bluuebunny/crossref_metadata_2025_split huggingface dataset:
p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1
Authors: Dufour Emmanuelle , Dirani‐Diab Rim El , Boulmé Florence , Fournier Michel , Nevinsky Georgyi , Tarrago‐Litvak Laura , Litvak Simon , Andreola Marie‐Line 
Published: March 2003

Human immunodeficiency virus type‐1 (HIV‐1) reverse transcriptase (RT) initiates reverse transcription from tRNALys3. HIV‐1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNALys3 was studied. Two recombinant forms of HIV‐1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani‐Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago‐Litvak, L. (1992) FEBS Lett. 301, 23−28]. Here we show that HIV‐1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNALys3 to the primer‐binding site of the HIV‐1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non‐nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form.Part of this work was devoted to the study of the complex between the recombinant forms of HIV‐1 RT and its primer tRNA. Each enzymatic form was cross‐linked to tRNALys3 in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 tRNA, the label was mainly found to migrate with the p66 subunit, although some cross‐linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNALys3. Moreover, cross‐linking of tRNALys3 with HIV‐1 RT p66/p51 in the presence of a DNA template containing the primer‐binding‐site sequence yielded an enzymatically active complex.
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.
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.
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.
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.
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.
The structure of the carbohydrate backbone of the core‐lipid A region of the lipopolysaccharide from a clinical isolate of Yersinia enterocolitica O:9
Authors: Müller‐Loennies Sven , Rund Sabine , Ervelä Elise , Skurnik Mikael , Holst Otto 
Published: March 2003

Yersinia enterocolitica O:9 strain Ruokola/71‐c‐ΦR1‐37‐R possesses mainly rough‐type lipopolysaccaride (LPS) and smaller amounts of S‐form LPS. Structural analysis of the former is reported here. After deacylation of the LPS, the phosphorylated carbohydrate backbone of the inner core‐lipid A region could be isolated by using high‐performance anion‐exchange chromatography. Its structure was determined by means of compositional and methylation analyses and 1H‐, 13C‐, and 31P‐NMR spectroscopy as:in which l‐α‐d‐Hep is lglycero‐α‐dmanno‐heptopyranose, d‐α‐d‐Hep is dglycero‐α‐dmanno‐heptopyranose, and Kdo is 3‐deoxy‐dmanno‐oct‐2‐ulopyranosonic acid. All hexoses are pyranoses.
Aspartyl proteases in Caenorhabditis elegans
Authors: Geier Gebhard , Banaj Hans‐Jörg , Heid Hans , Bini Luca , Pallini Vitaliano , Zwilling Robert 
Published: March 2003

Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin. We have purified enzymes responsible for this proteolytic activity by a single‐step affinity chromatography on pepstatin–agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N‐terminal microsequencing. On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N‐termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active‐site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.
An anti‐idiotypic antibody with an internal image of human interferon‐γ and human interferon‐γ‐like antiviral activity
Authors: Depraetere Hilde , Depla Erik , Haelewyn Joost , De Ley Marc 
Published: March 2003

D9D10, a monoclonal antibody that inhibits the biological activity of human interferon‐γ (IFN‐γ), was used to generate monoclonal anti‐idiotypic antibodies. After a first selection, the monoclonal anti‐idiotypic antibody AA1E5 was chosen to be fully characterized. To the best of our knowledge this is the first description of a monoclonal antibody with an IFN‐γ‐like antiviral activity; AA1E5 competed with IFN‐γ for binding to D9D10 indicating its anti‐idiotypic character. However, AA1E5 also fully mimics HuIFN‐γ as it not only binds to the HuIFN‐γ‐receptor, where it competes with HuIFN‐γ, but more importantly AA1E5 and its Fv fragment, cloned and expressed in Escherichia coli, mimic the antiviral activity of HuIFN‐γ. Indeed, 15 µg of AA1E5 and 2.5 µg of its Fv fragment had an effect comparable to that of 10 IU of HuIFN‐γ in an antiviral assay on A549 cells. Sequence comparison between the complementarity determination regions of the antibody and the sequence of HuIFN‐γ revealed that both the heavy chain variable domain, VH , and the kappa light chain variable domain, Vκ , have epitopes of 3–4 amino acids that are present in the HuIFN‐γ sequence, some of which contribute to receptor binding, as identified by Walter et al. [M. R. Walter, W. T. Windsor, T. L. Nagabhushan, D. J. Lundell, C. A. Lunn, P. J. Zauodny & S. K. Narula (1995) Nature376, 230–235].
F1 and F0 connections in the bovine mitochondrial ATP synthase
Authors: Xu Ting , Zanotti Franco , Gaballo Antonio , Raho Gabriella , Papa Sergio 
Published: March 2003

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross‐linking of oligomycin‐sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated.The results obtained show that the α subunit N‐terminus is essential for the correct, functional connection of F1 to F0. The α‐subunit N‐terminus contacts OSCP which, in turn, contacts the F0I‐PVP(b) and the F0‐d subunits. The N‐terminus of subunit α, OSCP, a segment of subunit d and the C‐terminal and central region of F0I‐PVP(b) subunits are peripherally located with respect to subunits γ and δ which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N‐terminus of subunit α, OSCP, subunit d and F0I‐PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the γ subunit uncovered, surround the γ and the δ subunits located in the central stalk.
Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana
Authors: Papenbrock Jutta , Schmidt Ahlert 
Published: March 2003

Sulfurtransferases transfer a sulfane atom from a donor substrate to a thiophilic acceptor molecule. Recently a sulfurtransferase specific for the substrate 3‐mercaptopyruvate was isolated from Arabidopsis thaliana[Papenbrock, J. & Schmidt, A. (2000) Eur. J. Biochem. 267, 145–154]. In this study a second sulfurtransferase from Arabidopsis was characterized and compared to the enzyme described previously. Sequences of the mature proteins had an identity of 77.7%. The plant sulfurtransferases formed a distinct group within the known eukaryotic sulfurtransferases. When Southern blots were hybridized with labelled cDNA fragments from each of the plant sulfurtransferases the same pattern of bands was obtained indicating the existence of only these two closely related sulfurtransferases. The new sulfurtransferase was expressed in Escherichia coli fused with an N‐terminal His6‐tag, purified and tested for enzyme activity. Like the first enzyme, the newly isolated protein preferred 3‐mercaptopyruvate to thiosulfate as substrate. The Km of both enzymes determined for 3‐mercaptopyruvate and cyanide were almost identical. As a result of database searches it became obvious that sulfurtransferase proteins from higher plants showed high similarities to small senescence‐ and stress‐induced proteins. To prove the involvement of sulfurtransferases in senescence‐associated processes 3‐mercaptopyruvate sulfurtransferase activity was determined in crude protein extracts from Arabidopsis plants of different ages. 3‐mercaptopyruvate sulfurtransferase activity and steady‐state RNA levels of sulfurtransferases increased with increasing age. However, steady‐state protein levels as measured by using an antibody against the sulfurtransferase protein expressed previously decreased. Putative roles of sulfurtransferases in senescence‐associated processes are discussed.
Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28
Authors: Stohwasser Ralf , Salzmann Ulrike , Giesebrecht Jan , Kloetzel Peter‐Michael , Holzhütter Hermann‐Georg 
Published: March 2003

The activation kinetics of constitutive and IFNγ‐stimulated 20S proteasomes obtained with homomeric (recPA28α, recPA28β) and heteromeric (recPA28αβ) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling.The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28α/RecP28β/RecP28α+ RecP28β) exhibit biphasic characteristics which can be attributed to a low‐level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28αβ complex is about two orders of magnitude higher than towards the homomeric PA28α and PA28β complexes.Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin‐like, trypsin‐like and peptidylglutamyl‐peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.
Phospholipases A2 from Callosellasma rhodostoma venom gland
Authors: Tsai Inn‐Ho , Wang Ying‐Ming , Au Lo‐Chun , Ko Tzu‐Ping , Chen Yi‐Hsuan , Chu Yi‐Fang 
Published: March 2003

Callosellasma rhodostoma (Malayan pitviper) is a monotypic Asian pitviper of medical importance. Three acidic phospholipases A2 (PLA2s) and one basic PLA2‐homolog were purified from its venom while 10 cDNAs encoding distinct PLA2s were cloned from venom glands of a Thailand specimen of this species. Complete amino‐acid sequences of the purified PLA2s were successfully deduced from their cDNA sequences. Among the six un‐translated PLA2 cDNAs, two apparently result from recombination of its Lys49‐PLA2 gene with its Asp49‐PLA2 genes. The acidic PLA2s inhibit platelet‐aggregation, while the noncatalytic PLA2‐homolog induces local edema. This basic PLA2‐homolog contains both Asp49 and other, unusual substitutions unique for the venom Lys49‐PLA2 subtype (e.g. Leu5, Trp6, Asn28 and Arg34). Three‐dimensional modelling of the basic protein revealed a heparin‐binding region, and an abnormal calcium‐binding pocket, which may explain its low catalytic activity. Oxidation of up to six of its Met residues or coinjection with heparin reduced its edema‐inducing activity but methylation of its active site His48 did not. The distinct Arg/Lys‐rich and Met‐rich region at positions 10–36 of the PLA2 homolog presumably are involved in its heparin‐binding and the cell membrane‐interference leading to edema and myotoxicity.