bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of four heat shock protein cDNAs from grapefruit peel tissue and characterization of their expression in response to heat and chilling temperature stresses
Authors: Rozenzvieg Dafna , Elmaci Cengiz , Samach Alon , Lurie Susan , Porat Ron
Published: June 2004

In order to continuously supply horticultural products for long periods, it is essential to store them after harvest in low temperatures. However, many tropical and subtropical fruits and vegetables, such as citrus, are sensitive to chilling. In previous studies, the authors have shown that a short hot water rinsing treatment (at 62°C for 20 s) increased chilling tolerance in grapefruit. In order to gain more insight into the molecular mechanisms involved in heat‐induced chilling tolerance, PCR cDNA subtraction analysis was performed which isolated four different PCR fragments whose expression was enhanced 24 h after the heat treatment, and that showed high sequence homology with various plant HSP18‐I, HSP18‐II, HSP22 and HSP70 genes. It was found that the short hot water treatment given at 62°C for 20 s, but not at lower temperatures of 20 or 53°C, increased the expression of the various HSP cDNAs in grapefruit peel tissue. However, when the fruits were kept at ambient temperatures, the increases in HSP mRNA levels following the hot water treatment were temporary and lasted only between 6 and 48 h. Similar temporary increases in the HSP mRNA levels were detected following exposure of the fruit to a hot air treatment at 40°C for 2 h. Nevertheless, when the fruits were treated with hot water but afterwards stored at chilling temperatures of 2°C, the mRNA levels of the various HSP18‐I, HSP18‐II, HSP22 and HSP70 cDNAs increased and remained high and stable during the entire 8‐week cold‐storage period, suggesting their possible involvement in heat‐induced chilling‐tolerance responses. The chilling treatment by itself increased the expression of the HSP18‐I cDNA, but had no effect on the mRNA levels of any of the other HSP cDNAs. Exposure of fruit to other stresses, such as wounding, UV irradiation, anaerobic conditions and exposure to ethylene, had no effect on the expression of the various HSPs. Overall, the study explored the correlation between the expression and persistence of various HSP cDNAs in grapefruit peel tissue during cold storage, on the one hand, and the acquisition of chilling tolerance, on the other hand, and the results suggest that HSPs may play a general role in protecting plant cells under both high‐ and low‐temperature stresses.
Involvement of Laser Photo‐CIDNP(Chemically Induced Dynamic Nuclear Polarization)‐Reactive Amino Acid Side Chains in Ligand Binding by Galactoside‐Specific Lectins in Solution
Authors: Sieber Hans‐Christian , Adar Rivka , Arango Rafael , Burchert Maria , Kaltner Herbert , Kayser Gian , Tajkhorshid Emadeddin , Von Der Lieth Claus‐Wilhelm , Kaptein Robert , Sharon Nathan , Vliegenthart Johannes F. G. , Gabius Hans‐Joachim
Published: June 2004

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular‐dynamics (MD) simulations and NMR spectroscopy. Using the laser photoCIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin‐1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin‐1/chicken galectins and of Trpl94 in murine galectin‐3. This feature derived from the crystal structure of bovine galectin‐1 is maintained in solution for the prototype human homologue, two avian galectins and the chimeratype murine galectin‐3, as the spectra corroborate the CIDNP‐inferable spatial parameters of the four calculated models for binding‐site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site‐directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single‐site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to moleculardynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non‐uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP‐results modelling of the binding‐site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single‐site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild‐type and mutant proteins.
DNA Helicase Activity of the Hepatitis C Virus Nonstructural Protein 3
Authors: Gwack Yousang , Kim Dong Wook , Han Jang H. , Choe Joonho
Published: June 2004

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA · DNA and RNA · RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA‐stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3′ to 5′. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall order of inhibitory capacity was: adenosine 5′‐O‐(3‐thiotriphosphate) > adenylyl‐imidodiphosphate and adenylyl‐(β,γ‐methylene)‐diphosphate > AMP. DNA helicase activity was inhibited strongly by ssDNA and ssRNA, but was little affected by dsDNA. The ATPase activity was stimulated greatly by ssDNA and ssRNA, but not by dsDNA. The NS3 protein could unwind up to 500 base pairs of duplex DNA. The possible multifunctional nature of the NS3 protein is discussed and compared with that of Simian virus 40 large T antigen.
Elucidation of the Structure of the Core Region and the Complete Structure of the R‐Type Lipopolysaccharide of Erwinia carotovora FERM P‐7576
Authors: Fukuoka Satoshi , Knirel Yuriy A. , Lindner Buko , Moll Hermann , Seydel Ulrich , Zähringer Ulrich
Published: June 2004

An R‐type lipopolysaccharide (LPS) from Erwinia carotovora strain FERM P‐7576 was studied after strong alkaline degradation and mild acid hydrolysis. The resulting products were analyzed by fast‐atom bombardment mass spectrometry, one‐ and two‐dimensional 1H and 13C NMR spectroscopy, dephosphorylation and methylation analysis. The following structure was proposed for the core region of the LPS:
image

where Hep is l‐glycero‐d‐manno‐heptose and Kdo is 3‐deoxy‐d‐manno‐octulosonic acid. Some LPS species lack the β‐d‐Galp residue or the β‐d‐Galp‐(1→7)‐α‐Hepp disaccharide.

With the known structures of lipid A [Fukuoka, S., Kamishima, H., Nagawa, Y., Nakanishi, H., Ishikawa, K., Niwa, Y., Tamiya, E. & Karube, I. (1992) Arch. Microbiol. 157, 311–318] and the core moiety, the complete LPS structure was established and confirmed by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry of the native and O‐deacylated LPS.
Coacervation Characteristics of Recombinant Human Tropoelastin
Authors: Vrhovski Bernadette , Jensen Sacha , Weiss Anthony S.
Published: June 2004

Coacervation of soluble tropoelastin molecules is characterized by thermodynamically reversible association as temperature is increased under appropriately juxtaposed ionic conditions, protein concentration and pH. Coacervation plays a critical role in the assembly of these elastin precursors in elastic fiber formation. To examine the effect of physiological parameters on the ability of tropoelastin molecules to associate, solutions of recombinant human tropoelastin were monitored spectrophotometrically by light scattering over a broad range of temperatures. Coacervation of recombinant human tropoelastin is strongly influenced by the concentration of protein and NaCl and to a lesser extent on pH. Trends towards maximal association are apparent when each of these parameters is varied. Remarkably, optimal coacervation is found at 37°C, 150mM NaCl and pH7–8. Using the data generated by time courses, estimates of thermodynamic parameters were made. These estimates confirm that Coacervation is endothermic and is marked by a strong entropic contribution. Circular dichroism of recombinant human tropoelastin revealed that, rather than being random, the structure is compatible with being largely that, of an all‐β protein (with secondary structure estimated to be 3%α‐helix, 41%β‐sheet, 21%β‐turn and 33% other), exhibiting a spectrum as previously seen for tropoelastin populations and soluble elastin from naturally‐derived sources.
Interaction of the Recombinant S100A1 Protein with Twitchin Kinase, and Comparison with Other Ca2+‐Binding Proteins
Authors: Heierhorst Jörg , Mann Richard J. , Kemp Bruce E.
Published: June 2004

The giant myosin‐associated twitchin kinase, a member of the Ca2+‐regulated protein kinase superfamily, is activated by the EF‐hand protein S100A1 in a Ca2+‐dependent and Zn2+‐enhanced manner. We used recombinant S100A1 to further characterize the interaction between the two proteins. Zn2+ enhanced the binding of Ca2+/S100A1 to twitchin kinase fragments (Kd < 50 nM) in assays using a BIAcore biosensor by reducing the S100A1 off rate. Other Ca2+‐binding proteins (S100A6, calmodulin, and the calmodulin‐like domain of Ca2+‐dependent protein kinase α) bound to the kinase but did not activate it. These results indicate that binding of Ca2+‐binding proteins alone is insufficient to trigger the intramolecular rearrangement of kinase autoinhibitory contacts required for twitchin kinase activation that is specifically elicited by the S100A1 protein. Kinase fragments that contained only the autoinhibited catalytic sequence or an additional immunoglobulin‐like domain had very similar properties, indicating that the tethered immunoglobulin‐like domain does not modulate kinase regulation.
Mechanism of NADH Transfer between Alcohol Dehydrogenase and Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: Arias Wilma Martínez , Pettersson Gösta
Published: June 2004

Steady‐state and transient‐state kinetic experiments have been performed to test the proposal that there is a direct (channelled) transfer of NADH from glyceraldehyde‐3‐phosphate dehydrogenase to alcohol dehydrogenase. The results lend no support to this proposal, but can be best explained in terms of a free‐diffusion mechanism for NADH transfer between the two enzymes.
Dependence of the Lytic Activity of the N‐Terminal Domain of Human Perforin on Membrane Lipid Composition
Authors: Rochel Natacha , Cowan James A.
Published: June 2004

The kinetics and thermodynamics of pore formation by the 22‐residue N‐terminal domain of human perforin‐(1‐22)‐peptide have been studied for a variety of model phospholipid membranes. Peptide binding and aggregation, and cell lysis were monitored through the change in the fluorescence of Trp, or vesicle‐encapsulated carboxyfluorescein, respectively. Peptide binding was analyzed in terms of a model that incorporates non‐ideal interactions and aggregation in a membrane bilayer. The minimum number of peptide monomers required to form an active pore averaged from four to six, according to the lipid composition of the vesicle. This combined kinetic and thermodynamic approach has provided quantitative information that allows a direct comparison of the binding behavior of the perforin‐(1‐ 22)‐peptide in different lipid vesicles and affords molecular insight on the factors controlling pore formation. Pore formation is most favorable in thinner membranes with low melting temperatures. No significant difference in activity is observed for different zwitterionic headgroups. Rather, the gel state of the lipid chain, which diminishes the incorporation and aggregation of the perforin‐(1‐22)‐peptide shows the strongest influence. This effect is observed in both the thermodynamic (incorporation isotherm) and kinetic (carboxyfluorescein release) studies. Insertion and aggregation are more facile in membranes with less densely packed lipids. The dependence of pore‐forming activity on lipid composition provides important clues to understanding the self‐protection mechanism employed by cytotoxic T lymphocytes (CTL) against perforin‐mediated lysis.
Guanine‐Nucleotide Binding and Hydrolyzing Kinetics of ORrab2, a Rice Small GTP‐Binding Protein Expressed inEscherichia Coli
Authors: Seo Hak Soo , Choi Cheol Hoon , Kim Ho Yeon , Jeong Jin Yong , Lee Sang Yeol , Cho Moo Je , Bahk Jeong Dong
Published: June 2004

TheORrab2gene encodes a GTP‐binding protein of 23.169 kDa. The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP‐binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%), Hrab4 (51.9%), Hrab1 (46.2%), YPT (40.7%), Hrab3B (40.0%), Hrab3A (38.1%), SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%). To analyze the biochemical properties of this protein, anORrab2cDNA was overexpressed inEscherichia coliand the protein purified by Ni2+‐nitrilotriacetic acid agarose and hydroxyapatite column chromatography. The molecular mass of the protein bearing a His‐tag is approximately 28.2 kDa. The guanine‐nucleotide binding and hydrolyzing activity of ORrab2 increased with non‐ionic C12E10(polyoxyethylene 10‐lauryl ether) and ionic Chaps detergent treatment. ORrab2 bound maximally 1.03 mol of [γ‐35S]GTP[S]/mol of protein with aKdvalue of 56.83 nM. The ratioskoff GDP/koff GTPof ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP. The rate (kcat) of Pirelease against [γ‐32P]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [γ‐32P]GTP (kGTPase) were calculated to be 432×10−4± 8×10−4min−1and 172×10−4± 2×10−4min−1, respectively, in the presence of 0.1 % C12E10and 1 mM MgSO4.
Purification and Nucleic‐Acid‐Binding Properties of a Saccharomyces Cerevisiae Protein Involved in the Control of Ploidy
Authors: Weber Viktoria , Wernitznig Andreas , Hager Gudrun , Harata Masahiko , Frank Peter , Wintersberger Ulrike
Published: June 2004

Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kühne, C. & Karwan, A. (1995) Yeast 11, 929–944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA‐binding proteins. In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA‐binding proteins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear‐export signal (NES) near its N‐terminus and a potential nuclear‐localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA‐cellulose and anti‐Scp160p affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single‐stranded and double‐stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.
Contribution of Arg288 of Escherichia Coli Elongation Factor Tu to Translational Functionality
Authors: Rattenborg Thomas , Pedersen Gitte Nautrup , Clark Brian F. C. , Knudsen Charlotte R.
Published: June 2004

The recently solved structure of the ternary complex formed between GTP‐bound elongation factor Tu and aminoacylated tRNA reveals that the elements of aminoacyl‐tRNA that interact with elongation factor Tu can be divided into three groups: the T stem; the 3'‐end CCA‐Phe; and the 5' end. The conserved residues Arg288, Lys89 and Asn90 are involved in the binding of the 5' end. In the active, GTP‐bound form of the elongation factor, Arg288 and Asn90 are involved in the formation of a network of hydrogen bonds connecting the switch regions I and II of domain 1 with the rest of the molecule. This network is disrupted upon formation of the ternary complex. Arg288 was replaced by alanine, isoleucine, lysine or glutamic acid, and the resulting mutants have been subjected to an in vitro characterisation with the aim of clarifying the function of Arg288. Unexpectedly, the mutants behaved like the wild‐type factor with regard to the association and dissociation of guanine nucleotides, and the intrinsic GTPasc activities are unchanged. Furthermore, the mutants were as efficient as the wild‐type factor in carrying out protein synthesis in vitro in the presence of an excess of aminoacyl‐tRNA. However, the mutants' abilities to bind aminoacyl‐tRNA and protect the labile aminoacyl bond were impaired, especially where the charge had been reversed.
Changes in Composition of Newly Synthesized Sphingolipids of HeLa Cells During the Cell Cycle
Authors: Yokoyama Kazuaki , Suzuki Minoru , Kawashima Ikuo , Karasawa Ken , Nojima Shoshichi , Enomoto Takemi , Tai Tadashi , Suzuki Akemi , Setaka Morio
Published: June 2004

Sphingolipid biosynthesis in synchronized HeLa cells was studied by pulse labeling with [14C]Ser or [l4C]Gal and a simple TLC method. The major HeLa cell sphingolipids are ceramide (Cer), sphingomyelin, glucosylceramide (GlcCer), lactosylceramide (LacCer), globotriaosylceramide (Gb,Cer), N‐acetylneuraminosylgangliotriaosylceramide (GM2) and sialylparagloboside (GM1.Glc.NAc). The sphingolipid biosynthetic profiles of HeLa cells in the G1, G1/S boundary, S and G2 phases were similar, but significant changes occurred during M phase, when incorporation of radioactivity into sphingomyelin, Gb3 Cer and a mixture of GM2 and GM1‐GlcNAc decreased, and those of Cer and GlcCer increased. These data indicate that transfer of phosphocholine and galactose to Cer and GlcCer, respectively, decreased in mitotic cells, resulting in accumulation of Cer and GlcCer. Analysis of LacCer synthase activity revealed that GlcCer accumulation was not due to reduced activity of this enzyme. The results suggest that Cer and GlcCer accumulation in mitotic cells resulted from suppression of sphingomyelin and LacCer synthesis, probably caused by vesiculation of membranous organelles, such as the endoplasmic reticulum and Golgi apparatus.
Comparative Study In Vivo and In Vitro of Uniformly 14C‐Labelled and 125I‐Labelled Recombinant Fibroblast Growth Factor 2
Authors: Colin Sylvie , Mascarelli Frederic , Jeanny Jean‐Claude , Vienet Raymond , Bouche Gérard , Courtois Yves , Labarre Jean
Published: June 2004

Recombinant bovine fibroblast growth factor (FGF2), uniformly labelled with 14C ([14C]FGF2), was purified and showed to be highly stable and to retain full biological activity. Organ distribution of [14C]FGF2 after intravenous injection of young rats was assessed by autoradiography of whole body sections and compared with those obtained with [125I]iodinated FGF2 (125I‐FGF2). Thyroid, stomach, intestine, bladder and skin were radioactively labelled only in the case of 125I‐FGF2. This tissue‐labelling is artefactual, probably due to free iodide binding not observed when using [14C]FGF2. High‐resolution autoradiography showed a complex tissue distribution of [14C]FGF2 in kidney and adrenal organs. Incubation of frozen eye sections with [14C]FGF2 showed a specific and high‐resolution labelling pattern of ocular tissues. After cellular internalization, [14C]FGF2 was processed into five distinct polypeptides of 16, 14, 8, 7, and 5.5 kDa. The 14‐kDa and 7‐kDa polypeptides are novel catabolic fragments not detected with radioiodinated FGF2. In terms of stability, tissue distribution specificity, and autoradiographic resolution, [14C]FGF2 proved to have more advantages than 125I‐FGF2 for pharmacokinetic and catabolism studies.
Characterization of A Stable Intermediate Trapped during Reversible Refolding of Bacillus Subtilisα‐Amylase
Authors: Haddaoui El Arbi , Leloup Laurence , Petit‐Glatron Marie‐Françoise , Chambert Régis
Published: June 2004

Bacillus subtilis exocellular α‐amylase is reversibly refolded after denaturation by guanidine hydrochloride at pH 7 and 370C. The unfolding‐folding transition monitored by intrinsic fluorescence changes and resistance to proteolysis was resolved into a two‐state transition. The first step (t1/2 < 1 s) led from D. the totally unfolded state, to C. a stable partially structured state of the protein. This folding intermediate was devoid of any enzyme activity and partially resistant to protease degradation. Calcium was required for the transition from C to N. the native state. This metal did not remain associated with the native form and could be replaced by barium or strontium, but not by magnesium. We discuss the hypothesis that C. the folding intermediate whose further transformation is under kinetic control, is the competent state involved in the secretion process of α‐amylase.
Mutagenesis and Ligand Modification Studies on Galanin Binding to its GTP‐Binding‐Protein‐Coupled Receptor GalR1
Authors: Berthold Malin , Kahl Ulrika , Juréus Anders , Kask Kalev , Nordvall Gunnar , Langel Ülo , Bartfai Tamas
Published: June 2004

In this study, a large number of receptor mutants were generated and several N‐terminally modified galanin analogues synthesized to refine the previously proposed binding site model for galanin to its GTP‐binding‐protein‐coupled receptor GalR1. In addition to ligand‐binding studies, the functionality of mutant receptors was evaluated by assessing their ability to mediate galaninergic inhibition of isoproterenol‐stimulated adenylyl cyclase activity. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remain functional albeit 20‐fold less efficient than the wild‐type receptor in mediating inhibition of stimulated cAMP production by galanin. The His267Ala mutant is, apart from being deficient in galanin binding, also severely impaired in functional coupling. While His264 and Phe282 seem to be important in forming the binding pocket for galanin, His267 might play a role in forming or stabilizing the active conformation of the GalRl receptor rather than directly participating in the formation of the binding pocket for galanin.

N‐terminal carboxylic acid analogues of galanin have low affinity to wild‐type GalR1, but substantially increased affinity to the Glu271Lys receptor mutant. This, together with the finding that an alanine substitution of Phe115 in TM III results in a tenfold decrease in affinity for galanin, suggests that the N‐terminus of galanin interacts with PhellS. In contrast to the Phe282Ala mutation in TM VII, a conservative mutation of Phe282 to tyrosine did not alter the affinity for galanin. Thus, the interaction between Tyr9 of galanin and Phe282 is likely to be of an aromatic‐aromatic nature.
Stabilisation of Halophilic Malate Dehydrogenase from Haloarcula Marismortui by Divalent Cations
Authors: Madern Dominique , Zaccai Giuseppe
Published: June 2004

Halophilic malate dehydrogenase is stable in a limited concentration range of MgCl2 or CaCl2. Thermal deactivation of the protein at low concentrations of these divalent salts is very different from that occurring at high concentrations. In low salt, stability always increases as the temperature is lowered. In high salt, stability shows bell‐shaped behaviour as a function of temperature: increasing to a maximum at 4°C, and subsequently decreasing as the temperature is lowered. This is in contrast to other salts, for which the deactivation behaviour depends on the salt type but not on its concentration. Cofactor addition or replacement of H2O by D2O modify only the deactivation at low MgCl2 or CaCl2 concentrations. A pH transition between pH 7 and pH 8, however, modified enzyme deactivation at both low and high MgCl2 or CaCl2 concentrations. The pH effect on stability was also observed in other salts. By comparing the effect of CaCl2, MgCl2, and NaCl, a strong correlation was found between the minimum salt concentration required for the stabilisation of halophilic malate dehydrogenase and the hydration of the cation.
Acceleration of Unisite Catalysis of Mitochondrial F1‐Adenosinetriphosphatase by ATP, ADP and Pyrophosphate
Authors: García José J. , Gómez‐Puyou Armando , Maldonado Ernesto , Tuena De Gómez‐Puyou Marietta
Published: June 2004

The effect of ATP, ADP and pyrophosphate (PPi) on hydrolysis and release of [γ‐32P]ATP bound to the high‐affinity catalytic site of soluble F1, from bovine heart mitochondria under unisite conditions [Grubmeyer, C., Cross, R. L. & Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092–12100] was studied. In accord with the previous data, it was observed that millimolar concentrations of ATP or ADP added to F. undergoing unisite hydrolysis of [γ‐32P]ATP accelerated its hydrolysis. PP1 also produced a hydrolytic burst of a fraction of the previously bound [γ‐32P]ATP; kinetic data suggested that for production of optimal hydrolysis by PP1; of the bound [γ‐32P]ATP, two binding sites with apparent Kd of 27 μM and 240 μM must be filled. The extent of the hydrolytic burst induced by MgPP1 was lower than that induced by ADP and ATP. In F1, in which PP1, had produced a hydrolytic burst of the bound [γ‐32P]ATP, the addition of ATP induced a second burst of hydrolysis. By filtration experiments and enzyme trapping, it was also studied whether ATP, ADP and PP1 produce release of the tightly bound [γ‐32P]ATP. At millimolar concentrations, ATP and ADP brought about release of about 25 % of the previously bound [γ‐32P]ATP. At micromolar concentrations, ADP accelerated the hydrolysis of the previously bound [γ‐32P]ATP but not its release. Hence, the hydrolytic and release reactions could be separated, indicating that the two reactions require the occupancy of different sites in F1. With PP1, no release of the tightly bound [γ‐32P]ATP was observed. The ADP induced hydrolysis and release of the F1,‐bound [γ‐32P]ATP were inhibited by sodium azide to the same extent (60%). Since release of ATP from a high‐affinity catalytic site of F1 represents the terminal step of oxidative phosphorylation, the data illustrate that the binding energy of substrates to F1, is critical to the ejection of ATP into the media. The failure of PP1 to induce release of [γ‐32P]ATP bound to F1, under unisite conditions is probably due to its lower binding energy.
Variability in Arabidopsis Thaliana Chromosomal High‐Mobility‐Group‐1‐Like Proteins
Authors: Stemmer Christian , Ritt Christoph , Igloi Gabor L. , Grimm Rudi , Crasser Klaus D.
Published: June 2004

The vertebrate high‐mobility‐group (HMG) protein HMG1 is an abundant non‐histone protein which is considered as an architectural element in chromatin. In the monocotyledonous plant maize, four different HMG1‐like proteins (HMGa, HMGc1/2, HMGd) have been identified, whereas other eukaryotes usually express only two different proteins of this type. We have examined here the HMG1‐like proteins of the dicotyledonous plant Arabidopsis thaliana. The isolation and analysis of cDNAs encoding five different so far uncharacterised HMG1‐like proteins (now termed HMGα, HMGβ1/2, HMGγ, HMGδ) from Arabidopsis indicates that the expression of multiple HMG1‐like proteins is a general feature of (higher) plants. The Arabidopsis HMG1‐like proteins contain an HMG domain as a common feature, but outside this conserved DNA‐binding motif the amino acid sequences are significantly different indicating that this protein family displays a greater structural variability in plants than in other eukaryotes. The five HMG1‐like proteins were expressed in Escherichia coli and purified. They bind with somewhat different affinity to linear double‐stranded DNA. The recognition of DNA structure is evident from their preferential interaction with DNA minicircles relative to linear DNA. Reverse‐transcribed PCR suggested that the five HMG1‐like genes are simultaneously expressed in Arabidopsis leaves and suspension culture cells.
Comparative Stability and Clearance of [Met30]Transthyretin and [Met119]Transthyretin
Authors: Longo Alves Isabel , Hays Marguerite T. , Saraiva Maria João Mascarenhas
Published: June 2004

[Met119]Transthyretin has been described as a non‐amyloidogenic transthyretin variant. In Portugal, it has also been found in compound heterozygotic individual carriers of [Met30]transthyretin, the most prevalent variant associated with familial amyloidotic polyneuropathy. In these individuals, the evolution of the disease seems to be more benign than in typical [Met30]transthyretin carriers, suggesting a protective effect of [Met119]transthyretin on the pathogenic effects of [Met30]transthyretin. To study the mechanisms of this protective effect, we performed comparative in vivo clearance studies. Heterotetrameric [Met119]transthyretin showed a slower clearance, whereas homotetrameric [Met30]transthyretin presented a faster clearance. These data correlate with the relative TTR levels present in carriers of these mutations. Comparative analyses of the resistance to dissociation into monomers of serum transthyretin by 4M urea isoelectric focusing suggested a higher tetrameric stability of transthyretin in [Met119]transthyretin carriers, in contrast to a lower stability in [Met30]transthyretin carriers. The compound heterozygotes presented a pattern similar to the normal individuals. Our results suggest that the protective clinical effect of the Met119 mutation possibly involves the stabilisation of the tetrameric structure of transthyretin. Whether this behaviour correlates with the different metabolism found for the two variants is not known. The approaches reported here open some possibilities for the study and development of future therapeutic agents of familial amyloidotic polyneuropathy.
The Myrosinase‐Binding Protein from Brassica Napus Seeds Possesses lectin Activity and has a Highly Similar Vegetatively Expressed Wound‐Inducible Counterpart
Authors: Taipalensuu Jan , Eriksson Susanna , Rask Lars
Published: June 2004

This communication demonstrates that proteins in the family of myrosinase‐binding proteins (MBP) present in seeds of Brassica napus possess lectin activity, binding most efficiently to p‐aminophenyl α L‐D‐mannopyranoside–agarose, and to some extent to N‐acetylglucosamine–agarose. A cDNA encoding a vegetatively expressed, wound‐inducible counterpart to these seed MBP was isolated and characterised. Upon wounding, this MBP transcript accumulated in old and young leaves, and was systemically expressed in the young plant. Additionally, the wound‐induced MBP transcript increased in abundance after treating the young plants with methyl jasmonate (MeJA), jasmonic acid (JA) or abscisic acid (ABA), and to some extent in response to the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid. Expression induced by wounding, ABA or JA was antagonised by simultaneous feeding of the plants with salicylic acid. MBP polypeptides accumulated in MeJA‐treated plants. The myrosinases redistributed from the soluble fraction into the insoluble fraction of a tissue extract after induction. The most abundant MBP (94 kDa) partitioned in the insoluble fraction, while two larger MBP (103 kDa and 108 kDa) were present only in the soluble fraction of extracts obtained from the control or MeJA‐treated plant tissues.
Purification and Spectroscopic Characterization of β‐Amyloid Precursor Protein from Porcine Brains
Authors: De La Fourniere‐Bessueille Laurence , Grange Delphine , Bucket René
Published: June 2004

Soluble and membrane‐bound isoforms of β‐amyloid protein precursor (APP) of Alzheimer's disease were extracted and purified from porcine brains. At least three types of soluble APP and membrane‐bound APP with different molecular masses, ranging from 86 kDa to 116kDa, were obtained. CD and infrared spectroscopies were used to determine the overall secondary‐structure content of APP. The infrared spectra of soluble and membrane‐bound APP (in dry and hydrated states) were similar in the amide‐I and amide‐II regions, suggesting that the overall secondary structures of the soluble and membrane isoforms were roughly identical. The amide‐I band is composed of at least five component bands, located at 1694, 1674, 1652, 1637 and 1618 cm‐1 for soluble APP, and located at 1687, 1674, 1651, 1637 and 1614–1606 cm‐1 for membrane‐bound APP, as evidenced by their respective second‐derivative infrared spectra. The 1651 – 165 2‐cm‐1 band was associated with a‐helix structures, while two types of β‐sheet structures are evidenced by two characteristic pairs of component bands. The 1674‐cm‐1 and 1637‐cm‐1 bands for soluble APP and membrane‐bound APP were tentatively associated to β‐sheet structures. The second pair of bands, located at 1694cm‐1 and at 1618cm‐1 for soluble APP and at 1687cm‐1 and 1614–1606 cm‐1 for membrane‐bound APP, were associated with intermolecular β‐sheet structures or aggregated strands, as confirmed by heat denaturation. CD spectra indicated the presence of α‐helix structures in soluble and membrane‐bound APP. The secondary‐structure content, estimated from CD spectra, was about 40–45%α‐helix and 15–20%β‐sheet structures for soluble and membrane‐bound APP.
Nucleotide Binding Sites in Wild‐Type Creatine Kinase and in W227Y Mutant Probed by Photochemical Release of Nucleotides and inFrared Difference Spectroscopy
Authors: Raimbault Cyrille , Perraut Catherine , Marcillat Olivier , Buchet René , Vial Christian
Published: June 2004

Structural changes induced by nucleotide binding to the wild‐type rabbit muscle creatine kinase (CK) and to its W227Y mutant were compared and probed by reaction‐induced difference spectroscopy (RIDS). The reaction was induced by the photorelease of nucleotide from the caged nucleotides ADP[Et(PhN02)] or ATP[Et(PhNO2)], producing the RIDS of CK. The concomitant addition of a saturated concentration of nucleotide and caged nucleotide modified the RIDS of CK, permitting structural changes caused by nucleotide binding in the wild‐type creatine kinase to be identified. The W227Y mutant was inactive and its nucleotide binding site was partially impaired as shown by the disappearance or decrease of several nucleotide‐sensitive bands in the RIDS of W227Y mutant. The magnitude of the decrease was not the same for each band, suggesting that distinct groups of W227Y mutant were affected differently during nucleotide binding. More precisely, the binding sites for γ‐phosphate and β‐phosphate of the nucleotide were not accessible in W227Y mutant as shown by the absence of the phosphate‐sensitive 1666–1667–cm‐1 and 1625‐cm‐1 bands in the RIDS of W227Y mutant. However the binding site of other parts of the nucleotide was partially accessible, since the 1638–1639‐cm‐1 phosphate‐insensitive band did not completely vanish in the RIDS of W227Y mutant. The RIDS of W227Y mutant with ADP[Et(PhNO2)] and creatine lacked the 1613‐cm‐1 and 1581‐cm‐1 bands, associated with vibrational modes of creatine, suggesting that coupling between the binding sites of the nucleotide and of creatine was altered in W227Y mutant. These results are in accordance with the earlier suggestions that residue W227 in CK is essential for preventing water molecules from penetrating into the active site and for orienting nucleotide in the binding site, by forming stacking interactions between its indole group and purine of the nucleotide and its indole group.
Haloalkanoate Dehalogenase II (DehE) of a Rhizobium sp. — Molecular Analysis of the Gene and Formation of Carbon Monoxide from Trihaloacetate by the Enzyme
Authors: Stringfellow Joseph M. , Cairns Stephen S. , Cornish Alex , Cooper Ronald A.
Published: June 2004

A 3‐kb EcoRI fragment of genomic DNA from a Rhizobium sp. cloned into pUC19 endowed Escherichia coli K‐12 with the ability to grow, albeit slowly, with 2‐chloropropionic acid as substrate. The construct expressed weakly a gene that encoded a non‐stereospecific 2‐chloropropionic acid dehalogenase (dehalogenase II;DehE). The dehE gene was not closely linked to the organism's other two dehalogenase genes, dehD and dehL. The derived amino acid sequence of DehE showed little identity with DehD or DehL, but there was significant identity to two other dehalogenases that act non‐selectively on 2‐chloropropionic acid. The fragment carried a truncated ORF upstream of dehE that was 51% identical to a positively acting regulatory protein, DehR, required for expression of a Pseudomonas putida dehalogenase gene. In its complete form this gene could encode the Rhizobium sp. dehalogenase‐regulatory protein. DehE dehalogenated tribromoacetic acid completely, forming stoichiometric amounts of carbon monoxide and carbon dioxide as the other products.
Human Endothelin Receptors ETA and ETB Expressed in Baculovirus‐Infected Insect Cells
Authors: Satoh Misako , Miyamoto Chikara , Terashima Hiromichi , Tachibana Yukako , Wada Kenji , Watanabe Takahide , Hayes Ashley E. , Gentz Reiner , Furuichi Yasuhiro
Published: June 2004

We expressed human endothelin receptors, ETA and ETB, in insect Sf9 cells infected by recombinant baculoviruses that contained the respective cDNAs. Ligand‐binding experiments showed that the two expressed receptors have the same affinities as observed for the receptors in mammalian cells, i.e. the ETA receptor showed an affinity order of ET‐1≥ET‐21≫ET‐3, and the ETa receptor remained nonselective for three isopeptide ligands. The ETB receptor was purified by affinity chromatography with K9‐biotinyl‐ET‐1 without losing the ligand‐binding activity from the membrane of infected Sf9 cells. Protein chemical analysis of the purified ETB receptor showed that it is glycosylated, and that the N‐terminal 38‐amino‐acid peptide is susceptible to proteolytic digestion, resulting in a small 35‐kDa receptor like that found in the human placenta. Surprisingly, the infected and unlysed cells showed a strong intracellular Ca2+ concentration increase ([Ca2+]i), which was generated by a unique signal‐transduction pathway consisting of the insect GTP‐binding protein and human endothelin receptors expressed in the late phase of virus infection. Due mainly to an efficient expression (over 200000 receptors/cell), to a low background owing to no endogenous homolog receptor in insect Sf9 cells, and to a sensitive fluorescent reagent Fura‐2, this insect Sf9 cell system can detect the [Ca2+]i induced by picomolar levels of endothelin‐receptor. We propose that this highly sensitive system be used to screen for potential antagonists/agonists of endothelin receptors.
Isolation and Characterization of Goldfish Y Box Protein, a Germ‐cell‐specific RNA‐binding Protein
Authors: Katsu Yoshinao , Yamashita Masakane , Nagahama Yoshitaka
Published: June 2004

Cyclin B is a regulatory subunit of maturation‐promoting factor. In goldfish (Carassius auratus) oocytes, cyclin B is synthesized de novo after stimulation by 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (maturation‐inducing hormone). In this study, we examined goldfish oocyte proteins bound to cyclin B mRNA. Using oligo(dT)‐cellulose affinity chromatography and northwestern blotting analysis, we identified a 54‐kDa cyclin B mRNA‐binding protein (p54). Southwestern blotting analysis showed the binding of p54 to the Y box DNA element (CTGATTGGCCAA), suggesting that p54 is a Y box protein in goldfish. We isolated two cDNA clones, GFYP1 and GFYP2, the latter of which encodes a germell‐specific Y box protein. An antibody against a GFYP2 protein recognized p54, suggesting that p54 is identical or highly similar to GFYP2 protein. This is also supported by the finding that a recombinant GFYP2 expressed in bacteria bound to both the Y box DNA element and the goldfish cyclin B mRNA synthesized in vitro. These results suggest that p54 is a germ‐cell‐specific Y box protein and is a potential masking protein of cyclin B mRNA in goldfish oocytes.
The Cell Wall Polymer of the Extremely Halophilic Archaeon Natronococcus occultus
Authors: Niemetz Ruth , Kärcher Uwe , Kandler Otto , Tindall Brian J. , König Helmut
Published: June 2004

The cell wall polymer of Natronococcus occultus (DSM 3396) consists of L‐glutamate, N ‐acetyl‐d‐glucosamine, N ‐acetyl‐d‐galactosamine, d‐galacturonic acid, d‐glucuronic acid and d‐glucose in a molar ratio of 5:7:1:8:0.5:0.3.

Partial acid hydrolysis of the cell wall polymer produced soluble fragments that could be separated by HPLC. A γ‐glutamyl dipeptide was isolated. In the intact cell wall polymer, the glutamate residues form a poly‐(γ‐glutamine) chain with a length of about 60 monomers, which corresponds to a relative molecular mass of approximately 7700 Da. Two other soluble dimeric fragments, composed of glutamate and either glucosamine or galactosamine in a molar ratio of 1:1, were purified from the hydrolysate, suggesting the presence of two different oligosaccharides linked to the poly‐(γ‐glutamine) chain of the intact polymer. The analysis of additional fragments, which were composed of an amino sugar and galacturonic acid or glucose indicated that one oligosaccharide consisted of a glucosamine pentamer in an α‐1,3 linkage at the reducing end and an oligomer with at least five β‐1,4‐linked galacturonic acid residues at the non‐reducing end. The second oligosaccharide was comprised of a galactosamine dimer in a β‐1,3 linkage at the reducing end and a maltose unit at the non‐reducing end. Both oligosaccharides were linked to the α‐amide group of the glutamine residues of the poly‐(γ‐glutamine) chain. The whole cell wall polymer, which represents a novel type of natural glycoconjugate, has a relative molecular mass of 54kDa.
In Vitro Assembly of Drosophila— Lamin Dm0 Lamin Polymerization Properties are Conserved
Authors: Sasse Bernd , Lustig Ariel , Aebi Ueli , Stuurman Nico
Published: June 2004

Vertebrate nuclear lamins exhibit polymerization properties that are remarkably different from those of vertebrate cytoplasmic intermediate filament (IF) proteins. Notably, under conditions where vertebrate cytoplasmic IF proteins form tetramers consisting of laterally associated dimers, nuclear lamin dimmers associate longitudinally into head‐to‐tail polymers. Also, in vitro, nuclear lamins readily form paracrystalline fibers, rather than stable 10‐nm filaments. To investigate whether these properties are also shared with invertebrate nuclear lamins, we analyzed in considerable detail the polymerization behavior of recombinant full‐length lamin Dm0 from the invertebrate Drosophila melanogaster. This lamin differs substantially from vertebrate lamins in its primary structure. We also analyzed lamin Dm0‐derived fragments lacking either the head domain (headless), the tail domain (tailless), or both (rod). Like vertebrate lamins, full‐length Drosophila lamin Dm0 assembled into head‐to‐tail polymers, with little or no formation of tetramers by lateral association of dimers. This longitudinal assembly was severely inhibited by deletion of the head domain. Removal of the tail domain led to increased formation of filamentous polymers. Under appropriate conditions, full‐length Drosophila lamin Dm0 as well as the three lamin Dm0‐derived fragments assembled into paracrystalline fibers. No steady‐state condition tested yielded assembly of 10‐nm filaments resembling those formed by vertebrate cytoplasmic IF proteins. These findings indicate that the in vitro assembly behavior of nuclear lamins is highly conserved but distinct from that of cytoplasmic IF proteins, thus evidencing its functional importance.
Complex Regulation of Transferrin Receptors during Erythropoietin‐Induced Differentiation of J2E Erythroid Cells
Authors: Busfield Samantha J. , Tilbrook Peta A. , Callus Bernard A. , Spadaccini Angelo , Kuhn Lukas , Klinken S. Peter
Published: June 2004

The regulation of transferrin‐receptor synthesis was studied in J2E erythroid cells induced to differentiate with erythropoietin. Nuclear run‐on assays demonstrated that transcription of the transferrin‐receptor gene rose markedly after erythropoietin treatment. In addition, transferrin‐receptor mRNA was stabilised and this was associated with an increase in the activity of the RNA‐binding protein IRP (iron regulatory protein). As a result of increased transcription and mRNA stabilisation, steady‐state RNA levels increased 10–20‐fold. However, despite these large increases in mRNA, translation only doubled; consequently, modest increases in total protein and surface transferrin receptors were observed. Moreover, this rise in transferrin receptors was transient, and correlated with a burst of proliferation shortly after erythropoietin treatment. The expected inverse relationship between transferrin receptors and ferritin did not occur during J2E maturation as translation of both ferritin subunits increased when transferrin‐receptor mRNA levels rose. Analysis of mutant J2E clones incapable of synthesising haemoglobin revealed that surface transferrin‐receptor levels were only 15–25% that of the parental erythroid line. We propose that the surface expression of transferrin receptors in J2E cells is governed by three factors: basal levels essential for normal growth in culture; elevated levels needed for haemoglobin synthesis; and a transient erythropoietin‐induced increase that is required for the final burst of proliferation. It was concluded that the regulation of transferrin‐receptor production in erythropoietin‐stimulated J2E cells is complex and that there are several sites of control.