bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipophosphoglycan of Leishmania donovani inhibits lipid vesicle fusion induced by the N‐terminal extremity of viral fusogenic Simian immunodeficiency virus protein
Authors: Martin Isabelle , Turco Salvatore J. , Epand Richard M. , Ruysschaert Jean‐Marie
Published: March 2003

Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania parasites, was recently shown to be a potent inhibitor of viral infection. The mechanism by which this natural membrane amphiphile compound inhibits membrane fusion was investigated in this study using a simple model membrane system and a synthetic peptide corresponding to the fusion peptide of simian immunodeficiency virus (SIV). At low concentration (< 10 μM), LPG inhibits SIV‐induced lipid mixing of large unilamellar vesicles composed of an equimolar mixture of egg phosphatidylcholine and egg phosphatidylethanolamine. Importantly, this inhibition was observed regardless of which LPG was inserted in the inner monolayer, the outer monolayer or both sides of the membrane, suggesting that the inner monolayer plays a determining role in membrane fusion. Fourier transform infrared spectroscopy revealed that LPG induced a conformational change of SIV fusion peptide without affecting its capacity to interact with the lipid membrane. This structural change was shown not to depend on the LPG localization and was observed even when LPG was exclusively associated to the inner lipid membrane.
Involvement of NF‐Y in transcriptional regulation of the phospholamban gene
Authors: Yabuki Masanori , Toyofuku Toshihiko , Otsu Kinya , Nishida Masashi , Kuzuya Tsunehiko , Hori Masatsugu , Tada Michihiko
Published: March 2003

To understand the transcriptional regulation of the phospholamban gene, we analyzed a 5′‐upstream region of the gene. Using a series of deletion constructs, we demonstrated that the region from −96 bp to −78 bp, containing the CCAAT sequence, is essential for transcription of this gene. This region specifically bound to nuclear proteins extracted from rat hearts, and gel‐shift assays using competitive oligonucleotides, antibodies and recombinant proteins showed that this region binds to the NF‐YA and NF‐YB, members of the CCAAT‐binding nuclear protein family. This region‐dependent transcription in cardiac myocytes transfected with antisense cDNAs encoding NF‐YA and NF‐YB was decreased to approximately 50 % of that seen in cells transfected with the same sense cDNAs. We, therefore, conclude that the region from −96 bp to −78 bp plays a critical role in expression of the phospholamban gene, which is regulated by binding of the nuclear protein NF‐Y.
Structural determinants in 5S RNA and TFIIIA for 7S RNP formation
Authors: Theunissen Oliver , Rudt Falko , Pieler Tomas
Published: March 2003

C2H2‐type zinc‐finger modules define a unique structural motif, which is capable of forming specific complexes with both DNA and RNA. While the principles governing DNA binding have been defined in great detail, the mode of RNA recognition remains only poorly understood. In the absence of information from three‐dimensional structural analysis of a zinc‐finger/RNA complex, we have performed a number of biochemical studies to gain further insight into the molecular details of the interaction of 5S ribosomal RNA with the zinc‐finger protein TFIIIA. Previous work had indicated that zinc finger 6 of TFIIIA contacts 5S RNA in close proximity or directly in the loop‐A region (nucleotides 10−13). Permutation analysis of this sequence reveals that three of the four nucleotides are of vital importance for RNA recognition. Exchange of unusual and therefore characteristic aromatic residues in finger 6 against aliphatic or other aromatic amino acids reveals that the aromatic character of tryptophan 177 is essential for RNA recognition. Association with helix V in 5S RNA appears to involve specific contacts with the phosphate backbone, as evidenced by ethylation‐interference assays. Introduction of multiple internal and 3′‐terminal as well as 5′‐terminal deletions accompanied by stabilizing sequence substitutions defines a minimal RNA fragment that is sufficient for TFIIIA binding. This RNA molecule includes a truncated/mutated helix I, helix II and helix V, as well as structurally intact loops A and E. Permutation analysis of the loop‐E region emphasizes its importance for TFIIIA recognition.
Multiple specificities of the staphylococcal and streptococcal fibronectin‐binding microbial surface components recognizing adhesive matrix molecules
Authors: Joh Danny , Speziale Pietro , Gurusiddappa Sivashankarappa , Manor Jeanette , Höök Magnus
Published: March 2003

Many pathogenic Gram‐positive bacteria express fibronectin (Fn)‐binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), most of which have a similar structural organization with a primary ligand‐binding domain consisting of 3−6 repeats of 40−50 amino‐acid‐residue motifs. The MSCRAMMs appear to preferentially bind to the N‐terminal region of Fn, which is composed of five type‐I modules. Here we report that the Fn‐binding MSCRAMM FnbpA of Staphylococcus aureus contains a second ligand‐binding domain located outside the repeat units. In addition, several sites in the Fn N‐terminus presented as recombinant type‐I module pairs bind to the repeat domain of the MSCRAMM. All of the MSCRAMMs analyzed, which include FnbpA of Staphylococcus aureus, Sfb of Streptococcus pyogenes, and FnbA and FnbB of Streptococcus dysgalactiae, were shown to bind to multiple sites in the N‐terminal domain of Fn. By dissecting the repeat domain of FnbpA using synthetic peptides and recombinant fragments, we show that discrete, different motifs are responsible for the binding to individual sites in Fn, rather than a common motif being able to bind to several pairs of type‐I Fn modules. The C‐terminal half of many of the MSCRAMM repeat units contain a common motif, which is shown here to bind to the type‐I module pair 4 and 5. In addition, some of the repeat units of FnbpA contain N‐terminal motifs which bound to the type‐I module pairs 1−2 and 2−3, respectively. These latter binding motifs appear to be partly overlapping and dependent on flanking sequences. Fluorescence polarization experiments using fluorescein‐labeled MSCRAMM peptides and recombinant type‐I Fn module pairs revealed dissociation constants of 1−13 μM. It was also shown that the fluorescein‐labeled peptides differed in their primary binding sites on Fn.
A new exon in the 5′ untranslated region of the connexin32 gene
Authors: Duga Stefano , Asselta Rosanna , Del Giacco Luca , Malcovati Massimo , Ronchi Severino , Tenchini Maria L. , Simonic Tatjana
Published: March 2003

The cloning and sequencing of two bovine connexin32 cDNAs are reported. Comparative analysis with known corresponding mammalian cDNA and protein sequences, besides confirming a high degree of similarity among these proteins, allowed us to identify some specific features of the bovine connexin32 gene. The latter include: the presence of a novel exon in the 5′ UTR which is alternatively spliced, giving rise to a new mRNA species; the presence of two potential hairpin loops in the 5′ and 3′ UTR; and the presence of an additional amino acid, glycine235, in the C‐terminal domain of the 284 residue protein. Among the common features, the presence of polypyrimidine clusters within the 3′ UTR, containing a consensus sequence for a cis‐acting element, is noteworthy. Expression of connexin32 mRNAs was analysed in 16 bovine tissues. Transcript analysis suggests the presence, in cattle, of an alternative downstream promoter.
Depletion of Escherichia coli 4.5S RNA leads to an increase in the amount of protein elongation factor EF‐G associated with ribosomes
Authors: Nakamura Kouji , Fujii Yasuyuki , Shibata Toshinori , Yamane Kunio
Published: March 2003

In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF‐G). To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis. Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF‐G–GDP from the ribosome. On the other hand, depletion of 4.5S RNA led to the retention of a significant amount of EF‐G on 70S ribosomes. In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF‐G‐binding site at positions 1068–1077 on 23S RNA. We therefore examined by gel mobility‐shift assay whether or not mutations in the domain‐IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF‐G to 23S RNA. Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity. Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF‐G with the ribosomes. Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten‐base sequence conserved between the two RNAs.
Aggregation of hsp70 and hsc70 in vivo is distinct and  temperature‐dependent and their chaperone function is  directly related to non‐aggregated forms
Authors: Angelidis Charalampos E. , Lazaridis Ioannis , Pagoulatos Gerassimos N.
Published: March 2003

We used non‐denaturing gradient analysis of cell extracts before and after heat treatment of the cells and showed that hsp70 and hsc70 aggregate in vivo in a temperature‐dependent fashion. Their aggregation profiles were found to be clearly distinguishable and sensitive to ATP depletion. Pore exclusion limit electrophoresis showed that these two proteins are mainly found in autoaggregated forms including dimers, trimers and oligomers. The addition of denatured luciferase to the cell extracts reversed the aggregation of both proteins towards their non‐aggregated forms. Immunoprecipitation and Western‐blot analysis showed that the non‐aggregated form is the only one bound to denatured luciferase. Our results suggest that aggregated hsp70 and hsc70 represent predominantly self‐associated molecules unable to exert chaperone activity. The cochaperone hsp40 was also found to be aggregated and, on addition of denatured luciferase, its aggregation was reversed to a non‐aggregated state. Immunoprecipitation analysis indicated that hsp40 forms a complex with the non‐aggregated form of hsc70 and denatured luciferase. These results confirm previous in vitro studies and support the suggestion that in vivo cytosolic hsp70 and hsc70 exist mainly in an oligomer–monomer equilibrium which is dependent on the environmental temperature, the levels of ATP and the presence of denatured proteins.
Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed inEscherichia coli
Authors: Seto Nina O. L. , Compston Catherine A. , Evans Stephen V. , Bundle David R. , Narang Saran A. , Palcic Monica M.
Published: March 2003

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H‐precursor structure Fucα(1–2)Galβ‐OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed inEscherichia coli. The purified hybrid glycosyltransferases were characterized by two‐substrate enzyme kinetic analysis using both UDP‐GalNAc and UDP‐Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP‐GlcNAc and UDP‐Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.
The mitochondrial adenine nucleotide translocator from  Dictyostelium discoideum
Authors: Bof Mireille , Brandolin Gérard , Satre Michel , Klein Gérard
Published: March 2003

The mitochondrial adenine nucleotide translocator (ANT) catalyses the exchange of ATP and ADP between the mitochondria and the cytosol. We have cloned and sequenced the gene encoding the Dictyostelium discoideum ANT (DdANT) and analysed its transcriptional regulation. The single copy D. discoideum ant gene encodes a protein of 309 amino acid residues with a predicted molecular mass of 33 469 Da and a pI of 9.85. These values are comparable to those of ANTs from mammals, insects and fungi. The long N‐terminal extension characteristic of plant ANT is absent in DdANT. The protein coding region of the D. discoideum ant gene is interrupted by three introns. Polyclonal antibodies directed against the beef heart mitochondrial ANT or its C‐terminal peptide recognized the D. discoideum protein. Northern blot analysis revealed that the expression of the D. discoideum ant gene decreased rapidly during the first hours of multicellular development but the amount of protein remained stable throughout differentiation.
Chemotaxin‐dependent translocation of immunoreactive ADP‐ribosyltransferase‐1 to the surface of human neutrophil polymorphs
Authors: Kefalas Panagiotis , Saxty Barbara , Yadollahi‐Farsani Masoud , MacDermot John
Published: March 2003

mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine‐specific ADP‐ribosyltransferase (ART1). A single 2.6‐kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872‐bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription–PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S‐transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl‐Met‐Leu‐Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half‐maximum response was 28.6 n
m. Similar responses were observed after exposure of the cells to platelet‐activating factor or interleukin‐8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane.
Pregnenolone esterification in Saccharomyces cerevisiae
Authors: Cauet Gilles , Degryse Eric , Ledoux Catherine , Spagnoli Roberto , Achstetter Tilman
Published: March 2003

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl‐CoA to the 3β‐hydroxyl group of pregnenolone. The corresponding enzyme, acetyl‐CoA:pregnenolone acetyltransferase (APAT) is specific for Δ5‐ or Δ4‐3β‐hydroxysteroids and short‐chain acyl‐CoAs. The apparent Km for pregnenolone is ≈0.5 µm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N‐terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62‐kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf2‐Δ mutation is introduced in a yeast strain devoid of the ATP‐binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3β‐hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.
Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris
Authors: Huecas Sonia , Villalba Mayte , Gonza´lez Eva , Martínez‐Ruiz Antonio , Rodríguez Rosalía
Published: March 2003

The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiaeα‐mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion‐exchange and size‐exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1‐specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high‐yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.
An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state
Authors: Casey Tammy M. , Dufall Kitiya G. , Arthur Peter G.
Published: March 2003

An improved method for the measurement of tissue metabolites associated with cellular energetic state by capillary electrophoresis is described. This method allows 17 compounds present in a mixture of standards to be determined simultaneously within 43 min with good reproducibility. ATP, ADP, AMP, UTP, IMP, inosine, hypoxanthine, creatine, phosphocreatine, UDP‐galactose, NAD and NADH were detected in samples of either rat heart tissue or rat neonatal cardiomyocytes. This method can detect compounds at concentrations of 5 µm in samples. Recoveries for ATP and phosphocreatine added to cardiomyocyte samples were 99.4 ± 2.1% and 103.1 ± 3.3%, respectively (mean ± SEM, n = 3). Our method has been comprehensively validated and is capable of measuring a wider range of tissue metabolites important in assessing cellular energy status than existing methods.
Interactions of fluorescent triacylglycerol analogs covalently bound to the active site of a lipase from Rhizopus oryzae
Authors: Zandonella Gerhild , Stadler Peter , Haalck Lutz , Spener Fritz , Paltauf Fritz , Hermetter Albin
Published: March 2003

Fluorescent triacylglycerol analogs were synthesized as covalent inhibitors of lipase activity. The respective 1(3),2‐O‐dialkylglycero‐3(1)‐alkyl‐phosphonic acid p‐nitrophenyl esters contain a fluorescent pyrenealkyl chain and a long‐chain alkyl residue bound to the sn‐2 and sn‐1(3) positions of glycerol, respectively. The phosphonic acid p‐nitrophenyl ester bond is susceptible to nucleophilic substitution by the active serine residue in the catalytic triad of a lipase, leading to inactivation of the enzyme. The fluorescent dialkylglycerophosphonates contain two chiral centers, the sn‐2 carbon of glycerol and the phosphorus atom. The (1‐O‐hexadecyl‐2‐O‐pyrenedecyl‐sn‐glycero)‐O‐(p‐nitrophenyl)‐n‐hexyl‐phosphonate, first peak during HPLC separation and the (3‐O‐hexadecyl‐2‐O‐pyrenedecyl‐sn‐glycero)‐O‐(p‐nitrophenyl)‐n‐hexyl‐phosphonate, second peak during HPLC separation were found to be potent lipase inhibitors. After incubation of an equimolar amount of these isomers with lipase from Rhizopus oryzae complete inactivation was observed. Stable conjugates containing a 1 : 1 molar ratio of lipid to protein were formed. The spatial proximity of the fluorescently labeled sn‐2 alkyl chain of the inhibitor and tryptophan residues of the lipase was assessed by fluorescence resonance energy transfer. The extent of tryptophan fluorescence quenching and the concomitant increase in pyrene fluorescence upon excitation of lipase tryptophans was found to be similar for the above‐mentioned isomers. Thus, the (labeled) sn‐2 alkyl chains of a triacylglycerol analog are likely to interact with the same binding site of the R. oryzae lipase, irrespective of their steric configuration. However, it was shown that the extent of resonance energy transfer is strongly influenced by the reaction medium, indicating conformational changes of the lipase in different environments.
Alteration of α‐spectrin ubiquitination due to age‐dependent changes in the erythrocyte membrane
Authors: Corsi Dario , Paiardini Mirko , Crinelli Rita , Bucchini Anahi , Magnani Mauro
Published: March 2003

Mammalian red blood cell α‐spectrin is ubiquitinated in vitro and in vivo[Corsi, D., Galluzzi, L., Crinelli, R., Magnani, M. (1995) J. Biol. Chem. 270, 8928–8935]. This process shows a cell age‐dependent decrease, with senescent red blood cells having approximately one third of the amount of ubiquitinated α‐spectrin found in young cells. In‐vitro ubiquitination of α‐spectrin was dependent on the source of the red cell membranes (those from older cells are less susceptible to ubiquitination than those from younger cells), on the source of ubiquitin‐conjugating enzymes (those from older cells catalyze the process at a reduced rate compared to those from younger cells) and on the ubiquitin isopeptidase activity (which decreases during red cell ageing). However, once α‐spectrin has been extracted from the membranes of young or old red blood cells, it is susceptible to ubiquitination to a similar extent regardless of source. This suggests that it is the membrane architecture, and not spectrin itself, that is responsible for the age‐dependent decline in ubiquitination. Furthermore, spectrin oligomers, tetramers and dimers are also equally susceptible to ubiquitination. As spectrin ubiquitination occurs on domains αIII and αV of α‐spectrin, and domain αV contains the nucleation site for the association of the α‐ and β‐spectrin chains, alterations in ubiquitination during red cell ageing could affect the stability and deformability of the erythrocyte membrane.
Transcription factors and their genes in higher plants
Authors: Liu Liansen , White Michael J. , MacRae Thomas H.
Published: March 2003

A typical plant transcription factor contains, with few exceptions, a DNA‐binding region, an oligomerization site, a transcription‐regulation domain, and a nuclear localization signal. Most transcription factors exhibit only one type of DNA‐binding and oligomerization domain, occasionally in multiple copies, but some contain two distinct types. DNA‐binding regions are normally adjacent to or overlap with oligomerization sites, and their combined tertiary structure determines critical aspects of transcription factor activity. Pairs of nuclear localization signals exist in several transcription factors, and basic amino acid residues play essential roles in their function, a property also true for DNA‐binding domains. Multigene families encode transcription factors, with members either dispersed in the genome or clustered on the same chromosome. Distribution and sequence analyses suggest that transcription factor families evolved via gene duplication, exon capture, translocation, and mutation. The expression of transcription factor genes in plants is regulated at transcriptional and post‐transcriptional levels, while the activity of their protein products is modulated post‐translationally. The purpose of this review is to describe the domain structure of plant transcription factors, and to relate this information to processes that control the synthesis and action of these proteins.
Methanol:coenzyme M methyltransferase from Methanosarcina barkeri ‐‐ substitution of the corrinoid harbouring subunit MtaC by free cob(I)alamin
Authors: Sauer Karin , Thauer Rudolf K.
Published: March 2003

Methyl‐coenzyme M formation from coenzyme M and methanol in Methanosarcina barkeri is catalysed by an enzyme system composed of three polypeptides MtaA, MtaB and MtaC, the latter of which harbours a corrinoid prosthetic group. We report here that MtaC can be substituted by free cob(I)alamin which is methylated with methanol in an MtaB‐catalysed reaction and demethylated with coenzyme M in an MtaA‐catalysed reaction. Methyl transfer from methanol to coenzyme M was found to proceed at a relatively high specific activity at micromolar concentrations of cob(I)alamin. This finding was surprising because the methylation of cob(I)alamin catalysed by MtaB alone and the demethylation of methylcob(III)alamin catalysed by MtaA alone exhibit apparent Km for cob(I)alamin and methylcob(III)alamin of above 1 mm. A possible explanation is that MtaA positively affects the MtaB catalytic efficiency and vice versa by decreasing the apparent Km for their corrinoid substrates. Activation of MtaA by MtaB was methanol‐dependent. In the assay for methanol:coenzyme M methyltransferase activity cob(I)alamin could be substituted by cob(I)inamide which is devoid of the nucleotide loop. Substitution was, however, only possible when the assays were supplemented with imidazole: ≈1 mm imidazole being required for half‐maximal activity. Methylation of cob(I)inamide with methanol was found to be dependent on imidazole but not on the demethylation of methylcob(III)inamide with coenzyme M. The demethylation reaction was even inhibited by imidazole. The structure and catalytic mechanism of the MtaABC complex are compared with the cobalamin‐dependent methionine synthase.
Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament
Authors: Ellouze Christine , Selmane Tassadite , Kim Hye‐Kyung , Tuite Eimer , Nordén Bengt , Mortensen Kell , Takahashi Masayuki
Published: March 2003

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein‐labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small‐angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA–DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA–DNA complex. Higher stability of the RecA‐DNA complex in the presence of adenosine 5′‐O‐3‐thiotriphosphate or guanosine 5′‐O‐3‐thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA–DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.
Structural difference in the H+,K+‐ATPase between the E1 and E2 conformations
Authors: Raussens Vincent , Pézolet M. , Ruysschaert Jean‐Marie , Goormaghtigh Erik
Published: March 2003

Conformational changes taking place in the gastric H+,K+‐ATPase when shifting from the K+‐induced E2 form to the E1 form upon replacing K+ ions by Na+ were investigated by different spectroscopic approaches. No significant secondary‐structure change or secondary‐structure reorientation with respect to the membrane plane could be measured by attenuated total reflection Fourier transform infrared spectroscopy of oriented films. Circular dichroism and Raman spectra obtained on tubulovesicle suspensions indicated no significant secondary structure or tyrosine and tryptophan side‐chain environment changes in tubulovesicle suspensions. The smallest observable structural changes are discussed in term of the number of amino‐acid residues involved for each technique.
Polymorphic forms of human ADP‐ribosyltransferase‐1
Authors: Yadollahi‐Farsani Masoud , Kefalas Panagiotis , Saxty Barbara A. , MacDermot John
Published: March 2003

Full length cDNA encoding ADP‐ribosyltransferase‐1 (ART1) was generated from human skeletal muscle. A single base variation from the published sequence was observed (C770→T), and was established as a polymorphism by the screening of a population of 50 Caucasians. The base variation predicted a nonconservative substitution of Leu for Pro at codon 257. Cell lines with stable and doxycycline‐inducible expression of the two polymorphic forms of ART1 were generated from Chinese hamster V79 cells, and exploited in studies to compare the activities of ART1‐Pro257 and ART1‐Leu257. The results revealed no differences in the capacity of phosphoinositide‐specific phospholipase C to cleave the two ART1 isoforms from the plasma membrane. Furthermore, the capacities of ART1‐Pro257 and ART1‐Leu257 to ADP‐ribosylate agmatine or fibroblast growth factor‐2 were similar. Differences in the catalytic activities of ART1‐Pro257 and ART1‐Leu257 were however, identified when measurements were made of their capacities to ADP‐ribosylate membrane‐associated proteins on the surface of V79 cells. Protein(s) of molecular mass 80–110 kDa were more extensively ADP‐ribosylated by ART1‐Pro257 than ART1‐Leu257, in accordance with the Vmax (59.5 ± 5.5 and 37.0 ± 3.0) and Km values (12.5 ± 4.5 and 5.0 ± 1.9) for ART1‐Pro257 and ART1‐Leu257, respectively.
Mechanism of metabolite transfer in coupled two‐enzyme reactions involving aldolase
Authors: Pettersson Henrik , Pettersson Gösta
Published: March 2003

Transient‐state kinetic experiments and analyses have been performed to examine the validity of hitherto unchallenged evidence proposed to be indicative of a channelled transfer of triose phosphates from aldolase to glyceraldehyde‐3‐phosphate dehydrogenase and glycerol‐3‐phosphate dehydrogenase. The results lend no support to such proposals, but show that the kinetic behaviour of the examined aldolase–dehydrogenase reactions is fully consistent with a free‐diffusion mechanism of metabolite transfer.
Structural requirements of para‐alkylphenols to bind to estrogen receptor
Authors: Tabira Yukiko , Nakai Makoto , Asai Daisuke , Yakabe Yoshikuni , Tahara Yoshiko , Shinmyozu Teruo , Noguchi Masato , Takatsuki Mineo , Shimohigashi Yasuyuki
Published: March 2003

Octyl‐ and nonylphenols in the environment have been proposed to function as estrogens. To gain insight into their structural essentials in binding to the estrogen receptor, a series of phenols with saturated alkyl groups at the para position, HO‐C6H4‐CnH2n+1 (n = 0–12), were examined for their ability to displace [3H]17β‐estradiol in the recombinant human estrogen receptor, which was expressed in Sf9 cells using the vaculovirus expression system. All tested para‐alkylphenols were found to bind fully to the estrogen receptors in a dose‐dependent manner. The interaction of alkylphenols with the receptor became stronger with increase in the number of the alkyl carbons and the activity was maximized with n = 9 of nonylphenol. Phenol (n = 0) exhibited weak but full binding to the receptor, whereas anisole with a protected phenolic hydroxyl group was completely inactive. Also, alkanes such as n‐octane, 2,2,4‐trimethylpentane corresponding to tert‐octane, and n‐nonane exhibited no binding. The results indicate that the binding of para‐alkylphenols to the estrogen receptor is due to the effect of covalent bonding of two constituents of the phenol and alkyl groups, which correspond to the A‐ring and hydrophobic moiety of the steroid structure, respectively. When alkylphenols were examined for their receptor binding conformation by 1H‐NMR measurements and ab initio molecular orbital calculations, it was suggested that nonbranched alkyl groups are in an extended conformation, while branched alkyl groups are in a folded conformation. These results suggest that branched and nonbranched alkyl moieties of alkylphenols interact differently with the lipophilic ligand binding cavity of the estrogen receptor when compared to the binding of 17β‐estradiol.
Interaction of elongation factor eEF‐2 with ribosomal P proteins
Authors: Bargis‐Surgey Patricia , Lavergne Jean‐Pierre , Gonzalo Philippe , Vard Cécile , Filhol‐Cochet Odile , Reboud Jean‐Paul
Published: March 2003

The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF‐2 was studied. Both recombinant proteins were found to be required for the ribosome‐dependent GTPase activity of eEF‐2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF‐2, with a higher affinity for P1 (Kd = 3.8 × 10−8 m) than for P2 (Kd = 2.2 × 10−6 m). Phosphorylation resulted in a moderate increase (two‐ to four‐fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF‐2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu‐C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF‐2. Our results suggest that the two proteins P1 and P2 both interact with eEF‐2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.
Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection
Authors: Flatmark Torgeir , Almås Bjørg , Knappskog Per M. , Berge Sissel V. , Svebak Randi M. , Chehin Rosana , Muga Arturo , Martínez Aurora
Published: March 2003

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)‐lerythro‐5,6,7,8‐tetrahydrobiopterin (H4biopterin) a time‐dependent change (burst) in enzyme activity was observed, with a half‐time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 ± 4 μm). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 ± 2 μm) and the negative cooperativity was amplified (h = 0.27 ± 0.03). The dimeric C‐terminal deletion mutant (Δ473–528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6‐methyl‐5,6,7,8‐tetrahydropterin (h = 0.9 ± 0.1; Km = 62.7 ± 5.7 μm) and 3‐methyl‐5,6,7,8‐tetrahydropterin (H43‐methyl‐pterin)(h = 1.0 ± 0.1; Km = 687 ± 50 μm). In the presence of 1 mm H43‐methyl‐pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real‐time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron‐free apoenzyme results in a saturable positive resonance unit (ΔRU) response with negative cooperativity (h = 0.52–0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo‐and the holo‐hTH1 on binding of H4biopterin and lerythro‐dihydrobiopterin (H2biopterin). Moreover, the ligand‐bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.
Identification of defensins in human lymphocyte nuclei
Authors: Blomqvist Maria , Bergquist Jonas , Westman Ann , Håkansson Kristina , Håkansson Per , Fredman Pam , Ekman Rolf
Published: March 2003

The cell nucleus plays an essential role in all aspects of cell function and regulation. Most of the nuclear proteins/peptides are synthesized in the cytoplasm and transported into the nucleus through the nuclear pore complexes. The nuclear proteins/peptides conjugate with each other and interact in transcriptional activation/inactivation. Several of the high molecular mass transcription factors (> 30 kDa) have been identified and characterized. However, the information on the low molecular mass proteins/peptides of the nucleus is limited. We have investigated these low molecular mass proteins/peptides from the nucleus of human peripheral blood lymphocytes using reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The HPLC fractions were further analysed by matrix assisted laser desorption/ionization time of flight (MALDI‐TOF) mass spectrometry, electrospray ionization time of flight (ESI‐TOF) mass spectrometry and electrospray ionization fourier transform ion cyclotron resonance (ESI‐FTICR) mass spectrometry for mass determination. Using this combination of mass spectrometry techniques and microsequence analysis, we have shown that human lymphocyte nuclei contain defensins, a mixture of human neutrophil granule peptide 1, 2 and 3.
Refolding of recombinant α and β subunits of the Rhodospirillum rubrum F0F1 ATP synthase into functional monomers that reconstitute an active α1β1‐dimer
Authors: Du Ziyun , Gromet‐Elhanan Zippora
Published: March 2003

The α subunit from the Rhodospirillum rubrum F0F1 ATP synthase (RrF1α) was over‐expressed in unc operon‐deleted Escherichia coli strains under various growth conditions only in insoluble inclusion bodies. The functional refolding of urea‐solubilized RrF1α was followed by measuring its ability to stimulate the restoration of ATP synthesis and hydrolysis in β‐less R. rubrum chromatophores reconstituted with pure native or recombinant RrF1β[Nathanson, L. & Gromet‐Elhanan, Z. (1998) J. Biol. Chem.273, 10933–10938]. The refolding efficiency was found to increase with decreasing RrF1α concentrations and required high concentrations of MgATP, saturating ≈ 60% when 50 µg protein·mL−1 were refolded in presence of 50 mm MgATP. Size‐exclusion HPLC of such refolded RrF1α revealed a 50–60% decrease in its aggregated form and a parallel appearance of its monomeric peak. RrF1β refolded under identical conditions appeared almost exclusively as a monomer. This procedure enabled the isolation of large amounts of a stable RrF1α monomer, which stimulated the restoration of ATP synthesis and hydrolysis much more efficiently than the refolded α mixture, and bound ATP and ADP in a Mg‐dependent manner. Incubation of both RrF1α and β monomers, which by themselves had no ATPase activity, resulted in a parallel appearance of activity and assembled α1β1‐dimers, but showed no formation of α3β3‐hexamers. The RrF1‐α1β1‐ATPase activity was, however, very similar to the activity observed in isolated native chloroplast CF1‐α3β3, indicating that these dimers contain only the catalytic nucleotide‐binding site at their α/β interface. Their inability to associate into an α3β3‐hexamer seems therefore to reflect a much lower stability of the noncatalytic RrF1α/β interface.
Transforming growth factor β regulates clusterin gene expression via modulation of transcription factor c‐Fos
Authors: Jin Ge , Howe Philip H.
Published: March 2003

Transforming growth factor‐β (TGFβ) induces gene expression of the glycoprotein clusterin in a variety of cell types via a consensus AP‐1 binding site. Here, we demonstrate, by supershift analysis, that JunB, JunD, Fra1, Fra2, and c‐Fos bound to AP‐1 but that prior treatment of the cells with TGFβ reduced dramatically c‐Fos binding, suggesting that c‐Fos might be playing a negative regulatory role in clusterin gene expression. Transient cotransfection assays in mink lung epithelial (CCL64) cells, using a human c‐Fos expressing plasmid together with a clusterin promoter/reporter construct or the artificial TGFβ‐inducible reporter construct 3TPLux, revealed that c‐Fos was indeed repressive for TGFβ‐induced promoter transactivation. Further, we demonstrate that in stable c‐Fos‐overexpressing cell lines, TGFβ induction of endogenous clusterin mRNA, as well as clusterin promoter transactivation are blocked. Co‐transfection with c‐Fos deletion constructs revealed that the C‐terminal region, including the homologue box 2 motif and the extreme C‐terminal serine phosphorylation sites (Ser362 and Ser374) are required for repression of clusterin and 3TPLux transactivation. TGFβ treatment of CCL64 cells resulted in the induction of c‐Fos mRNA but caused no alternation in total c‐Fos protein levels. The results suggest that the c‐Fos represses clusterin gene expression, maintaining a low basal level in the absence of TGFβ, and that TGFβ, presumably through its effects on c‐Fos protein synthesis and/or stability, abrogates the repression of c‐Fos, thereby resulting in gene expression.
Characterization of a novel cdk1‐related kinase
Authors: Détivaud Lénaïck , Pettit George R. , Meijer Laurent
Published: March 2003

The p13suc1/p9CKShs proteins bind tightly to the cyclin‐dependent kinases cdk1 and cdk2. The distantly related protein, p15cdk‐BP, binds cdk4/6, cdk5 and cdk8. We now show that immobilized p15cdk‐BP binds both an HMG‐I kinase and a 35‐kDa protein that cross‐reacts with anti‐PSTAIRE antibodies (PSTAIRE is a totally conserved motif located in subdomain III of cdk). This ‘cdkX’ and the HMG‐I kinase also bind to an immobilized inhibitor of cdks (HD). Several properties clearly distinguish cdkX, and its associated HMG‐I kinase, from known anti‐PSTAIRE cross‐reactive cdks: (a) cdkX migrates, in SDS/PAGE, in a position intermediate between prophase phosphorylated cdk1 and metaphase dephosphorylated cdk1; (b) in contrast with cdk1, cdkX and associated HMG‐I kinase activity do not decrease following successive depletions on p9CKShs1–sepharose; (c) cdkX and associated HMG‐I kinase activity, but not cdk1, decrease following depletions on immobilized inhibitor; (d) cdkX is expressed during the early development of sea urchin embryos; in contrast with cdk1/cyclin B kinase, the p15cdk‐BP‐bound HMG‐I kinase is active throughout the cell cycle; compared with cdk1 it is active later in development; (e) p15cdk‐BP‐bound HMG‐I kinase is essentially insensitive to powerful inhibitors of cdk such as purvalanol, roscovitine, olomoucine, p21cip1 and p16INK4A; HD is only moderately inhibitory. Altogether these results suggest the existence of a new cdk1‐related kinase, possibly involved in the regulation of early development. The presence of this kinase in all organisms investigated so far, from plants to mammals, calls for its definitive identification.
Purification and characterization of fetal bovine serum β‐N‐acetyl‐D‐galactosaminyltransferase and β‐D‐glucuronyltransferase involved in chondroitin sulfate biosynthesis
Authors: Tsuchida Kazunori , Lind Thomas , Kitagawa Hiroshi , Lindahl Ulf , Sugahara Kazuyuki , Lidholt Kerstin
Published: March 2003

β‐N‐Acetylgalactosaminyltransferase II and β‐glucuronyltransferase II, involved in chondroitin sulfate biosynthesis, transfer an N‐acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through β‐linkages to an acceptor chondroitin oligosaccharide derived from the repeating disaccharide region of chondroitin sulfate. They were copurified from fetal bovine serum approximately 2500‐fold and 850‐fold, respectively, by sequential chromatographies on Red A‐agarose, phenyl‐Sepharose, S‐Sepharose and wheat germ agglutinin‐agarose. Identical and inseparable chromatographic profiles of both glycosyltransferase activities obtained through the above chromatographic steps and gel filtration suggest that the purified enzyme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; β‐N‐acetylgalactosaminyltransferase and β‐glucuronyltransferase, reminiscent of the heparan sulfate polymerase reaction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions recently developed for the chondroitin‐synthesizing system, derived from human melanoma cells, each monosaccharide transfer took place, but no polymerization occurred. These results may suggest that the purified serum enzyme preparation contains both β‐N‐acetylgalactosaminyltransferase II and β‐glucuronyltransferase II activities on a single polypeptide or on the respective polypeptides forming an enzyme complex, but is different from that obtained from melanoma cells in that it transfers a single GalNAc or GlcA residue but does not polymerize chondroitin.