bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermodynamic Analysis of the Binding of 5‐fluoro‐2′‐deoxyuridine 5′‐monophosphate to Thymidylate Synthase Over a Range of Temperatures
Authors: García‐Fuentes Luis , Reche Pedro , López‐Mayorga Obdulio , Santi Daniel V. , González‐Pacanowska Dolores , Barón Carmen
Published: March 2005

The binding of 5‐fluoro‐2′‐deoxyuridine 5′‐monophosphate (FdUMP) to Lactobacillus casei recombinant thymidylate synthase has been studied by isothermal titration microcalorimetry at pH 7.1 over the temperature range 16–35°C. Calorimetric measurements in various buffer systems with different heats of ionization suggest that a proton uptake is involved in the binding process of the nucleotide. In the temperature range investigated, the mol protons bound/mol nucleotide increases as the temperature decreases.

A model of two equal and independent sites fits well with the binding isotherms for thymidylate synthase. The binding constants, the changes in Gibbs energy, enthalpy, and entropy/site for FdUMP binding were calculated at each temperature. The results show that the binding is driven by both enthalpy and entropy contributions in the range 16–35°C. The enthalpy changes become more negative as the temperature increases, with CP=–170±20 J K1 (mol FdUMP bound)−1. The behavior of the system supports the observation that FdUMP binds to thymidylate synthase without producing profound conformational changes in the protein dimer.
Expression of the Subunits of Protein Kinase CK2 During Oogenesis in Xenopus laevis
Authors: Wilhelm Vivian , Rojas Patricio , Gatica Marta , Allende Catherine C. , Allende Jorge E.
Published: March 2005

Northern‐blot analysis of RNAs from different tissues demonstrated that the mRNA for the protein kinase CK2α subunit is very abundant in the ovary of Xenopus laevis. The competitive reverse‐PCR technique has been used to quantitate the mRNA for both CK2α and CK2β subunits during oogenesis. The results obtained using eight different animals consistently show an increment of 2–3‐fold in the mRNA for both subunits in vitellogenic oocytes (stages II‐VI). Each stage‐VI oocyte contains approximately 5 × 10−7 molecules CK2α mRNA and 1 × 10−7 molecules CK2β mRNA. These amounts are considerably higher than many other mRNAs analyzed in these cells. Activity measurements of CK2 using casein or a specific model peptide revealed increments of about 10–12‐fold during oogenesis, and also indicated that the amount of enzyme in the nucleus accounted for 15–30% of the total enzyme in the oocyte at all stages. Western‐blot analysis of CK2α indicated that the amount of this protein subunit also increased during oogenesis in a parallel fashion with the increment of enzymic activity.
Molecular Cloning and Characterization of Anionic and Cationic Variants of Trypsin from Atlantic Salmon
Authors: Male Rune , Lorens James B. , Smalås Arne O. , Torrissen Krisna R.
Published: March 2005

Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin‐specific probes. Five clones containing near full‐length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate‐binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP‐I, SalTRP‐IA and SalTRP‐IB) possessed very similar sequences and may represent allelic variants encoded by the same gene locus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP‐II and SalTRP‐III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X‐ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins.
Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor
Authors: Takagi Junichi , Aoyama Toshihiro , Ueki Shoko , Ohba Hirotsugu , Saito Yuji , Lorand Laszlo
Published: March 2005

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100‐kDa propeptide, called the propolypeptide of von Willebrand factor (pp‐vWF), is generated during biosynthesis. We found that pp‐vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311–123161. As such, it could form cross‐linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp‐vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861–867].

In this work, we identified the Gln residues in pp‐vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme‐reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti‐dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp‐vWF, only four could be modified in the factor‐XIIIa‐catalyzed reaction.
Cloning and Characterization of cDNA for Adenosine Kinase from Mammalian (Chinese Hamster, Mouse, Human and Rat) Species
Authors: Singh Bhag , Hao Weihua , Wu Zai‐chuan , Eigl Bernhard , Gupta Radhey S.
Published: March 2005

The enzyme adenosine kinase constitutes the major purine nucleoside phosphorylating activity in mammalian cells. In view of its central role in adenosine metabolism, which is an important physiological regulator, an understanding of the primary structure of adenosine kinase is of much interest. Using micro‐sequence information from peptides derived from purified Syrian hamster liver enzyme, we have succeeded in isolating full length cDNA clones encoding adenosine kinase from Chinese hamster ovary cells and mouse 3T3 cells. The open reading frames in these clones consist of 334 and 335 amino acids and encode proteins of molecular masses 37364 Da and 37489 Da, respectively. In addition, the coding and upstream sequences for adenosine kinase from human (HeLa cells) and rat liver have also been cloned and sequenced. Transfection of an adenosine‐kinase‐deficient mutant (selected for resistance to the adenosine analog toyocamycinj of Chinese hamster ovary cells with a plasmid containing the cloned adenosine kinase cDNA, leads to regaining of adenosine kinase activity in the transformed cell. The adenosine kinase transformants also simultaneously lost their toyocamycin resistance and became similarly sensitive to the analog as the parental wild‐type Chinese hamster ovary cells. The cloned adenosine kinase cDNA was also used to examine structural changes in mutants affected in adenosine kinase. In Chinese hamster ovary cells, one type of mutant that lacks adenosine kinase activity and displays high degree of resistance to various adenosine analogs, is obtained at an unusually high spontaneous frequency 10−4 ‐ 10−3). Results of Southern and northern‐blot analysis provide evidence that this group of mutants involves gross structural alterations affecting the adenosine kinase gene. Such structural alterations are not observed in another type of mutant which exhibits increased resistance only to C‐adenosine analogs. Sequence similarity searches indicate that several of the bacterial and yeast sugar kinases (ribokinase, fructokinase and inosine‐guanosine kinase) exhibit limited but significant similarity to the mammalian adenosine kinase. The sequence similarity data support the possibility that adenosine kinase shares a common evolutionary ancestor with these protein sequences.
Identification of the Proteolytic Thrombin Fragments Formed after Cleavage with Rat Mast Cell Protease 1
Authors: Pejler Gunnar , Karlström Anders R. , Berg Lotta
Published: March 2005

We have previously identified rat mast cell protease 1 (RMCP‐1), a chymotrypsin‐like secretory granule serine protease, as a potent inactivator of thrombin. The present study outlines the cleavage pattern obtained after degradation of thrombin by RMCP‐1. The cleavage sites in thrombin were identified by N‐terminal amino acid sequence analysis of recovered thrombin fragments. Incubation of thrombin with RMCP‐1 resulted in the rapid formation of a 37‐kDa fragment, due to cleavage of the Phe1G‐Gly1F bond in the thrombin A chain (numbering of amino acid residues according to topological equivalencies with chymotrypsinogen). Further incubation resulted in cleavage of the Trp148‐Thr149 bond in the B chain, along with the formation of fragments of 27 kDa and 15 kDa. When the RMCP‐1/thrombin mixtures were incubated further, successive degradation of the 37‐kDa, 27‐kDa and 15‐kDa fragments was observed, along with cleavage of the Tyr117‐Ile118 bond in the B chain and the formation of fragments of 12, 9 and 6 kDa. No residual thrombin activity was detected after the degradation process had proceeded to this stage. Heparin was shown to markedly enhance the rate of thrombin degradation by RMCP‐1.
Expression and Purification of a Soluble Functional form of the Platelet αIIbβ3 Integrin
Authors: Gulino Danielle , Martinez Pascal , Delachanal Elisabeth , Concord Evelyne , Duperray Alain , Alemany Mònica , Marguerie Gérard
Published: March 2005

Platelet glycoproteins αIIb and β3 are membrane proteins that associate to form a Ca2+‐dependent heterodimer which constitutes an inducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, αIIb and β3 were both deleted of their transmembrane and cytoplasmic domains and were expressed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and compared to those of wild‐type αIIbβ3. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that observed for the wild‐type αIIbβ3 in COS cells or in human megakaryocytes. The truncated β3 subunit associated with the Pro‐truncated form of the αIIb subunit. This precursor form was not secreted. After proteolytic cleavage of the Pro‐truncated αIIb, the mature heterodimer was secreted into the culture supernatant. To quantify the molar ratio of the various secreted soluble forms, an immunocapture assay was designed. All secreted tr‐αIIb subunits associated with tr‐β3. In contrast, tr‐β3 was produced and secreted in excess as the free form. Immunoreactivity of the wild‐type and soluble truncated complexes was identical since all the monoclonal antibodies used reacted with surface‐located epitopes on both complexes. This indicated that the soluble truncated heterodimer adopted a native conformation. To purify this soluble heterodimer, tr‐αIIbβ3‐containing culture supernatant was adsorbed on an RGDW‐affinity column and eluted with a solution of the free peptide RGDW. In the RGD‐eluted material, the amount of each subunit was stoichiometric, suggesting that the complex was not disrupted during purification. The capacity of the wild‐type and truncated RGD‐eluted complexes to interact with soluble fibrinogen was compared using a solid‐phase immunocapture assay. tr‐αIIbβ3 and platelet αIIbβ3 exhibited similar fibrinogen‐binding capacity. For both complexes, these interactions were mediated by RGD and γ fibrinogen signals.
Solvent‐induced Structural Distortions of Horse Metmyoglobin
Authors: Glandières Jean‐Marie , Calmettes Patrick , Martel Peter , Zentz Christian , Massat Alain , Ramstein Jean , Alpert Bernard
Published: March 2005

Structural and dynamic constraints produced by the surrounding solvent on the aquometmyoglobin molecule were investigated by means of circular dichroism and Fourier‐transform infrared spectroscopies, tritium/hydrogen exchange kinetics and small‐angle neutron‐scattering experiments. Formamide and ethanol were chosen as cosolvents because they are known to increase and decrease protein activity, respectively. The CD measurements in the Soret region show that no changes occur in the heme molecular structure nor in the protein near the heme. The results of proton‐exchange kinetics experiments indicate that the conformational dynamics of aquometmyoglobin is only marginally affected by the cosolvents. However, the small‐angle neutron‐scattering spectra strongly suggest that these cosolvents induce some distortions of the tertiary conformation. According to the ultraviolet CD and Fourier‐transform infrared data, the alteration of the tertiary conformation results from changes in both the number of intrachain hydrogen bonds and the structures of β turns of type I' for formamide and of type II for either of the two cosolvents. The use of several techniques allows the present approach to demonstrates that the myoglobin structure is extremely sensitive to its environmental conditions.
Identification and Localization of a Synaptosomal Membrane Prolyl Endopeptidase from Bovine Brain
Authors: O'Leary Rhona M. , O'Connor Brendan
Published: March 2005

Prolyl endopeptidase, which has long been recognised for its importance in the degradation of several neuropeptides such as thyroliberin, luteinising hormone releasing hormone, angiotensin, substance P and neurotensin, has been widely characterised as a cytosolic enzyme. However, in this paper, we report the presence of a prolyl endopeptidase activity in the particulate fractions of bovine brain, which is distinct from that in the cytoplasm. This previously uncharacterised activity was found to reside in the synaptosomal membranes, a location which is highly significant for the inactivation of neuropeptides in brain. Following vigorous salt washing and osmotic shock, the prolyl endopeptidase activity was released from the membranes by treatment with the detergent Triton X‐100, and was partially purified by gel filtration on a Sephacryl S‐200HR column. This prolyl endopeptidase activity was shown to have a molecular mass (87 kDa) higher than the cytosolic prolyl endopeptidase but, from initial investigation, appears to demonstrate a similarly broad substrate specificity towards proline‐containing neuropeptides. The partially purified enzyme was inhibited by certain thiol‐protease inhibitors and was also found to be sensitive to the metal chelator 1,10‐phenanthroline.
Characterization of Activators and Inhibitors of Protein Kinase Cμ
Authors: Johannes Franz‐Josef , Prestle Jürgen , Dieterich Sabine , Oberhagemann Petra , Link Gisela , Pfizenmaier Klaus
Published: March 2005

In order to investigate regulatory mechanisms and to identify potential substrates of a novel member of the protein kinase C (PKC) family, PKCμ, specific antibodies have been raised against unique amino‐ and carboxy‐terminal regions. PKCμ kinase activity was studied upon immunoprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogeneous PKCμ gene. Cell fractionation revealed that PKCμ is predominantly found in the particulate fraction, suggesting an association with the membrane or membrane‐bound structures.In vitrokinase assays with immunoprecipitated PKCμ demonstrated a Ca2+independent enhancement of constitutive autophosphorylation activity by phosphatidylserine. Despite a limitedin vitrophorbol ester response, an apparent phorbol ester activation of PKCμ was observed when cell cultures, instead of immunoprecipitated enzyme, were treated with either phorbol 12‐myristate 13‐acetate or 1,2 dioleoyl‐sn‐glycerol. Bothin vitroautophosphorylation and substrate phosphorylation of myelin basic protein and histone III were enhanced under these conditions. However, long‐term treatment with the phorbol ester did not result in downregulation of PKCμ protein levels and kinase activity. Studies with several protein kinase inhibitors revealed a novel sensitivity profile of PKCμ, with no inhibition by calphostin C, reduced sensitivity to staurosporine but, compared to other PKCs, an approximately 60‐fold higher sensitivity to the selective PKA inhibitor H89. Together, the data presented here show that localization of PKCμ and regulation of its kinase activity differ from that of other PKCs suggesting a novel function of PKCμ in intracellular signal pathways.
A Seven‐iron Ferredoxin from the Thermoacidophilic Archaeon Desulfurolobus ambivalens
Authors: Teixeira Miguel , Batista Rita , Campos António P. , Gomes Cláudio , Mendes Joaquim , Pacheco Isabel , Anemuller Stefan , Hagen Wilfred R.
Published: March 2005

A seven‐iron ferredoxin was isolated from aerobically grown cells of the hyperthermoacidophilic archaeon Desulfurolobus ambivalens (DSM 3772). The protein is monomeric, with an apparent molecular mass of 15 kDa and contains 7 iron atoms/molecule. The N‐terminal sequence shows a large similarity (70% identity) with that of the ferredoxin isolated from the archaeon Sulfolobus acidocaldarius. The EPR characteristics in both the native (oxidized) and dithionite‐reduced states of this protein allowed an unequivocal identification of a [3Fe‐4S]1+/0 center, with a reduction potential of −270 ± 20 mV, at pH 7.5. The protein also contains a [4Fe‐4S]2+/1+ center with a very low reduction potential (Eo = ‐540 mV, pH 7.0), which yields a rhombic EPR spectrum upon reduction with sodium dithionite at high pH. The reduction potentials of both centers are slightly pH dependent between pH 6 and 9. The [3Fe‐4S] ferredoxin center is able to accept electrons from pyruvate oxidase and NADH oxidase isolated from D. ambivalens. This ferredoxin is present in large amounts (at least 130 mg/kg wet cells), which allowed the unequivocal observation of oxidized [3Fe‐4S] clusters in intact D. ambivalens cells.
The Contribution of Glucose Cycling to the Maintenance of Steady‐State Levels of Lactate by Hepatocytes During Glycolysis and Gluconeogenesis
Authors: Phillips John W. , Clark Dallas G. , Henly Debra C. , Berry Michael N.
Published: March 2005

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 min after which steady‐state concentrations of these metabolites became established. The rate of glycolysis, determined with [6‐3H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one‐third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20–80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose.

Rates of glycolysis were also measured using 40 mM [6‐3H]glucose and [U‐14C]glucose added alone, or in combination with a steady‐state lactate concentration (3 mM). There was no effect on the rate of glycolysis determined with [6‐3H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflects the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 μmol C3 equivalents · min−1· g−1 respectively. Identical studies employing [U‐14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady‐state conditions and that the formation of lactate from [U‐14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production.
Kinetics of β‐1,3 Glucan Interaction at the Donor and Acceptor Sites of the Fungal Glucosyltransferase Encoded by the BGL2 Gene
Authors: Goldman Robert C. , Sullivan Patrick A. , Zakula Dorothy , Capobianco John O.
Published: March 2005

Formation of branched glucan, glucan‐glucan cross links, and glucan‐chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor β‐1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor β‐1,3 glucan, yielding a linear glucan containing a β‐1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429–441], An apparent Km of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent Km of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H2O competing for reaction at the activated donor site, thus resulting in hydrolysis.

However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for β‐1,3 glucan as acceptor, in as much as no transfer was detected when either hexa‐N‐acetyl‐chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of β‐1,3 glucan into the previously existing cell wall structure.
The Role of an Enolase‐Related Molecule in Plasminogen Binding to Cells
Authors: Redlitz Alexander , Fowler Bruce J. , Plow Edward F. , Miles Lindsey A.
Published: March 2005

The α isoform of enolase is a candidate plasminogen receptor on U937 monocytoid cells [Miles, L. A., Dahlberg, C. L., Plescia, J., Felez, J., Kato, K. & Plow, E. F. (1991) Biochemistry 30, 1682–1691]. In the present study, an enolase‐related molecule was detected on the surfaces of peripheral blood monocytes and neutrophils by fluorescence‐activated cell sorting. A mRNA transcript encoding a unique membrane form of an enolase‐related molecule was not detected by Northem‐blotting and primer‐extension analyses, consistent with the cell‐surface protein being authentic α‐enolase. Both the α and β isoforms of purified enolase, bound plasminogen with an affinity similar to that of the cell surface. Moreover, immunopurified α‐enolase enhanced plasminogen activation by tissue plasminogen activator and blocked the binding of plasminogen to α2‐antiplasmin, mimicking functions arising from the association of plasminogen with cells. The interaction of the enolase isoforms with plasminogen was dependent upon recognition of the C‐terminal lysyl residue of the enolases by the lysine‐binding sites of plasminogen, as the interaction was blocked by (a) peptides with C‐terminal lysine residues and (b) an antibody to the C‐terminal aspect of enolase. A monoclonal antibody was developed, characterized and utilized to quantify the enolase molecules present on the surface of U937 cells. A substantial number of molecules, 1.8×106/cell, was present, accounting for approximately 10% of the plasminogen‐binding capacity of these cells. These studies clearly establish the role of enolase as a cell‐surface plasminogen‐binding site with profibrinolytic functions.
Characterization of 45‐kDa/54‐kDa HSP27 Kinase, a Stress‐Sensitive Kinase Which may Activate the Phosphorylation‐Dependent Protective Function of Mammalian 27‐kDa Heat‐shock Protein HSP27
Authors: Huot Jacques , Lambert Herman , Lavoie Josée N. , Guimond Alain , Houle François , Landry Jacques
Published: March 2005

Heat‐shock protein 27 (HSP27) is a major target of phosphorylation upon cell stimulation with a variety of agents and has been suggested to have a phosphorylation‐regulated function at the level of actin filaments. Here we investigated comparatively the mechanisms of HSP27 phosphorylation by oxidative stresses, exposures to tumor necrosis factor (TNF), heat shock and growth factors. Extracts of Chinese hamster or human cells exposed to H2O2, xanthine/xanthine oxidase, menadione or TNF contained up to 15‐fold more HSP27 kinase activity than comparable extracts obtained from control cells. Induction of HSP27 kinase activity by TNF or H2O2 was completely inhibited by first treating the cells with the antioxidant N‐acetyl‐l‐cysteine, suggesting that generation of reactive oxygen metabolites was the key triggering element of this induction. In contrast, prior treatment with acetylcysteine had no or little effect on the induction by thrombin, serum and heat shock. The kinase activity in extracts of cells stimulated by healt shock, H2O2, sodium arsenite, TNF or growth factors was identified by in‐gel renaturation and purified ≈8000‐fold by sequential chromatography. In all cases, the induced kinase activity was entirely associated with two polypeptides of 45 kDa and 54 kDa, identified as mitogen‐activated‐protein kinase‐activated protein (MAPKAP) kinase‐2 based on its reactivation in vitro by 42/44‐kDa MAP kinases, its antigenic properties and its substrate specificity. The 45/54‐kDa HSP27 kinase may play an important role in the cell response to oxidative stress. Overexpression of the wild‐type HSP27 but not of a non‐phosphorylatable form of human HSP27 in Chinese hamster cells conferred resistance to actin fragmentation by oxidative stress generated by H2O2. It is concluded that activation of the 45/54‐kDa HSP27 kinase is a coimmon mechanism of HSP27 phosphorylation to which converge both oxyradical‐dependent and oxyradical‐independent pathways and which may participate in a homeostatic response to stress at the level of actin microfilament.
Interaction Studies Between Elongation Factor Tu and Anthraniloyl‐fluorescent Analogues of Guanyl Nucleotides
Authors: Giovane Alfonso , Balestrieri Ciro , Balestrieri Maria Luisa , Servillo Luigi
Published: March 2005

A fluorescent analogue of GDP, the 3′‐O‐anthraniloyl‐GDP (anl‐GDP) was demonstrated to bind to the elongation factor Tu (EF‐Tu) with an affinity even higher than that of the parent nucleotide. As a consequence of the binding, an increase in fluorescence anisotropy and an emission band arising from non‐radiative energy transfer among the protein intrinsic fluorophores and the labelled nucleotide were observed. Therefore, it was possible to study the exchange kinetics and the equilibrium between the protein‐bound labelled GDP and the natural nucleotide through modifications, occurring during the course of the reaction, of fluorescence anisotropy and non‐radiative energy transfer. In this way, it was also easily proven that, in the presence of aurodox (N ‐methylkirromycin), an antibiotic impairing EF‐Tu biological function, the exchange kinetics between the protein‐bound labeled GDP and the natural nucleotide was faster. Moreover, it was also found that the labelled nucleotide is recognized as a substrate by pyruvate kinase, being converted by this enzyme, in the presence of phosphoenolpyruvate, into anl‐GTP. Pyruvate kinase is also able to convert, in the presence of phosphoenolpyruvate, the complex EF‐Tu · anl‐GDP into the complex EF‐Tu · anl‐GTP. The fluorescence properties of the 3′‐O‐anthraniloyl‐labeled guanyl nucleotides and their feature as excellent acceptors of fluorescence arising from protein intrinsic fluorophores, may make these compounds useful for structural and binding studies on guanosine‐nucleotide‐binding proteins.
A Two‐Dimensional NMR Study of Bovine Cu, Co Superoxide Dismutase
Authors: Sette Marco , Paci Maurizio , Desideri Alessandro , Rotilio Giuseppe
Published: March 2005

Bovine Cu, Co superoxide dismutase has been investigated by two‐dimensional NMR with regard to the resonances arising from protons that surround the copper site. These protons have magnetic properties that are intermediate between those belonging to residues coordinated to the paramagnetic metal center and the diamagnetic residues. Nuclear Overhauser enhancement spectroscopy spectra have been recorded by using a new procedure and with different mixing times and different spectral widths, in order to observe dipolar connectivities between isotropically shifted and diamagnetic resonances. Scalar‐correlated spectra were obtained with both correlation spectroscopy and total‐correlation spectroscopy experiments. The original X‐ray coordinates of the Cu, Co enzyme were used in order to obtain the appropriate interproton distances. The data allowed us to assign more than 20 new resonances to protons which cover a wide region around the copper ion, including two protons belonging to the catalytically important Arg141 residue. The results represent significant progress in the effort to elucidate the three‐dimensional features of the region surrounding the active site of Cu, Zn superoxide dismutase in solution and a tool for a deeper investigation of the reaction mechanism of the enzyme.
Primary Structure and Tissue Distribution of Human 4‐Aminobutyrate Aminotransferase
Authors: De Biase Daniela , Barra Donatella , Simmaco Maurizio , John Robert A. , Bossa Francesco
Published: March 2005

cDNA encoding human 4‐aminobutyrate aminotransferase (aminobutyrate:2‐oxoglutarate aminotransferase) was prepared by polymerase chain reaction using mRNA from human neuroblastoma cells as the template and oligonucleotides synthesized on the basis of the information obtained from direct protein sequencing. The cDNA‐deduced sequence enabled peptides, sequenced by automated Edman degradation, to be aligned for confirmation of the complete primary structure. The results are compared with the recently published sequence of the rat enzyme deduced entirely from DNA sequencing [Medina‐Kauwe, L. K., Tillakaratne, N. J. K., Wu, J.‐Y. & Tobin, A. J. (1994) J. Neurochem. 62, 1267–1275]. Although the sequences are almost identical for most of their length, they differ in a segment of 36 residues. Almost complete identity of the two sequences is established if it is assumed that a frame‐shift error was introduced into the reported rat cDNA sequence. The human cDNA was used to probe for the presence of 4‐aminobutyrate aminotransferase mRNA in human tissues and a significant transcript was found in heart, placenta and in tissues usually associated with the expression of this enzyme.
Inhibition by Modified Oligodeoxynucleotides of the Expression of Type‐1 Plasminogen Activator Inhibitor in Human Endothelial Cells
Authors: Cierniewski Czeslaw S. , Babinska Anna , Swiatkowska Maria , Wilczynska Malgorzata , Okruszek Andrzej , Stec Wojciech J.
Published: March 2005

Antisense phosphodiester and phosphorothioate oligodeoxynucleotides (23‐residue or 24‐residue oligodeoxynucleotides) were constructed for sequences of type‐1 plasminogen‐activator‐inhibitor mRNA to assess their capability to modulate type‐1 plasminogen‐activator‐inhibitor‐mediated fibrinolysis. Antisense oligodeoxynucleotides were targeted at the mRNA sequence coding a signal peptide, at a part of the reactive center Ile342–Pro349, and at an internally translated segment Asn265–Leu272. The effect of antisense oligonucleotides on the concentration of type‐1 plasminogen activator inhibitor in conditioned media and human endothelial cells was determined by the activity test with fibrin as a substrate, and by immunoprecipitation after metabolic labelling of cells with [35S]methionine. Three phosphorothioate oligodeoxynucleotides were specifically inhibitory while phosphodiester oligodeoxynucleotides with the same sequence did not show any activity. Phosphorothioate oligodeoxynucleotides 2, 4 and 6 inhibited the synthesis of type‐1 plasminogen activator inhibitor in endothelial cells in a time‐dependent and concentration‐dependent manner. These data suggest that antisense oligodeoxynucleotides may be useful in the design of antithrombolytic therapeutics.
Expression and Purification of the High‐affinity Phosphate Transporter of Saccharomyces cerevisiae
Authors: Berhe Abraham , Fristedt Ulrika , Persson Bengt L.
Published: March 2005

The plasma membrane high‐affinity phosphate permease of Saccharomyces cerevisiae has been overproduced as a stable membrane‐bound chimeric protein in Escherichia coli. Construction of a chimera between the permease and a peptide containing 10 consecutive histidine residues allowed selective binding of the chimera to a chelating column charged with Ni2+, and elution with imidazole in a high state of purity. Approximately 5 mg purified His10‐permease was obtained from 3 g (wet mass) cells. The purified phosphate permease chimera catalyzes uncoupler‐sensitive phosphate transport after reconstitution into proteoliposomes.
Activation of the Double‐Stranded‐RNA‐Activated Protein Kinase and Induction of Vascular Cell Adhesion Molecule‐1 by Poly (I) · Poly (C) in Endothelial Cells
Authors: Offermann Margaret K. , Zimring James , Mellits Kenneth H. , Hagan M. Kelly , Shaw Reneé , Medford Russell M. , Mathews Michael B. , Goodbourn Stephen , Jagus Rosemary
Published: March 2005

Double‐stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM‐1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM‐1 is also induced by the cytokine interleukin 1β (IL‐1β), activation of the dsRNA‐activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL‐1β. Incubation of HUVE cells with the synthetic dsRNA, poly (I) · poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2α, and increased activation of the transcription factor NF‐кB. Promoter analysis in HUVE cells using a VCAM‐1 promoter linked to CAT reporter gene demonstrates that poly (I) · poly (C) responsiveness resides in the minimal VCAM‐1 promoter that contains two NF‐кB sites, and deletion of the NF‐кB sites eliminates basal and poly (I) · poly (C)‐induced CAT activity, supporting the importance of NF‐кB in the poly (I) · poly (C)‐mediated induction of VCAM‐1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating IкBα (the inhibitory subunit of NF‐кB) in a dsRNA‐dependent manner. This suggests that phosphorylation of IкBα by PKR could be an initial step in the activation of NF‐кB by dsRNA. NF‐кB is also activated by IL‐1β in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2α phosphorylation. Poly (I) · poly (C) induces VCAM‐1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL‐1β. Although phosphorylation of eIF2α interferes with protein translation, sufficient VCAM‐1 mRNA translation occurs in response to poly (I) · poly (C) to yield VCAM‐1 protein levels that are similar to levels that are induced by IL‐1bT. This suggests that the higher, sustained VCAM‐1 mRNA levels that occur in response to incubation with poly (I) · poly (C) compensate for the partial translational block resulting from increased eIF2α phosphorylation. These studies indicate that transcrip‐tional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM‐1 gene expression in HUVE cells.
Localized Chemical Reactivity in Double‐Stranded DNA Associated with the Intercalative Binding of Benzo[e]Pyridoindole and Benzo[g]Pyridoindole Triple‐Helix‐Stabilizing Ligands
Authors: Bailly Christian , Marchand Christophe , Nguyen Chi Hung , Bisagni Emile , Garestier Thérèse , Hélène Claude , Waring Michael J.
Published: March 2005

Footprinting with methidiumpropyl‐EDTA · FeII has been used to map the binding sites on duplex DNA of two closely related benzopyridoindole derivatives which selectively stabilize triple‐helical DNA–oligonucleotide complexes. Both ligands bind to many sites, including certain oligopurine · oligopyrimidine tracts, with a weak preference for some (but not all) sequences rich in A · T base pairs. This indifference to primary sequence, with evidence of binding to the commonly disfavoured (A)n· (T)ntracts, may at least partially explain why the ligands stabilize triplex structures composed of T · A · T pairings. Neither 3‐methoxy‐7H‐8‐methyl‐11‐[(3′amino)propylamino]benzo[e]pyrido[4, 3‐b]indole (BePI) nor 3‐methoxy‐7‐[3′‐diethylamino)propylamino]‐10–methyl‐11H‐benzo[g]pyrido[4,3‐b]indole (BgPI) affect the reaction of dimethyl sulphate or potassium tetrachloropalladinate with the N7 of purines in the major groove, but both enhance the reactivity of purines (mostly adenine residues) towards diethylpyrocarbonate, both proximal and distal to their identified binding sites. With potassium permanganate and osmium tetroxide/pyridine, probes for the accessibility of the 5,6 double bond of pyrimidine residues, BgPI has a more potent effect than BePI and, generally, the reaction with KMnO4 is more pronounced than that with OsO4. BgPI conspicuously potentiates the oxidation of pyrimidines in the triplet sequences 3′‐ATA, 3′‐GTA and 3′‐GCA, whereas BePI enhances the reactivity of OsO4 towards thymine in sequences 3′‐ATYR, with no effect on cytosine residues. Thus, despite their structural homology and common lack of specific sequence preferences, the two benzopyridoindole derivatives induce distinct conformational changes in duplex DNA, not just within the sites where footprints can be detected.
Molecular Cloning and Functional Expression of the Gene Encoding the Human Proteinase‐Activated Receptor 2
Authors: Nystedt Sverker , Emilsson Kjell , Larsson Anna‐Karin , Strömbeck Bodil , Sundelin Johan
Published: March 2005

We previously reported the molecular cloning of a mouse guanosine‐nucleotide‐binding‐protein‐coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase‐activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern‐blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase‐activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.
Conserved Alternative Splicing in the 5′‐Untranslated Region of the Muscle‐Specific Enolase Gene
Authors: Oliva Daniele , Venturella Silvana , Passantino Rosa , Feo Salvatore , Giallongo Agata
Published: March 2005

We report here the isolation and characterization of cDNAs covering the 5′‐end region of mouse and rat mRNAs that encode the β or muscle‐specific isoform of the glycolytic enzyme enolase. As previously determined for humans, two classes of β‐enolase transcripts with distinct sequences in their 5′‐untranslated regions are present in both mouse and rat muscles. A mechanism of alternative splicing, conserved from mouse to man, generates the two forms of mRNA. Secondary‐structure predictions indicated that, in all cases, a more stable secondary structure could exist in the 5′ end of the message with the longer leader. In vitro transcripts containing defined human or mouse 5′‐untranslated sequences were obtained by fusion of the different cDNA clones and tested for their relative translational efficiencies in rabbit reticulocyte lysates. Transcripts containing the human long and short leader sequences showed differences in the translational rate, suggesting a role for the 5′‐untranslated region in the regulation of translation. No detectable difference was found between transcripts with the two distinct mouse leader sequences. In addition, both transcripts are bound to polysomes and are equally distributed along differently sized polysomes in C2C12 myogenic cells. The relative expression of the two spliced forms in developing and adult muscle tissues by means of reverse transcription and polymerase chain reaction did not show a stage‐specific or a tissue‐type‐specific pattern. A putative functional role for the 5′‐untranslated sequences of β‐enolase transcripts is discussed.
α2‐Macroglobulin Receptor Mediates Binding and Cytotoxicity of Plant Ribosome‐Inactivating Proteins
Authors: Cavallaro Ugo , Nykjaer Anders , Nielsen Morten , Soria Marco R.
Published: March 2005

It has been proposed that unconjugated type I ribosome‐inactivating proteins (RIP) enter cells through passive mechanisms such as fluid‐phase pinocytosis. However, some observations, such as the difference in sensitivity to type I RIP among different cell types, and the organ‐specific toxicity of type I RIP, indicate a specific mechanism for the entry of these proteins into target cells. The α2‐macroglobulin receptor (α2MR) is responsible for the binding and endocytosis of several ligands, including α2‐macroglobulin/proteinase complexes, plasminogen‐activator‐inhibitor complexes, apoE‐enriched β‐very low density lipoproteins, and lipoprotein lipase. Here we demonstrate that saporin, a potent type I RIP, binds specifically to purified α2MR and the binding is prevented by some α2MR ligands. Moreover, the occupancy of specific ligand‐binding sites on cell surface α2MR decreases the cytotoxicity of saporin. The A chain of ricin, a type II RIP, also interacts with α2MR. This, and the fact that saporin and ricin A chain both interact also with α2‐macroglobulin, indicates a general mechanism of complex interactions between RIP and cellular membranes that is mediated by α2‐macroglobulin and the α2MR system.
Reversible Binding and Inhibition of Catalase by Nitric Oxide
Authors: Brown Guy C.
Published: March 2005

The interactions between nitric oxide (NO), H2O2, and catalase were investigated. H2O2 did not cause detectable breakdown of NO in the absence of catalase, but did cause NO breakdown in the presence of catalase. Catalase bound NO, and NO rapidly and reversibly inhibited catalase with a Ki of 0.18 μM. The significance of these results for NO cytotoxicity is discussed.
Solution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum
Authors: Bertini Ivano , Donaire Antonio , Feinberg Benjamin A. , Luchinat Claudio , Picciolp Mario , Yuan Huaiping
Published: March 2005

Following the recently developed approach to the solution structure of paramagnetic high‐potential iron‐sulfur proteins, the three‐dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H‐NMR. The X‐ray structure is not available. The protein contains 55 amino acids and two [4Fe‐4S] clusters. In the oxidized state, the clusters have S= 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non‐selective T1 smaller than 25 ms.

The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN‐Hα, H‐bond constraints and dihedral angle constraints on the Cys X2 angles have been generated by using a recently derived Karplus‐type relationship for the hyperfine shifts of cysteine βCH2protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root‐mean‐square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.‐M. (1994) J. Mol Biol. 243, 683–695]. The two proteins are very similar in the overall folding, secondary structure elements and side‐chain orientations. The Cα root‐mean‐square deviation values between the X‐ray‐determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3–53). Discrepancies in residues 26–28 may arise from the disorder observed in the X‐ray structure in that region.
Fasciola Hepatica Cathepsin L Proteinase Cleaves Fibrinogen and Produces a Novel Type of Fibrin Clot
Authors: Dowd Andrew J. , McGonigle Sharon , Dalton John P.
Published: March 2005

A cathepsin L proteinase secreted by the parasitic helminth Fasciola hepatica can cleave fibrinogen and produce a fibrin clot with a specific activity of 4.7 National Institutes of Health thrombin‐equivalent U/mg. This is the first report of a fibrinogen‐clotting activity aside that of thrombin and the snake venom proteinases, which are all serine proteinases. Clot formation by cathepsin L is not inhibited by the thrombin inhibitor hirudin or by the anti‐polymerant H‐Gly‐Pro‐Arg‐Pro‐OH. The enzyme exerts its activity on fibrinogen in a unique manner. Although the cleavage of fibrinogen may involve the initial removal of fibrinopeptides, additional proteolysis of the α, β and γ fibrinogen polypeptides takes place. SDS/PAGE analysis of the cathepsin‐l‐produced clots revealed that cleavage of the α polypeptide (66kDa) precedes that of the β (52kDa) and γ (46.5kDa) polypeptides. Concurrent with the cleavage of these polypeptides is the appearance of components of 120, 100 and 25 kDa. The appearance of higher molecular‐sized components in the cathepsin L clots suggests that polymerisation involves the formation of molecular interactions that are resistant to boiling in mercaptoethanol and SDS.