+3) on scuPA binding would result from a conformational change, and that this steric event might trigger the signal transduction reported for scuPA/uPAR binding.
The structure of the melittin tetramer at different temperatures
Authors: Iwadate Mitsuo , Asakura Tetsuo , Williamson Michael P.
Published: March 2003

The structure of the bee venom peptide melittin has been determined in water at pH 6.0, 50 mM sodium phosphate, at various temperatures. At all temperatures studied, the peptide is tetrameric, and consists of two helical regions (residues 2−11 and 13−23) with an unstructured C‐terminal region. The connection between the helices (residues 11−13) is well defined. At 30 +C, the structure of the monomeric unit has been characterised using NOEs, and a family of structures is presented (root‐mean‐square deviation to the mean structure 1.4 Å over the structured residues), with low NOE violations and good stereochemistry. The angle between the helices is 46 ± 13+, and the structure is very similar to the previously determined crystal structure of the aqueous tetramer. The peptide forms a tetramer that is made up from a dimer of dimers.

The structure of the dimeric unit has been determined, using a novel joint refinement of intermonomer NOEs and chemical shifts. The relative position of the monomeric units in the dimer is different from that in the crystal, with less direct contact between monomers. As the temperature is raised to 70 +C, the peptide remains tetrameric, but the monomer units start to separate, as shown by a reduction in intermonomer NOE intensities and chemical shifts. The structural changes have been characterised : over the temperature range studied, the monomers separate by approximately 2.0 Å. This movement may have implications for the mechanism by which melittin inserts into membranes.
Under respiratory growth conditions, Bcl‐x(L) and Bcl‐2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor
Authors: Clow Angela , Greenhalf William , Chaudhuri Bhabatosh
Published: March 2003

We have reported earlier that cytosolic expression of the full‐length human apoptosis inducer Bax‐α (Bax) in the yeast Saccharomyces cerevisiae suppresses growth and induces mortality in cells containing functional mitochondria. Human Bcl‐x(L) overcomes this toxicity. Here we describe that a mutant Bax protein, with a missing membrane anchor region (BaxΔ), also inhibits growth and causes cell death in yeast. However, the death inhibitory proteins Bcl‐x(L) and Bcl‐2 fail to rescue BaxΔ‐mediated growth inhibition under conditions promoting respiration, although they bind BaxΔ in the cell. Results in Jurkat T‐cells corroborate that Bcl‐x(L) is much less efficient at rescuing mammalian cells from the effect of BaxΔ than from full length Bax. We have also inquired if the respiration‐dependent toxicity of Bax and BaxΔ in yeast is nullified by Bcl‐x(L)Δ and Bcl‐2Δ, molecules which lack membrane anchors but bind Bax in the yeast two‐hybrid system. It appears that, under conditions which facilitate respiration in yeast, Bcl‐x(L)Δ and Bcl‐2Δ are incapable of rescuing both Bax‐containing and BaxΔ‐containing cells. Our results open up the interesting possibility that there might exist proteins, unrelated to the Bcl‐2 family, which could negate death induced by a membrane anchor‐free form of Bax.
The solution NMR structure of a blue‐green algae hepatotoxin, microcystin‐RR
Authors: Trogen Gull‐Britt , Edlund Ulf , Larsson Göran , Sethson Ingmar
Published: March 2003

The microcystin‐RR structures are compared with the structures of microcystin‐LR in solution as well as in the crystal structure of the complex with protein phosphatase. The gross structures of the two peptides are similar, but with a more accentuated and compact saddle structure for microcystin‐RR. The structural differences affect the hydrogen‐bond pattern in the peptides and the location of the side chain of N‐methyldehydroalanine, both of which are important for the ability of the peptide to form a tight complex with protein phosphatase. These structural differences may contribute to the observed differences in toxicity of microcystin‐RR and microcystin‐LR.
Identification and characterization of human PMP34, a protein closely related to the peroxisomal integral membrane protein PMP47 of Candida boidinii
Authors: Wylin Tine , Baes Myriam , Brees Chantal , Mannaerts Guy P. , Fransen Mark , Van Veldhoven Paul P.
Published: March 2003

The cDNA of a human protein displaying 33 % identity to the peroxisomal integral membrane protein PMP47 of Candida boidinii (CbPMP47) was cloned. It encodes a protein of 307 amino acids, with a calculated molecular mass of 34 566 Da. The protein, which we name HsPMP34, contains six membrane spans and belongs, like PMP47, to the family of mitochondrial solute carriers. Based on overlapping available expressed sequence tags (ESTs) combined with the sequencing of one EST, the amino acid sequence of the mouse counterpart (molecular mass of 34 422 Da) could also be deduced. By means of reverse‐transcription PCR, the presence of PMP34 mRNA was revealed in liver of man and in liver and other tissues of mouse and rat. The human gene is localized to chromosome 22q13. Given the similarity of HsPMP34 not only to CbPMP47 but also to known or putative mitochondrial transporters, its subcellular localization was investigated. Fluorescence microscopy of HepG2 cells or mouse fibroblasts transfected with a plasmid encoding a HsPMP34/green fluorescent fusion protein revealed a punctate pattern, reminiscent of a peroxisomal distribution. By indirect immunofluorescence, the HsPMP34/green fluorescent fusion protein clearly colocalized with the dimeric peroxisomal thiolase. Upon transfection of mouse fibroblasts lacking functional peroxisomes due the knock‐out of PEX5 [Baes, M., Gressens, P., Baumgart, E., Carmeliet, P., Casteels, M., Fransen, M., Evrard, P., Fahimi, D., Declercq, P. E., Collen, D., Van Veldhoven, P. P. & Mannaerts, G. P. (1997) Nat. Genet. 17, 49−53], fluorescence was associated with larger and less‐abundant structures. Taken together, the data indicate that HsPMP34 is a peroxisomal membrane protein and is either the human counterpart of CbPMP47 or closely related to it. According to its structure, the protein is most probably involved in transport.
Does an asparaginyl‐specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings ?
Authors: Senyuk Vitalyi , Rotari Vitalyi , Becker Claudia , Zakharov Alexander , Horstmann Christian , Müntz Klaus , Vaintraub Iosif
Published: March 2003

An asparaginyl‐specific cysteine endopeptidase which was named ’legumain‐like proteinase' (LLP) and has an apparent molecular mass of 38.1 kDa was isolated from cotyledons of kidney bean (Phaseolus vulgaris L.) seedlings and partially characterized. It is, to our knowledge, the first known proteinase which in vitro extensively degrades native phaseolin, the major storage globulin of this grain legume.

Phaseolin that in vitro had been partially degraded by LLP (Pvitro) and phaseolin that was isolated after partial in vivo breakdown 6 days after the start of seed imbibition (Pvivo) showed similar fragment patterns on SDS/polyacrylamide gels. The fragments had identical cleavage sites in Pvitro and Pvivo as determined by partial amino acid sequencing. In both types of partially degraded phaseolin, these cleavage sites have asparagine in the P1 position. Two of the cleavage sites are located in the β‐barrel domain of the C‐terminal module and only one cleavage site was found in the β‐barrel domain of the N‐terminal module according to the consensus structural model of phaseolin subunits. These results suggest that very likely LLP could in vivo be responsible for the initiation of phaseolin proteolysis.

Two different legumain‐specific clones named cp6b and p21b were isolated from a cDNA library of germinated bean cotyledons. Cp6b encodes LLP, while p21b encodes a VPE‐like enzyme. Southern‐blot analysis revealed a single gene copy for Pv‐VPE and, presumably, at least two gene copies for LLP in the kidney bean genome. Northern‐blot analysis indicated that mRNAs for both clones appear de novo during seed germination. However, the developmental patterns of the transcript levels corresponding to the two clones differed significantly. The temporal pattern of phaseolin degradation and of LLP polypeptide levels agreed well with the suggestion that LLP plays a key role in the mobilization of phaseolin during and after kidney bean germination.
Genes of succinyl‐CoA ligase from Saccharomyces cerevisiae
Authors: Przybyla‐Zawislak Beata , Dennis Richard A. , Zakharkin Stanislav O. , McCammon Mark T.
Published: March 2003

Succinyl‐CoA ligase (succinyl‐CoA synthetase) catalyzes the nucleotide‐dependent conversion of succinyl‐CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone‐body breakdown in animals. The enzyme is composed of α and β subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl‐CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl‐CoA ligase α subunit, and this band was absent in Δlsc1 deletion strains. Succinyl‐CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl‐CoA ligase was shown to utilize ATP but not GTP for succinyl‐CoA synthesis.