bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncoupled changes in the expression of the jun family members during myeloid cell differentiation
Authors: MOLLINEDO Faustino , NARANJO Jose R.
Published: March 2005

The differentiation into macrophages of the U937 and HL60 human cell lines induced by 4β‐phorbol 12‐myristate 13‐acetate (PMA) was accompanied by induction of the expression of the proto‐oncogenes c‐jun, jun B and jun D. However, expression of the three jun genes was regulated differently during induction of cell differentiation in both U937 and HL60 cells, with the three jun family members being expressed distinctly at different stages of cell differentiation. Whereas jun B transcription was strongly stimulated following treatment with PMA for 30 min, jun D mRNA levels were only increased 6 h after PMA treatment and the content of c‐jun mRNA was elevated maximally only 24 h after PMA treatment. The rapid induction of the jun B mRNA level suggests a putative role for this proto‐oncogene in the early triggering step of U937 and HL60 cell differentiation induced by PMA. Interestingly, a weak induction of jun B and jun D mRNA levels, but no induction of the c‐jun mRNA level, was detected during Me2SO‐induced granulocytic HL60 differentiation. These data suggest a different role for each jun proto‐oncogene in regulating gene activity and that different transcriptional complexes involving distinct jun proto‐oncogenes can be formed during macrophage and granulocytic differentiation.
A novel mechanism of Sarcophaga lectin gene expression
Authors: SUGIYAMA Hiromi , NATORI Shunji
Published: March 2005

The Sarcophaga lectin gene, an acute‐phase‐responsive protein of Sarcophaga, was found to be activated when the fat body was incubated in phosphate‐buffered insect saline (10 mM NaH2PO4/Na2HPO4, pH 6.2, 2 mM NaHCO3, 1 mM MgCl2, 5 mM KCl, 1 mM CaCl2 and 120 mM NaCl). This activation was selectively suppressed by addition of 2‐mercaptoethanol. 2‐Mercaptoethanol did not affect the transcription directly, suggesting that the oxidation of the sulfhydryl group of a fat‐body protein is required for activation of the Sarcophaga lectin gene. This oxidation reaction seemed to occur immediately when the fat body was incubated in vitro, and once this reaction was initiated, 2‐mercaptoethanol no longer inhibited transcription of the Sarcophaga lectin gene. This gene is known to be activated when the larval‐body wall of Sarcophaga is injured. It is suggested that the Sarcophaga lectin gene is activated via this novel mechanism on its acute‐phase expression in vivo.
Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver microsomes
Authors: MAURICE Manuelle , EMILIANI Stephane , DALET‐BELUCHE Isabelle , DERANCOURT Jean , LANGE Reinhard
Published: March 2005

Antibodies raised against cytochrome P450, which is overexpressed in mouse hepatic tumors, (P450tu) crossreact with two human liver microsomal proteins (49 kDa and 52 kDa). We have quantified these proteins in 60 human liver samples and found great interindividual variability in both of them. The concentration of the 49‐kDa protein varies up to 144 fold in the various samples and represents typically 10% of the total mincrosomal P450 content. Its immunologically determined concentration correlates well (R= 0.78) with the microsomal coumarin‐7‐hydroylase (COH) activity. This activity is strongly and completely inhibited by anti‐P450tu antibody (IC50= 0.13 mg IgG/mg microsomal protein). The crossreacting 49‐kDa protein shows an unusually high substrate specificity towards coumarin; it presents all human COH and part of 7‐ethoxycoumarin O‐deethylase (ECOD). Besides these two activities, we did not find any activity with other typical P450 substrates. In primary cultures of human hepatocytes, it is inducible by phenobarbital and dexamethasone, but not by pyrazole and β‐naphthoflavone. We isolated this protein from human liver microsomes and purified it to homogeneity by a combination of aminooctyl‐amino‐Sepharose chromatography and immunoaffinity chromatography. The protein was identified as a cytochrome P450 of the IIA subfamily. Its N‐terminal amino‐acid sequence was identical with the first 20 residues deduced from the nucleotide sequence of P450IIA6.
Conformational and epitope mapping of herpes‐simplex‐virus type‐1 thymidine kinase using synthetic peptide segments
Authors: ZIMMERMANN Norbert , BECK‐SICKINGER Annette G. , FOLKERS Gerd , KRICKL Sabine , MÜLLER Ingrid , JUNG Günther
Published: March 2005

Adjacent peptide segments covering the complete sequence of herpes‐simplex‐virus type‐1 thymidine kinase (HSV1‐TK) of 376 amino acids were synthesized in order to experimentally verify the three‐dimensional structure of the HSV1‐TK active site, which was previously determined by molecular modeling. 26 peptides have been prepared by multiple solid‐phase synthesis using the 9‐fluorenylmethoxycarbonyl strategy. The purified peptides were linked covalently to bovine serum albumin. The peptide/ELISA of the synthesized bovine‐serum‐albumin conjugates using polyclonal rabbit anti‐(HSV1‐TK)serum resulted in ten epitopes, which correlate excellently with the computer‐proposed active site of HSV1‐TK. CD spectra of the HSV1‐TK peptides were recorded in trifluoroethanol/water (9:1 by vol.). An eigenvalue method based on CD spectra of 15 well known protein structures was used to calculate the relative percentage of secondary structures from the CD data. The computer model of the HSV1‐TK showed full conformity with the folding pattern determined by CD of the synthetic peptide segments. Therefore, conformational peptide mapping with CD‐based secondary structures combined with epitope mapping from the peptide/ELISA is an efficient and reliable method to support computer‐aided protein design.
Monoclonal antibodies against core and cellulose‐binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I
Authors: AHO Sirpa , OLKKONEN Vesa , JALAVA Taina , PALOHEIMO Marja , BÜHLER Rolf , NIKU‐PAAVOLA Marja‐Leena , BAMFORD Dennis H. , KORHOLA Matti
Published: March 2005

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose‐binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western‐blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross‐reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.
A second pathway of activation of the Torpedo acetylcholine receptor channel
Authors: OKONJO Kehinde O. , KUHLMANN Jürgen , MAELICKE Alfred
Published: March 2005

We have studied the interaction of the reversible acetylcholine esterase inhibitor (—)physostigimine (d‐eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligandinduced ion flux into nAChR‐rich membrane vesicles and of equilibrium binding. We find that (—)physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as d‐tubocurarine, α‐bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FK1 and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 μM range, there exists a further class of low‐affinity (Kd˜ mM) sites from which eserine acts as a direct blocker of the acetylcholine‐activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel.
Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021‐7
Authors: MASOUD Hussein , WEINTRAUB Susan T. , WANG Rong , COTTER Robert , HOLT Stanley C.
Published: March 2005

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021‐7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR‐type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric‐acid‐positive material (3‐deoxy‐d‐manno‐octulosonic acid equivalents) and four neutral sugars: glucose, galactose, d‐glycero‐d‐manno‐heptose and l‐glycero‐d‐manno‐heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3‐hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6‐glucosamine disaccharide esterified at C4′ by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non‐reducing glucosamine were both acylated by 3‐myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3‐hydroxymyristic acid. Hydroxyl groups at C4 and C6′ in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine‐sensitized mice, comparable to that of Salmonella.
Expression and characterization of protein kinase C‐δ
Authors: OLIVIER Andrée R. , PARKER Peter J.
Published: March 2005

A cDNA encoding protein kinase C‐δ (PKC‐δ) was isolated from a rat brain library. The coding region was subcloned into the expression vector pmt2 and transfected into COS‐1 cells. Expression of the protein led to an 11‐fold increase in activity as determined with a synthetic peptide based on the PKC‐δ pseudosubstrate site. The Mr of PKC‐δ as determined by SDS/PAGE and immunoblot analysis using anti‐(PKC‐δ C‐terminal) antibodies was 77000. The enzyme was purified to near homogeneity and showed total dependency on phospholipid and diacylglycerol (or phorbol esters) for activity. Like PKC‐ɛ, PKC‐δ displays no Ca2+ dependence for activation. The substrate specificity of PCK‐δ is similar to that of PKC‐ɛ but quite different from other PKCs.
The plasma membrane H+ ‐ATPase from yeast
Authors: WACH Achim , GRÄBER Peter
Published: March 2005

The H+ ‐ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated.

The pH dependence of the rate of ATP hydrolysis forms a bell‐shaped curve with a maximum at pH 6 and half‐maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding.

Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.
The 5′ untranslated regions of acetyl‐coenzyme A carboxylase mRNA provide specific translational control in vitro
Authors: LÓPEZ‐CASILLAS Fernando , KIM Ki‐Han
Published: March 2005

Acetyl‐coenzyme A carboxylase (ACC) catalyzes the rate‐limiting step in the biosynthesis of long‐chain fatty acids. Transcription of the single‐copy ACC gene from two independent promoters, together with the differential splicing of the transcripts, gives rise to mature ACC mRNA having the same open reading frame (ORF), but exhibiting heterogeneity in their 5′ untranslated region (5′‐UTR). Class 1 ACC mRNA are transcribed from the inducible promoter 1 and their 5′‐end leading sequences are provided by exon 1. Class 2 ACC mRNA are transcribed from the constitutively expressing promoter 2 and their leading sequences are derived from exon 2.

In order to understand the role of different 5′ UTR of ACC mRNA we have synthesized in vitro transcripts with defined ACC mRNA 5′ UTR and examined their relative translational efficiencies in rabbit reticulocyte lysates. The major translation product of both forms of ACC mRNA was initiated at the first AUG of the ORF. Class 1 transcripts had a 6–9‐fold better translational efficiency than class 2 transcripts, based on the quantity of major peptide produced by a given amount of transcript. The poor translational efficiency of class 2 transcripts can be improved by the removal of sequences contributed by exon 2, suggesting that they play an inhibitory role in the translation of class 2 types of ACC mRNA. In addition to their higher translational efficiency, the class 1 transcripts can also initiate translation at in‐frame non‐AUG codons, located in exon 1, i.e. upstream to the starting AUG of the common ACC mRNA ORF. This results in novel ACC peptides with extended N termini. These observations are consistent with the hypothesis that the 5′ UTR heterogeneity in the ACC mRNA may be involved in post‐transcriptional control, at the level of translation, of the ACC gene expression.
Structural analysis of the entire proopiomelanocortin gene ofXenopus laevis
Authors: DEEN Peter M. T. , TERWEL Dick , BUSSEMAKERS Marion J. M. , ROUBOS Eric W. , MARTENS Gerard J. M.
Published: March 2005

In the pars intermedia of the pituitary the prohormone proopiomelanocortin (POMC) is tissue‐specifically processed to, among other peptides, α‐melanotropin (αMSH). In the South African clawed toadXenopus laevisthis hormone mediates the process of background adaptation: release of α‐MSH causes darkening of the animal, while inhibition of α‐MSH release results in a pale toad. Elevated release of α‐MSH coincides with a higher rate of POMC gene transcription. The present study aims to find possible transcriptional regulatory elements in theXenopusPOMC gene. For that purpose the complete nucleotide sequence of the POMC gene and its 5′‐ and 3′‐flanking regions were determined and analyzed. TheXenopusPOMC gene promoter contains several regions which may be regulatory DNA elements in view of their similarity with corresponding regions of mammalian POMC gene promoters. In the rat POMC gene promoter, many of these regions represent protein‐binding sequences. Besides the promoter sequence and the protein‐coding sequences, no other segments with significant identity between theXenopusand human POMC genes were found. Intron A of theXenopusPOMC gene contains a simple sequence, (TATC)76, and a JH12 repetitive element, while the 3′‐flanking region contains a repetitive‐EcoRI‐monomer‐2 element. Comparison of the JH12 sequence of the POMC gene with JH12 sequences from otherXenopusgenes revealed a 335‐bp consensus sequence which is flanked by a 30‐bp inverted repeat. This JH12 consensus sequence is significantly larger than the previously reported JH12 core region. Alignment of intron B of theXenopusPOMC gene with database sequences revealed a consensus sequence of a novelXenopusrepetitive element of 330 bp flanked by a nearly perfect inverted repeat. indicating that this element may be a transposonlike element.
Molecular symmetry and arrangement of subunits in extracellular hemoglobin from the nematode Ascaris suum
Authors: DARAWSHE Saleh , DANIEL Ezra
Published: March 2005

The arrangement of subunits and molecular symmetry of extracellular hemoglobin from the nematode Ascaris suum, an 11.7S molecule mass 332 kDa and composed of eight identical subunits, was studied.

Dissociation of the molecule at alkaline and acid pH yielded 4.6S and 2.7S components, identified as polypeptide‐chain dimers and monomers, respectively. Cross‐linking with glutardialdehyde followed by SDS/PAGE resulted in a maximum number of eight bands identified in order of decreasing mobility as monomeric and 2–8 cross‐linked‐polypeptide‐chain species. Comparison with values predicted from theory shows that the distribution of protein among the various cross‐linked species, obtained after different extents of exposure to cross‐linker, is consistent with a two‐layered arrangement of subunits involving one type of interaction between subunits from different layers and another between subunits within the same layer. Electron micrographs of the molecule showed two profiles, a square and a rectangle. We propose a model for the molecule which is eight subunits arranged in two layers, stacked in an eclipsed orientation. The proposed model is consistent with the results from sedimentation, cross‐linking and electron microscopy. Taken together, our findings indicate D4 symmetry for Ascaris hemoglobin.
Crystal structure of the Tyr45Trp mutant of ribonuclease T1 in a complex with 2′‐adenylic acid
Authors: KOELLNER Gertraude , GRUNERT Hans‐Peter , LANDT Olfert , SAENGER Wolfram
Published: March 2005

The recombinant Tyr45Trp mutant of Lys25‐ribonuclease T1 was overexpressed and purified from an Escherichia coli strain. The mutant enzyme, which shows reduced activity towards GpA and increased activity towards pGpC, pApC and pUpC compared with wild‐type RNase T1, was co‐crystallized with 2′‐adenylic acid by microdialysis. The space group is P212121 with unit cell dimensions a = 4.932(2), b = 4.661(2), c = 4.092(1) nm. The crystal structure was solved using the coordinates of the isomorphous complex of wild‐type RNase T1 with 2′‐AMP. The refinement was based on Fhkl of 7726 reflexions with Fo≥ 1σ (Fo) in the resolution range of 2.0–0.19 nm and converged with an R factor of 0.179. The adenosine of 2′‐AMP is not bound to the guanosine binding site, as could be expected from the mutation of Tyr45Trp, but is stacked on the Gly74 carbonyl group and the His92 imidazole group which form a subsite for substrate binding, as already observed in the wild‐type 2′‐AMP complex. The point mutation of Tyr45Trp does not perturb the backbone conformation and the Trp‐indole side chain is in a comparable position to the phenolic Tyr45 of the wild‐type enzyme.
Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium‐transport enzyme
Authors: RONZANI Nelly , STEPHAN Lore , HASSELBACH Wilhelm
Published: March 2005

The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium‐transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding.

The vanadate‐free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7–14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure‐binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure‐binding relationship indicates that the description of vanadate binding requires at least a two‐step reaction sequence. The volume increments which predominate at lower pressure values, range from 200–400 ml · mol−1 depending on the composition of the reaction medium containig 5 μM and 20 μM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20–40 ml · mol−1. Vanadate binding is reduced in the presence of 30 μM calcium, and simultaneously both binding volumes are diminished by 100 ml · mol−1 and 20 ml · mol−1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.
In vivo31P‐NMR studies of Desulfovibrio species
Authors: SANTOS Helena , FARELEIRA Paula , PEDREGAL Carmen , LeGALL Jean , XAVIER António V.
Published: March 2005

The phosphorus metabolism of sulfate‐reducing bacteria was, for the first time, probed by in vivo31P NMR. A novel phosphoric anhydride diester compound was detected in Desulfovibrio desulfuricans ATCC 27774 at intracellular concentration up to 5 mM. The compound has been extracted and partially purified by anion‐exchange chromatography and analysed by 31P, 13C and 1H NMR. These studies show that the novel phosphorus‐containing compound is formed by five carbon atoms and is probably cyclic, with a Mr of approximately 300. Various Desulfovibrio strains were examined in vivo for the presence of this phosphorus‐contianing compound. Detectable amounts of the novel metabolite were found in D. desulfuricans ATCC 27774 when grown on lactate/sulfate lactate/thiosulfate or pyruvate/sulfate. The phosphorus‐containing compound was not detected when this strain of D. desulfuricans was grown on lactate/nitrate or pyruvate; neither was it detected in two other strains which, like D. desulfuricans ATCC 27774, have the capability of utilizing nitrate as a terminal electron acceptor.
The influence of transferin binding to L2C guinea pig leukemic lymphocytes on the endocytosis cycle kinetics of its receptor
Authors: SAINTE‐MARIE Josette , VIDAL Michel , BETTE‐BOBILLO Pascale , PHILIPPOT Jean R. , BIENVENÜE Alain
Published: March 2005

The parameters regulating the internalization and recycling of transferrin‐specific receptors were determined in guinea pig leukemic B lymphocytes, in the absence or presence of ligand. We show that after the cells were purified, 45–56% of the total receptors were on the cell surface. In the absence of transferin, unoccupied receptors are quickly internalized (rate constant, 0.12 min−1) whereas their recycling is much slower (rate constant, 0.026 min−1). This difference between endocytosis and recycling rates leads to a balanced receptor distribution with only 22% of the total receptors outside after incubation of the cells for 20–30 min at 37°C. The internalization rate of occupied receptors, measured in the presence of transferrin is faster (rate constant, 0.21 min−1) than that of unoccupied receptors calculated in the absence of transferrin (0.12 min−1; see above). On the other hand, mere binding of transferrin to its receptor, without internalization, arrested by cytoplasm acidification, is sufficient to induce a large increase (by a factor of seven) in the recycling rate of unoccupied internal receptors from 0.026 min−1 to 0.17 min−1. Thus, in these lymphocytes, transferrin mobilizes internal receptors by modifying the kinetic rates of internalization and recycling, leading to a new equilibrium between external and internal receptors.
Purification and properties of a high‐molecular‐mass complex between Val‐tRNA synthetase and the heavy form of elongation factor 1 from mammalian cells
Authors: MOTORIN Yury A. , WOLFSON Alexey D. , LÖHR Dirk , ORLOVSKY Alexander F. , GLADILIN Kirill L.
Published: March 2005

In extracts of various mammalian tissues obtained in the presence of protease inhibitors Val‐tRNA synthetase exists exclusively as a complex with a molecular mass of about 800 kDa. This complex was purified by gel filtration and two HPLC steps and contained five different polypeptides with molecular masses of 140, 50, 50, 40 and 30 kDa. The complex seems to have no tissue or species specificity, because preparations with identical polypeptide composition were obtained by the same method from rabbit liver and reticulocytes, and rat and beef liver. Four low‐molecular‐mass polypeptides were identified by two‐dimensional electrophoresis as subunits of the heavy form of elongation factor 1 (EF‐1H). The complex possesses the activity of EF‐1 in the poly(U)‐directed translation system, indicating that EF‐1H is an integral part of the complex. Gel filtration of the tissue extracts reveals three different peaks of EF‐1 activity, corresponding to EF‐1α, EF‐1H and the high‐molecular‐mass complex of Val‐tRNA synthetase and EF‐1H. All activity of Val‐tRNA synthetase and about 25% of EF‐1 activity are associated with the complex. Different forms of EF‐1 revealed no significant differences in the nucleotide‐binding properties, but the complex of Val‐tRNA synthetase with EF‐1H was 10 times more active in the poly(U)‐directed binding of Phe‐tRNA phe to ribosomes than EF‐1H. These result strongly suggest that the complex of Val‐tRNA synthetase with EF‐1H is a novel functionally active individual form of EF‐1.
Conformational change of the haemolymph juvenile‐hormone‐binding protein from Galleria mellonella (L)
Authors: WIECZOREK Elźbieta , KOCHMAN Marian
Published: March 2005

A juvenile‐hormone‐binding protein (juvenile‐hormone carrier), isolated fromGalleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile‐hormone‐binding activity of this protein. With SDS/PAGE it was shown that juvenile‐hormone‐binding protein, a 32‐kDa peptide, is first slowly converted into a 30‐kDa molecule, then into two or three smaller‐molecular‐mass species (20–25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30‐kDa peptide has a 2.4‐times‐higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20–25‐kDa peptides. The rate of proteolysis of juvenile‐hormone‐binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non‐denaturing conditions, free binding‐protein molecules could be separated from juvenile‐hormone–binding‐protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross‐linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is conclude that juvenile‐hormone binding induces a conformational transition of its carrier protein. This hormone‐induced change might have a physiological significance for signal transmission.
Pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase
Authors: MARAS Bruno , DOMINICI Paola , BARRA Donatella , BOSSA Francesco , VOLTATTORNI C. Borri
Published: March 2005

The complete amino acid sequence of pig kidney 3,4‐dihydroxyphenylalanine (Dopa) decarboxylase is reported. The sequence was determined from analysis of peptides isolated after tryptic and cyanogen bromide cleavages of the enzyme. Each subunit is made up of 485 residues, corresponding to a molecular mass of 53858 Da. The N‐terminus of the polypeptide chain is an acetylated methionyl residue. A number of structural features, previously shown to be important for the structure and function of the enzyme, could be localized along the polypeptide chain. Comparison of the primary structure with the known cDNA‐deduced sequences of other Dopa decarboxylases (i.e. the human, bovine, rat, guinea‐pig andDrosophila enzymes) reveals 50% identity. The identity increases to 73%, if the comparison is restricted to the mammalian sequences. Comparison with other aromatic and non‐aromatic decarboxylases allows some consideration to be made in terms of structure/function and evolutionary relationships in this class of enzymes.
Expression of mammalian tyrosine aminotransferase in Saccharomyces cerevisiae and Escherichia coli
Authors: DIETRICH Jean Bernard , LORBER Bernard , KERN Daniel
Published: March 2005

Rat liver tyrosine aminotransferase has been expressed inSaccharomyces cerevisiae andEscherichia coli. In yeast, the extent of production is 20‐fold higher than that in rat liver after induction by dexamethasone, and reaches 250‐fold higher in anE. coli strain carrying the T7 RNA polymerase transcription system. About 250 mg pure and homogeneous enzyme was obtained from 50 g transformedE. coli cells. Determination ofMr and pI, as well as analysis of N‐ and C‐terminal amino acids, suggest that the isolated protein is native. The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post‐translational modifications in the mammalian cells are not essential for activity. Pyridoxal 5′‐phosphate strongly protects the enzyme against thermal inactivation. After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5′ ‐dithiobis(2‐nitrobenzoic acid) whereas only five or six are accessible under native conditions. Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5′‐phosphate partially protects them in holoenzyme. The results are interpreted in the light of the structure/function relationship in this enzyme.
Effects of ions on vanadate‐induced photocleavage of myosin subfragment 1
Authors: MUHLRAD Andras , PEYSER Y. Michael , RINGEL Israel
Published: March 2005

Myosin subfragment 1 (S1) is cleaved by near‐ultraviolet irradiation in the presence of vanadate at three sites located at 23, 31 and 74 kDa from the N‐terminus. Since vanadate is considered to be a good structural analogue of phosphate, it is assumed that the cleavage sites participate in forming the phosphate‐binding site(s) of S1. In this work, the effect of various ions on the vanadate‐induced photocleavage of S1 was studied. Monovalent anions were found to inhibit photocleavage in the 50–200 mM range. The inhibition is more expressed at a site 74 kDa from the N‐terminus than at the 23‐kDa and 31‐kDa sites. The inhibitory effect of the monovalent anions increases in the order acetate = F− < CI− < Br− < I−= SCN−. The order of the inhibitory effect is identical to the protein‐structure‐damaging effect of monovalent anions in the von Hippel series [von Hipel, P. H. & Wong, K. Y.(1964) Science 145, 577–581]. Therefore, it is assumed that decreased photocleavage is due to local perturbations of structure, especially at the 74‐kDa site, in addition to increased ionic strength. Divalent anions, sulfate and thiosulfate, strongly inhibit photocleavage at 2 mM. The inhibition is very pronounced at the 23‐kDa and 31‐kDa sites, while the 74‐kDa site is hardly affected. Since photocleavage at the 23‐kDa and 31‐kDa sites is regulated jointly and independently from cleavage at the 74‐kDa site, it is assumed that S1 has two distinct phosphate‐binding sites: the regions of the 23‐kDa and 31‐kDa cleavage sites, which are proximal to each other in the spatial structure, participate in forming the first phosphate‐binding site, while the 74‐kDa site is part of the second binding site. Sulfate was also found to inhibit the trapping of vanadate and to facilitate its release from the S1‐MgADP‐Vi (Vi, inorganic vanadate) complex. Photocleavage of S1 takes place at all three sites, both in the presence or absence of divalent cations, indicating that these, including Mg2+, are not essential for cleavage.
Neutral endopeptidase activity in the interaction ofN‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine with human polymorphonuclear leukocytes
Authors: YULI Itzhak , LELKES Peter I.
Published: March 2005

Human polymorphonuclear leukocytes (PMN) hydrolyze the synthetic chemoattractantN‐formyl‐l‐methionly‐l‐leucyl‐l‐phenylalanine (fMet‐Leu‐Phe) at nanomolar concentrations in an autocatalytic‐like manner that deviates from classical Michaelis‐Menten kinetics[Yuli, I. & Snyderman, R. (1986) J. Biol. Chem. 261, 4902–4908]. By using inhibitors of distinct classes of endoproteases, this particular fMet‐Leu‐Phe degradation was attributed exclusively to an exoplasmic metalloendoprotease that matches the ubiquitous neutral endopeptidase (NEP). Membrane‐bound NEP hydrolyzes non‐chemotactic substrates according to a classic Michaelis‐Menten mechanism. By competitive inhibition with non‐chemotactic substrates, fMet‐Leu‐Phe was found to interact with membrane NEP through a single active site, in a non‐cooperative mode with an apparentKm in the order of 1 mM. The discrepancy between the ordinary hydrolysis of the micromolar and millimolar concentrations of fMet‐Leu‐Phe, reported by others, and the particular degradation of the nanomolar fMet‐Leu‐Phe, could not be accounted for by any coherent correlation between NEP activity/inhibition and modulation of fMet‐Leu‐Phe binding to its receptor, and/or induction of fMet‐Leu‐Phe‐receptor‐mediated inflammatory responses. Based on these and previously reported results, a novel model is proposed in which the fMet‐Leu‐Phe‐induced inflammatory stimulation of PMN involves both NEP and the fMet‐Leu‐Phe receptor. By this model, NEP and the fMet‐Leu‐Phe receptor are distinct membrane entities which can form dynamic binary and tertiary complexes; thus accounting for the unusual kinetic features of fMet‐Leu‐Phe degradation, as well as the two receptor states. The complex of NEP and the fMet‐Leu‐Phe receptor might be conceived as a chemotactic‐perception mechanism that combines the high affinity of the receptor and the rapid turnover of NEP.
Identification of a novel UDP‐Gal:GalNAcβ1‐4GlcNAc‐R
Authors: MULDERB Hans , SCHACHTER Harry , DE JONG‐BRINK Marijke , VAN DER VEN Jos G. M. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain galactosyltrans ferase activity capable of transferring Gal from UDP‐Gal in β1‐3 linkage to terminal GalNAc of GalNAcβ1‐4GlcNAc‐R [R =β1‐2Manα1‐O(OH2)8 COOMe, β1‐OMe, or α,β1‐OH]. Using GalNAcβ1‐4GlcNAcβ‐2Manα‐1‐O(CH2)8 COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X‐100, and no activation effect was found. The enzyme was active with GalNAcβ1‐4GlcNAcβ1‐2Manα1‐O(CH2)8 COOMe (Vmax 140 nmol · h−1· mg protein−1; Km 1.02 mM), GalNAcβ‐4GlcNAc (Vmax 105 nmol · h−1· mg protein−1; km 0.99 mM), and GalNAcβ‐4GlcNAcβ1‐OMe (Vmax 108 nmol · h−1· mg protein−1; Km1.33 mM). The products formed from GalNAcβ1‐4GlcNAcβ1‐2Manα1‐O(CH2)8COOMe and GalNAcβ1‐4GlcNAcβ1‐OMe were purified by high performance liquid chromatography, and identified by 500‐MHz1H‐NMR spectroscopy to be Galβ1‐3GalNAcβ1‐4GlcNAcβ1‐2Manα1‐O(CH2)8COOMe and Galβ‐3GalNAcβ1‐4GlcNAcβ1‐OMe, respectively. The enzyme was inactive was inactive towards GlcNAc, GalNAcβ1‐3Galα‐OMe, GalNAcα1‐OC6H5, GalNAcα1 – ovine‐submaxillary‐mucin, lactose andN‐acetyllactosamine. This novel UDPGal:GalNAcβ1‐4GlcNAc‐R β1‐3‐galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.
Isolation, purification and partial characterization of a 30‐kDa chlorophyll‐a/b‐binding protein from spinach
Authors: IRRGANG Klaus‐D. , RENGER Gernot , VATER Joachim
Published: March 2005

A 30‐kDa chlorophyll‐a/b‐binding protein was purified from photosystem II membrane fragments using Ca2+‐chelating Sepharose 6B chromatography. The protein binds approximately four chlorophyllamolecules, one chlorophyllbmolecule and carotenoids. Its 77‐K fluorescence‐emission spectrum exhibits a maximum at 680−+1 nm. The protein has a high tendency to form dimer in the presence of Ca2+. Ca2+binding affects the low‐temperature fluorescence‐emission maximum, leading to a decrease in its intensity and a blue shift of 1 nm. Similar spectral changes were obtained in the presence of Mg2+, possibly indicating a common binding domain for both cations. We interpret these observations as cation‐induced conformational changes of the protein, which were reversible upon subsequent incubation in EDTA. Evidence is presented for the involvement of carboxyl groups in the coordination sphere of the bivalent cations. The possible structural and functional role of the protein is discussed.
NMR studies of the activation of the Escherichia coli trp repressor
Authors: HYDE Eva I. , RAMESH Vasudevan , FREDERICK Ronnie , ROBERTS Gordon C. K.
Published: March 2005

The Escherichia coli trp repressor binds to the trp operator in the presence of tryptophan, thereby inhibiting tryptophan biosynthesis. Tryptophan analogues lacking the α‐amino group act as inducers of trp operon expression. We have used one‐ and two‐dimensional 1H‐NMR spectroscopy to compare the binding to the repressor of the corepressors l‐tryptophan, d‐tryptocphan and 5‐methyl‐dl‐tryptophan with that of the inducer indole‐3‐propionic aicd. We have determined the chemical shifts of the indole ring protons of the ligands when bound to the protein, principally by magnetization‐transfer experiments. The chemical shifts of the indole NH and C4 protons differ between corepressors and inducer. At the same time, the pattern of intermolecular NOE between protons of the protein and those of the ligand also differ between the two classes of ligand. These two lines of evidence indicate that corepressors and inducers bind differently in the binding site, and the evidence suggests that the orientation of the indole ring in the binding site differs by approximatley 180° between the two kinds of ligand. This is in contrast to a previous solution study [Lane, A. N. (1986) Eur. J. Biochem. 157, 405–413], but consistent with recent X‐ray crystallorgraphic work [Lawson, C. L. & Sigler, P. B. (1988) Nature 333, 869–871]. d‐Tryptohan and 5‐methyltryptophan, which are more effective corepressors than l‐tryptophan, bind similarly to l‐tryptophan. The indole ring of d‐tryptphan appears to bind in essentially the same orientation as that of the l isomer. There are, however, some differences in chemical shifts and NOE for 5‐methyltryptophan, which indicate that there are significant differences between the two corepressors l‐tryptophan and 5‐methyltryptophan in the orientation of the indole ring within the binding site.
Purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius
Authors: MOLL Ralf , SCHÄFER Günter
Published: March 2005

A succinate dehydrogenase complex was isolated in a three‐step purification from plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It consists of four subunits; a, 66 kDa; b, 31 kDa; c, 28 kDa and d, 12.8 kDa. In the 141‐kDa native protein, the four subunits are present in an equimolar stoichiometry. The complex contains acid‐non‐extractable flavin, iron and acid‐labile sulphide. Maximal succinate dehydrogenase activities were recorded at pH 6.5, which coincides with the internal pH of Sulfolobus cells. The temperature optimum of 81°C defines the Sulfolobus succinate dehydrogenase as a thermophilic enzyme complex. The Km for succinate was found to be 1.42 mM (55°C). Similar to the mitochondrial soluble succinate dehydrogenase, this enzyme is capable of transferring electrons to artifical electron acceptors, for instance phenazine methosulfate, N,N,N′,N′‐tetramethyl‐p‐phenylenediamine and ferricyanide. In contrast to the mitochondrial succinate dehydrogenase, the archaebacterial enzyme reduces 1,4‐dichloroindophenol also in the absence of phenazine methosulfate. Calderiella quinone, the physiological electron mediator in the Sulfolobus respiratory chain, was only slowly reduced under adjusted conditions. The succinate–phenazine methosulfate‐(1,4‐dichloroindophenol) oxidoreductase of the isolated complex was strongly inhibited by tetrachlorobenzoquinone. In plasma membranes the complex reduces molecular oxygen in a cyanide‐sensitive reaction. Polyclonal Sulfolobus anti‐a antibodies crossreacted with 66–67‐kDa polypeptides from membranes of Thermoplasma acidophilium, Sulfolobus solfataricus and beef heart submitochondrial particles.
Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments
Authors: WOLBRING Gregor , COOK Neil J.
Published: March 2005

A rapid FPLC procedure for the purification of protein kinase C from bovine rod outer segments is described. The enzyme is essentially homogenous after purification and exhibits a molecular mass of approximately 85 kDa, as determined by SDS/PAGE. From its chromatographic behaviour on hydroxyapatite, and from Western‐blotting experiments using isoenzyme‐specific antibodies, we were able to identify the bovine rod outer segment protein kinase C as being of the α or type‐III form. The purified protein kinase C has a specific activity of 1066 nmol 32P · min−1· mg protein−1, and shows a 30‐fold activation upon the addition of the effectors Ca2+, PtdSer and 1,2‐diacylglycerol. Arachidonic acid and linoleic acid were also found to enhance significantly the activity of the purified enzyme.
Analysis of the complex transcription termination region of the Escherichia coli rrn B gene
Authors: OROSZ András , BOROS Imre , VENETIANER Pál
Published: March 2005

The complex terminator region of the Escherichia coli rrnB gene was analyzed by subcloning the terminators T1 and T2 and the inverted repeats IR1 and IR2 individually, or in various combinations, in a normal or inverted orientation into a terminator probe vector. The in vivo terminating efficiency was assayed by measuring the galacokinase activity encoded by the downstream galK gene. Termination efficiencies of all fragments were compared in two construcs, differing in the presence or absence of readthrough translation over the investigated terminator signal. The following main conclusions were drawn. (a) T1 and T2 are both efficient terminators in isolated forms. (b) IR1 and IR2 have some terminating effect (much lower than the proper terminators), especially in the inverted orientation. Their presence modifies the effect of the proper terminators in a quite unpredictable way, especially if these regions are translated. (c) The terminators are not symmetrical; in the inverted orientation T1 is practically inactive and T2 termination is reduced. (d) Translation radically decreases the efficiency of the terminators. (e) Several sequences in the rrnB gene, upstreams of the terminator region (one in the 16S RNA and one in the 5S RNA coding region), are very efficient in vivo terminators in the inverted orientation.
Purification and characterization of two highly different group II phospholipase A2 isozymes from a single viperid (Eristocophis macmahoni) venom
Authors: SIDDIQI Abdur Rehman , ZAIDI Zafar H. , JÖRNVALL Hans
Published: March 2005

Two phospholipase A2 isozymes have been purified from leaf‐nosed viper by gel permeation chromatography followed by reverse‐phase HPLC and cation‐exchange FPLC. Both enzymes contain seven pairs of half‐cystine, typical of group II phospholipase A2. Surprisingly large difference, affecting both N‐ and C‐terminal regions, exist between the two isozymes purified from the same snake venom. Exchanges occur at no less than 27 of 121 positions (22%), suggesting the possible existence of two genes for phospholipase A2. The residue identity with the enzymes from other Viperidae species is also low, only 44–48%, indicating extensive of this protein structure at large. Functionally, the present isozymes do not possess the cationic regions ascribed to myotoxicity and anti‐coagulant effects of the enzyme.
Assignment of the 600‐MHz 1H‐NMR spectrum of amicyanin from Thiobacillus versutus by two‐dimensional NMR methods provides information on secondary structure
Authors: LOMMEN Arjen , WIJMENGA Sybren , HILBERS Cees W. , CANTERS Gerard W.
Published: March 2005

The nearly complete assignment (pH6.8; T310 K) of the 1H‐NMR spectrum of reduced amicyanin from Thiobacillus versutus is reported. Experimental evidence is presented, that the structure of the amicyanin contains two β‐sheets, a feature common to plastocyanins and azurins. The loops joining the β‐strands have also been identified. The loop F‐G (Thr94–Phe98), together with the flanking residues Cys93 and Met99, comprises three of the four copper ligandes and is short compared to similar loops in plastocyanin and azurin. His96 turns out to be the copper ligand that can be protonated. Amicyanin resembles plastocyanin in overall structure but differs from it on account of an N‐terminal strand of 22 amino acids in front of strand A, shorter loops A‐B, D‐E and F‐G and the absence of any α‐helical segments.
The molecular action of tumor necrosis factor‐α
Authors: CAMUSSI Giovanni , ALBANO Emanuele , TETTA Ciro , BUSSOLINO Federico
Published: March 2005

Tumor necrosis factor‐α (TNF‐α) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin‐1 and, in an autocrine manner, TNF itself. The net biological effect of TNF‐α may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF‐α may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF‐α are triggered by the binding to specific cell surface receptors. The formation of TNF‐α‐receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine‐nucleotide‐binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF‐α. The full understanding of the molecular mechanisms of TNF‐α will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.