bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of 1,5‐Anhydroglucitol from 1,5‐Anhydrofructose in Erythroleukemia Cells
Authors: Suzuki Miho , Kametani Shunichi , Uchida Keisuke , Akanuma Hiroshi
Published: July 2004

The pyranoid polyol 1,5‐anhydroglucitol (1,5AnGlc‐ol) occurs in a wide variety of organisms. In humans, it is present as one of the major monosaccharide components in body fluids and serves as an indicator for glycemic control in diabetic care. However, its metabolic origin and fate have been poorly understood. Here we demonstrate that 1,5AnGlc‐ol is produced from glucose in erythroleukemia cells, K‐562. We show the occurrence of 1,5‐anhydrofructose (1,5AnFru), a derivative of 1,5AnGlc‐ol oxidized at the C2 position, in K‐562 cells. In addition, several pieces of evidence indicated that 1,5AnFru, rather than glucose, was the immediate precursor in 1,5AnGlc‐ol production in erythroleukemia cells: exogenous 1,5AnFru was readily taken up into the cells and reduced to 1,5AnGlc‐ol, but the reverse reaction, oxidation of l,5AnGlc‐ol to l,5AnFru, was scarcely observed. The apparent Kmof the overall cellular reduction for 1,5AnFru was estimated as 70 mg/l. This reduction was markedly inhibited by glucose in the culture medium but not by 1,5AnGlc‐ol or glucitol. Since 1,5AnFru arises from α‐1,4‐glucans through lyase reactions in fungi and algae, we suggest the possibilities that glycogen is the precursor of 1,5AnFru and, therefore, 1,5AnGlc‐ol originates from glycogen in mammals.
Phosphoregulatory Tyrosine of Xenopus Mitogen‐Activated Protein Kinase is out of the Reach of the Enzyme Catalytic Center After Autophosphorylation
Authors: Tokmakov Alexander A. , Sahara Setsuko , Sato Ken‐Ichi , Nishida Eisuke , Fukami Yasuo
Published: July 2004

Autophosphorylation of the recombinant mitogen‐activated protein kinase (MAPK) from Xenopus laevis has been studied to detect the conformational changes in the region of regulatory phosphorylation upon enzyme activation. Slow autophosphorylation of Xenopus MAPK occured predominantly on tyrosine, the major phosphoregulatory site of MAPKs, through an intramolecular mechanism and was accompanied by a low magnitude stimulation of the catalytic activity towards an exogenous substrate, myelin basic protein. Autophosphorylated but not unphosphorylated enzyme was shown to interact with the protein substrate. In constrast to the previously reported reversibility of many tyrosine kinase reactions, the tyrosine phosphorylation of Xenopus MAPK was found to be irreversible in the presence of high ADP concentrations, although ADP could competitively inhibit both autophosphorylation and myelin basic protein phosphorylation. We concluded, therefore, that the phosphoregulatory tyrosine is no more accessible to an intramolecular phosphotransferase reaction and is out of the reach of the enzyme catalytic center after phosphorylation. The conformational changes in the region of regulatory phosphorylation resulted in a reduced immunoprecipitation of autophosphorylated and MAPK‐kinase‐phosphorylated forms of the enzyme by a polyclonal antibody raised against a synthetic peptide corresponding to residues 173–197 of Xenopus MAPK which includes the sites of regulatory phosphorylation. The reduced recognition was not due to the phosphorylation itself, since the antibody efficiently immunoprecipitated SDS‐denatured forms of the phosphorylated enzyme. The antibody was not a neutralizing antibody, allowing unphosphorylated MAPK to undergo autophosphorylation while in the immune complex. However, autophosphorylation caused a release of phosphorylated enzyme from the immune complex, suggesting that dramatic conformational changes, which could even overcome the antibody constraints, took place in the phosphoregulatory region of MAPK upon enzyme activation.
The Unglycosylated Extracellular Domain of Type‐II Receptor for Transforming Growth Factor‐β
Authors: Goetschy Jean‐François , Letourneur Odile , Cerletti Nico , Horisberger Michel A.
Published: July 2004

The activation of the human transforming growth factor (TGF‐β) system begins with the cytokine‐induced association of the extracellular domains of two structurally related receptor subunits. To study the protein‐protein interactions between TGF‐β and the ligand‐specific receptor subunit, the extracellular domain of the human TGF‐β receptor type II (TβR‐II) has been expressed as an intracellular protein in insect cells using the baculovirus expression system. The cDNA construct was engineered to encode amino acids 24–159 (the signal sequence 1–23 was lacking) preceded by one initiator methionine residue and six histidine residues added at the carboxy terminus. The soluble receptor accumulated in the cytoplasm of infected cells and was purified by one‐step nickel‐chelate affinity chromatography. The purified protein was not glycosylated; it migrated as a single band of apparent mass 19.5 kDa in SDS/polyacrylamide gels, and had a homogenous N‐terminal sequence. We have established a solid‐phase binding assay using radioiodinated TGF‐β3 and capture antibodies to immobilize the soluble receptor. In this assay, the apparent dissociation constant of the TGF‐β type‐II receptor ectodomain for TGF‐β3 was approximately 150 nM (this value is approximately 1000‐fold higher than that of the cell‐membrane receptor complex of living cells). The affinity of TGF‐β3 for the unglycosylated ectodomain of TβR‐II from insect cells was lower than the affinity for the recombinant glycosylated ectodomain TβR‐II from mouse cells. The novel assay has been used to characterize affinities and specificities of TGF‐β3, TGF‐β2, corresponding mutants and hybrid proteins, as well as a related protein, BMP‐2. The assay could also be used to search for inhibitors.
The Cys292→Ala Substitution in Protein R1 of Class I Ribonucleotide Reductase from Escherichia Coli has a Global Effect on Nucleotide Binding at the Specificity‐Determining Allosteric Site
Authors: Ormö Mats , Sjöberg Britt‐Marie
Published: July 2004

Ribonucleotide reductase from aerobically grown Escherichia coli is allosterically regulated, both with respect to general activity and substrate specificity. Protein R1, the homodimeric enzyme component which harbours binding sites for allosteric effectors (nucleoside triphosphates) as well as substrates (ribo‐nucleoside diphosphates), has been engineered at Cys292 close to the dimer interaction area. This residue was earlier shown to be specifically photoaffinity labelled with the allosteric nucleotide dTTP. In this study the effect of the Cys292→Ala substitution is shown to be an overall diminished nucleotide binding at the specificity site reflected in Kd values for dTTP, dGTP and dATP higher by more than one order of magnitude with respect to wild type. The mutant protein's interaction with other protein components of the ribonucleotide reductase system was unaffected by the mutation. These results show that Cys292 in protein R1 of class I ribonucleotide reductase from E. coli is located in the allosteric specificity site.
Kinetic Characterization of the Neutral Protease Vimelysin from Vibrio Sp. T1800
Authors: Kunugi Shigeru , Koyasu Akira , Kitayaki Moto , Takahashi Saori , Oda Kohei
Published: July 2004

The kinetics of the hydrolysis of dipeptide and tripeptide substrates by the recently discovered neutral protease from Vibrio species T1800 (vimelysin) were studied. In the pH dependence of the apparent second‐order rate constant, the pKa2 value of vimelysin (≈6.5) was significantly lower than thermolysin (8.3). although the pKa2 (≈ 5.1) values were comparable (5.0). The kcat/Km(lim) parameter for hydrolysis of Fua‐Gly‐PheNH2 (Fua = furylacryloyl) was more than sevenfold greater than for Fua‐Gly‐LeuNH2. This higher specificity for Fua‐Gly‐PheNH2 was deduced for both kcat and Km parameters. Fua‐Phe‐PheNH2 showed the highest kcat/Km(app) value of the six substrates studied. The discrimination between Phe/Leu at the P1′ site was most evident when the P1 site was not sufficiently filled.

Reflecting the characteristically high proteolytic activity of vimelysin at lower temperatures [Oda, K., Okayama, K., Okutomi, K., Shimada, M., Sato, R. & Takahashi, S. (1996)Biosci. Biotech. Biochem. 60, 463–467], the Arrhenius plot of the apparent second‐order rate constant for the hydrolysis of Fua‐Gly‐LeuNH2 showed an inverse temperature dependence; higher reaction rates were observed at lower temperatures. This was not merely due to the pKa shift nor to thermal denaturation of the enzyme coupling, but rather to the kcat(app) parameter, which alone showed an inverse temperature dependence. A model containing two temperature‐dependent forms of the active enzyme was postulated to explain this unique temperature dependence.
Inhibition of Zinc‐Induced Metallothionein mRNA Accumulation by Gonadotropin‐Releasing Hormone in Human Hepatocarcinoma Cell Line HepG2
Authors: Pati Debananda , Habibi Hamid R. , Gedamu Lashitew
Published: July 2004

Recently we have demonstrated that the human hepatocellular carcinoma‐derived cell lines, HepG2 and HuH7, contain gonadotropin‐releasing hormone (gonadoliberin) receptors and respond to various molecular forms of gonadoliberin in terms of suppressed proliferation in vitro. This study provides the first demonstration that gonadoliberin inhibits the zinc‐induced production of metallothionein mRNA in HepG2 and HuH7 cells. Administration of gonadoliberin agonist (gonadoliberin‐A) inhibited the Zn‐induced metallothionein mRNA level in a time‐related and dose‐related manner. The effect of gonadoliberin‐A was found to be specific, because concomitant treatment with a gonadoliberin antagonist (gonadoliberin‐ANT) blocked gonadoliberin‐A inhibition of metallothionein mRNA accumulation. Furthermore, the gonadoliberin‐A‐induced inhibition of Zn‐mediated metallothionein accumulation was found to correlate closely with suppresion of cell proliferation and [3H] thymidine uptake in these cells. It is known that the metal‐binding protein metallothionein plays an important role in tumor cell pathobiology and resistance to chemotherapeutic drugs. The present findings may have important implications in the development of an effective chemotherapy for treatment of human liver cancer, in part, by improving the sensitivity of tumor cells through suppression of metallothionein production by gonadoliberin peptides.
Molecular Basis of the Variable Mitochondrial and Peroxisomal Localisation of Alanine‐Glyoxylate Aminotransferase
Authors: Oatey Paru B. , Lumb Michael J. , Danpure Christopher J.
Published: July 2004

The molecular basis of the variable species‐specific peroxisomal andor mitochondrial targeting of the enzyme alanine‐glyoxylate aminotransferase 1 (AGT) has been studied in human fibroblasts by confocal immunofluorescence microscopy after intranuclear microinjection of various human, rabbit, marmoset, and feline ACT cDNA constructs. The expression of full‐length human and rabbit AGT cDNA led to an exclusively peroxisomal distribution of ACT. However, the distribution of feline and marmoset AGT depended on the cDNA construct injected. In both species, injection of the short cDNAs (from transcripts that occur naturally in marmoset liver but not in feline liver) led to an exclusively peroxisomal distribution. However, injection of the long cDNAs (from transcripts that occur naturally in both species) led to most of the AGT being targeted to the mitochondria and only a small, yet significant, fraction to the peroxisomes. Reintroduction of the ‘ancestral’ first potential translation initiation site into human AGT cDNA led to an ‘ancestral’ distribution of AGT (i.e. both mitochondrial and peroxisomal). Deletion of the second potential translation start site from the long feline cDNA led to a distribution that was almost entirely mitochondrial, which suggests that most peroxisomal AGT encoded by the long cDNA results from internal translation initiation from this site with the consequent loss of the N‐terminal mitochondrial targeting sequence. Expression of rabbit cDNA and the short marmoset and feline cDNAs in cells selectively deficient in the import of peroxisomal matrix proteins showed that peroxisomal AGT in all these species is imported via the peroxisomal targeting sequence type 1 (PTS1) import pathway. The almost complete functional dominance of the N‐terminal mitochondrial targeting sequence over the C‐terminal PTS, which was not due to any direct interference of the former with peroxisomal import, was maintained even when the unusual PTSl of AGT (KKL in human) was replaced by the prototypical PTSl SKL. The results demonstrate that the major determinant of alanine‐glyoxylate aminotransferase subcellular distribution in mammals is the presence or absence of the mitochondrial targeting sequence rather than the peroxisomal targeting sequence. Various strategies have arisen during the evolution of mammals to enable the exclusion of the mitochondrial targeting sequence from the newly synthesised polypeptide, all of which involve the use of alternative transcription and/or translation initiation sites.
Changes in the Levels of Glycerophosphoinositols During Differentiation of Hepatic and Neuronal Cells
Authors: Falasca Marco , Marino Maria , Carvelli Angela , Iurisci Cristiano , Leoni Silvia , Corda Daniela
Published: July 2004

Glycerophosphoinositols are metabolites formed by a phosholipase A2 and a lysolipase specifically acting on membrane phosphoinositol lipids. High levels of these compounds characterize epithelial cells and fibroblasts transformed by ras and other cellular oncogenes. Here we have analyzed the glycerophosphoinositol levels in cells that are considered models of cell differentiation. Using rat hepatocytes at different stages of liver development we have shown that the glycerophosphoinositol basal levels of fetal cells were up to fourfold higher than in adult hepatocytes. No changes in glycerophosphoinositol were observed in regenerating rat liver, a model of differentiated cells proliferating in a synchronous manner, where only glycerophosphoinositol 4‐phosphate increased by 80%. Similarly to fetal hepatocytes, a modest but significant increase (30%) in the levels of glycerophosphoinositols was observed in undifferentiated NG‐108‐15 cells as compared to the same cells induced to differentiate by CAMP. In a different neuronal cell line, PC12 cells, increased glycerophosphoinositol levels characterized the differentiated cells. Based on these observations we suggest that high glycerophosphoinositol levels characterize cellular phenomena associated with the activation of ras/mitogen‐activated protein kinase pathways.
Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1
Authors: Kjøller Lars , Martensen Pia M. , Sottrup‐Jensen Lars , Justesen Just , Rodenburg Kees W. , Andreasen Peter A.
Published: July 2004

We have studied conformational changes of type‐1 plasminogen‐activator inhibitor (PAI‐1) during a temperature‐dependent inhibitor–substrate transition by measuring susceptibility of the molecule to non‐target proteinases. When incubated at 0°C instead of the normally used 37°C, a tenfold decrease in the specific inhibitory activity of active PAI‐1 was observed. Accordingly, PAI‐1 was recovered in a reactive‐centre‐cleaved form from incubations with urokinase‐type plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA) at 0°C, but not at 37°C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI‐1 was exposed to a variety of non‐target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in β‐sheet A (sSA) had a temperature‐dependent proteolytic susceptibility, while the P17–P16 (E332‐S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0° and 37°C. In latent and reactive‐centre‐cleaved PAI‐1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti‐PAI‐1 monoclonal antibody maintained the inhibitory activity of PAI‐1 and prevented reactive centre cleavage at 0°C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of s5A and the RCL, but it did not affect cleavage of the P17–P16 bond by V8 proteinase. Our observations suggest that temperature‐dependent conformational changes of β‐sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI‐1 is fully extracted from β‐sheet A in the inhibitory as well as in the substrate form, favoring a so‐called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into β‐sheet A.
Role of C‐Jun and Proximal Phorbol 12‐Myristate‐13‐Acetate‐(PMA)‐Responsive Elements in the Regulation of Basal and PMA‐Stimulated Plasminogen‐Activator Inhibitor‐1 Gene Expression in HepG2
Authors: Arts Janine , Grimbergen Jos , Bosma Piter J. , Rahmsdorf Hans J. , Kooistra Teake
Published: July 2004

Experiments were designed to clarify the role of c‐Jun/c‐Fos and of putative phorbol 12‐myristate‐13‐acetate‐(PMA)‐responsive elements (TREs) in the induction of plasminogen‐activator inhibitor 1 (PAI‐1) gene transcription in the human hepatoma cell line HepG2 by activators of protein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c‐Jun and c‐Fos mRNA and protein levels prior to PAI‐1 induction. This induction of PAI‐1 gene transcription was found to be dependent on ongoing protein synthesis. An essential role of cJun and c‐Fos in basal and PMA‐stimulated transcription of the PAI‐1 gene is demonstrated by our finding that antisense c‐jun and c‐fos oligodeoxynucleotides both strongly reduced basal and PMA‐stimulated PAI‐1 synthesis. Since it has already been shown that two TREs between positions ‐58 and ‐50 and between ‐79 and ‐72 of the PAL1 promoter are essential for basal and PMA‐induced PAI‐1 promoter activity ([16]), we examined binding of nuclear proteins to these elements. The protein‐binding activity to the TRE between positions ‐79 and ‐72 shows very strong PMA induction of an unknown factor, which is not related to c‐Jun or c‐Fos. The TRE binding between positions ‐58 and ‐50 forms two complexes, both containing c‐Jun protein. The faster migrating complex primarily contains c‐Jun homodimers. The amount of the faster migrating complex is enhanced more than 30‐fold in PMA‐treated cells, due to a strongly increased binding of c‐Jun homodimers and, to a minor extent, to binding of c‐Junk‐Fos heterodimers. Dissociation experiments suggest that the c‐Junk‐Fos heterodimers bind with much lower affinity compared to binding of c‐Jun homodimers. Together with the finding that both antisense c‐jun and antisense c‐fos oligodeoxynucleotides reduced the amount of cJun homodimer, we conclude that binding of c‐Jun homodimer to the TRE at positions ‐58 to ‐50 is important in the basal activity and PMA activation of the PAI‐1 promoter in HepG2 cells.
Transgenic Mice Carrying the Human β2‐Adrenergic Receptor Gene with its Own Promoter Overexpress β2‐Adrenergic Receptors in Liver
Authors: André Claudine , Erraji Loubna , Gaston Jesintha , Grimber Gisèle , Briand Pascale , Guillet Jean‐Gérard
Published: July 2004

Up to now, transgenic mice models created to study the physiological impact of alterations in the human β‐adrenoceptor system have only focused on cardiac tissues and carried hybrid transgenes with strong cardiac promoters. We have developed a transgenic mouse strain (F28) carrying the human β2‐adrenoceptor gene with its natural promoter region with the aim of producing a model that more closely reproduces the natural human β2‐adrenoceptor tissue expression pattern. By means of northern blot analyses, using the appropriate probes, we have obtained evidence that (a) the human β2‐adrenoceptor's structural gene is transcribed in several tissues of F28 mice; (b) the tissue distribution pattern of human β2‐adrenoceptor mRNA in F28 mice completely differs from that of mouse β2‐adrenoceptor mRNA; and (c) the tissue distribution pattern of mouse β2‐adrenoceptor mRNA in F28 mice is very similar to that observed in their non‐transgenic littermates. Like humans, F28 mice express human β2‐adrenoceptor mRNA in liver, lung, brain, heart, and muscle. However, unlike humans, F28 mice do not accumulate human β2‐adrenoceptor mRNA in kidney and spleen. By using [125I]idocyanpindolol to label all β‐adrenoceptors and ICI 118,551 to discriminate between the binding to β2‐ and β1‐adrenoceptors we have demonstrated that the β2‐adrenoceptor binding activity increases over control values in F28 mouse tissues that accumulate transgenic mRNA. Accordingly, the number of β2‐adrenoceptors increased slightly over the control values in muscle, heart, brain, and lung of F28 mice, while in liver these receptors were strongly overexpressed. We further showed that transgene β2‐adrenoceptors couple to GTP‐binding proteins, mediate P‐adrenoceptor agonist‐stimulated adenylyl cyclase activation, and cause a strong enhancement of this response in liver membranes of F28 versus control mice. Finally, F28 mice show a phenotype of depressed ponderal development and perturbed hindquarter movements. This unique model should be useful to further investigate β2‐adrenoceptor causal relationships with human pathologies.
Mössbauer, Electron‐Paramagnetic‐Resonance and X‐ray‐Absorption Fine‐Structure Studies of the Iron Environment in Recombinant Human Tyrosine Hydroxylase
Authors: Meyer‐Klaucke Wolfram , Winkler Heiner , Schünemann Volker , Trautwein Alfred X. , Nolting Hans‐F. , Haavik Jan
Published: July 2004

Isoforms (1 ‐4) of human tyrosine hydroxylase (TH) have been expressed in Escherichia coli and purified as apoenzymes (metal‐free). Apo‐human TH binds 1.0 atom Fe(II)/enzyme subunit, and iron binding is associated with an immediate and dramatic (40‐fold) increase in specific activity. For X‐ray absorption fine structure (XAFS) and electron paramagnetic resonance (EPR) measurements the apoenzyme was reconstituted with 56Fe and for Mossbauer measurements with 57Fe. XAFS measurements at the Fe‐K edge of human TH were performed on the native form [Fe(II)‐human TH], as well as after addition of stoichiometric amounts of the substrate tetrahydropterin, the inhibitor dopamine and of H2O2. The addition of dopamine or H2O2 oxidizes the ferrous iron of the native human TH to the ferric state. In both redox states the iron is octahedrally coordinated by low‐Z backscatterers, thus sulfur coordination can be excluded. From the multiple scattering analysis of the EXAFS region is was surmised that part of the iron coordination is due to (3 ± 1) imidazols. Addition of tetrahydropterin does not significantly change the iron coordination of the Fe(II) enzyme. The Mössbauer results confirm the valence states and the octahedral coordination of iron as well as the exclusion of sulfur ligation. Both the EPR spectra and the Mossbauer magnetic hyperfine pattern of dopamine‐ and H2O2‐treated native human TH, were analyzed with the spin‐Hamiltonian formalism. This analysis provides significantly different features for the two forms of human TH: the ferric iron (S= 5/2) of the H2O2‐treated form exhibits a rhombic environment while that of the dopamine‐treated form exhibits near‐axial symmetry. The specific spectroscopic signature of dopamine‐treated human TH, including that of an earlier resonance‐Raman study [Michaud‐Soret, I., Andersson, K. K., Que, L. Jr & Haavik, J. (1995)Biochemistry 34, 5504–5510] is most likely due to the Biolentate binding of dopamine to iron.
The Escherichia Coli‐Derived Fab Fragment of the IgM/κ Antibody IN‐1 Recognizes and Neutralizes Myelin‐Associated Inhibitors of Neurite Growth
Authors: Bandtlow Christine , Schiweck Wolfram , Tai Hsin‐Hsiung , Schwab Martin E. , Skerra Arne
Published: July 2004

A recombinant Fab fragment was prepared from the monoclonal IgM/k antibody IN‐1, which neutralizes central nervous system myelin‐associated neurite growth inhibitors both in vitro and in vivo. The variable domain gene sequences were amplified and cloned after cDNA synthesis from the hybridoma RNA. After insertion into the tet promoter vector pASK85, which provided the constant domains of class IgG1/κ, equipped with a Hish tag, large amounts of the Fab fragment were produced in Escherichia coli by medium cell density fermentation. The Fab fragment was purified to homogeneity by immobilized metal‐affinity chromatography and its biochemical activity was compared with the original IN‐1 antibody. In an assay for neurite outgrowth and fibroblast spreading, the Fab fragment showed a similar neutralizing effect on inhibitory substrate properties of central nervous system myelin as the unpurified IgM, although an approximately tenfold higher concentration was necessary. Immunoprecipitation experiments revealed a more selective antigen‐binding behaviour for the Fab fragment. The Fab fragment was also successfully applied for antigen detection in immunohistochemical analyses. Therefore, the recombinant Fab fragment of IN‐1 shows full functionality in vitro and appears to be well suited for replacing the monoclonal IgM in investigations on fiber tract regeneration in vivo.
Two‐Dimensional 1H‐NMR of Transmembrane Peptides from Escherichia Coli Phosphatidylglycerophosphate Synthase in Micelles
Authors: Morein Sven , Trouard Theodore P. , Hauksson Jón B. , Rilfors Leif , Arvidson Gösta , Lindblom Göran
Published: July 2004

Two 28‐residue peptides, PTLLTLFRVILIPFFVLVFYKKKGKKKG [Pgs‐(6–25)‐peptidyl‐KKKGKKKG; Pgs peptide A] and VEYAGIALFFVAAVLTLWSMLQYLSAAR [Pgs‐(149–176)‐peptide, Pgs peptide E], were synthesized and studied by CD and two‐dimensional 1H‐NMR spectroscopy. The first 20 amino acid residues of Pgs peptide A are identical to one predicted transmembrane segment (Pro6‐Tyr25) of the integral membrane protein phosphatidylglycerophosphate synthase (Pgs) of Escherichiu coli. Pgs peptide E is identical to another predicted transmembrane segment (Val149–Arg176), which is located in the C‐terminal end of this lipid synthase. Pgs peptides A and E were dissolved in methanol or trifluoroethanol or were incorporated into solvent‐free micelles of fully deuterated SDS. In all these systems, CD spectra of both peptides indicated an α‐helical secondary structure. However, peptides that were solubilized in micelles exhibited the highest content of α‐helix as judged from comparison of the CD spectra. Thermodynamically stable isotropic solutions at high peptide concentrations (1–3 mM) could only be obtained with the peptide incorporated in micelles; in organic solvents, significant peptide aggregation occurred. Relatively sharp peaks were obtained with 1H‐NMR spectroscopy of the peptides in SDS micelles, which indicates rapid tumbling of the peptides in the micellar environment. Translational‐diffusion coefficients of the micelles with and without peptide, determined by pulsed‐field‐gradient NMR, showed that the micellar size was unaffected by the solubilized peptide. The radius of the hydrated micelles was estimated to be about 2.7 nm (i.e. the mass of the aggregate is almost 30 kDa). Two‐dimensional NMR spectroscopy of both peptides solubilized in the micelles indicated an α‐helical conformation. This observation is strengthened by an investigation of the hydrogen exchange of the peptide amide protons, where significantly less exchange of the amide protons was observed in the middle of the peptides compared with the ends.
Synthesis, Cytotoxic Properties and Effects on Early and Late Gene Induction of a Chimeric Diphtheria Toxin‐Leukemia‐Inhibitory Factor Protein
Authors: Negro Alessandro , Super Stephen D.
Published: July 2004

Leukemia‐inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria‐toxin‐related LIF gene fusion in which the native receptor‐binding domain of diphtheria toxin was replaced with a gene encoding human LIE The fusion protein expressed from the chimeric tox gene was designated DT‐(1–389)‐LIF‐(2–184)‐peptide. This fusion protein has a deduced molecular mass of 65 980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2–184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single‐step purification of the fusion protein by Ni2+‐resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation‐free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT‐(1–389)‐LIF‐(2–184)‐peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF‐responsive neuroblastoma SH‐N‐BE cells. DT‐(1–389)‐LIF‐(2–184)‐peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT‐(1–389)‐LIF‐(2–184)‐peptide, like that of native diphtheria toxin, required receptor‐mediated endocytosis, passage through an acidic compartment, and delivery of an ADP‐ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT‐(1–389)‐LIF‐(2–184)‐peptide increased c‐fos mRNA expression in SH‐N‐BE cells, only LIF induced proenkephalin and c‐fos promoter activities in cells transiently transfected with c‐fos‐chloramphenicol acetyltransferase and proenkephalin‐chloram‐phenicol acetyltransferase fusion genes. Mutational analysis suggested that the C‐terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT‐(1–389)‐LIF‐(2–184)‐peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF β receptor.
The Role of a Trans‐Proline in the Folding Mechanism of Ribonuclease T1
Authors: Schindler Thomas , Mayr Lorenz M. , Landt Olfert , Hahn Ulrich , Schmid Franz X.
Published: July 2004

Protein folding is often retarded by the cis⇌trans isomerizations of prolyl peptide bonds both in vitro and in vivo. An important role for the folding mechanism is well established for the prolyl peptide bonds that are cis in the native protein, but not for those that are trans. Here we investigated the role of trans‐Pro73 for the folding of ribonuclease T1 (which additionally contains two cis‐prolines) by comparing the wild‐type protein with the Pro→734 Val variant. The Pro73→Val substitution led to a destabilization of the folded protein by 8.5 kJ/mol, which is explained by the strong, 25‐fold increase in the rate of unfolding. In contrast, the rates and amplitudes of the fast and slow refolding reactions were virtually unchanged. trans‐Proline residues remain largely trans after unfolding, and therefore their contributions to the observed folding kinetics should indeed be insignificant for proteins which also contain one or more cis prolines. The cis‐proline residues dominate the kinetics of refolding, because almost all slow‐folding molecules contain the respective incorrect (trans) isomers, and because trans‐cis isomerizations are slower than cis→trans isomerizations. The inability to detect contributions from a trans‐proline to the kinetics of folding does not imply that this proline is non‐essential for folding in the sense that its cis⇌trans isomerization is energetically uncoupled from conformational folding.
Site‐Directed Mutagenesis of Putative Active‐Site Residues in Squalene‐Hopene Cyclase
Authors: Feil Corinna , Süssmuth Roderich , Jung Günther , Poralla Karl
Published: July 2004

Squalene‐hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to sterols. Sequence analysis of SHC revealed a highly conserved aspartate‐rich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclases, which is supposed to be part of the active site. In order to determine the importance of the motif in squalene cyclization, the conserved residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclobacillus acidocaldarius were individually replaced by glutamate, glutamine, glycine, and arginine. With the exception of the [Glu376]SHC mutant, all other substitutions resulted in almost or complete loss of enzyme activity. Compared to that of the wild‐type enzyme, the specific activity of the [Glu376]SHC mutant enzyme was reduced to 10%, accompanied by a significant decrease in the apparent Vmax, whereas the apparent Km remained unchanged. CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp377 are crucial for catalysis and may act as point charges to stabilize intermediate cations. Moreover, for squalene‐hopene cyclase, a high content of α‐helical conformation could be found, providing the first structural information for a triterpene cyclase.
A New Scorpion Venom Toxin Paralytic to Insects that Affects Na+ Channel Activation
Authors: Borchani Lamia , Mansuelle Pascal , Stankiewicz Maria , Grolleau Françoise , Cestèle Sandrine , Karoui Habib , Lapied Bruno , Rochat Hervé , Pelhate Marcel , El Ayeb Mohamed
Published: July 2004

A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetunus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and α‐type insect‐selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As α‐type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike α‐type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross‐antigenic reactivity with depressant toxins. (d) Voltage and current‐clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage‐clamp conditions, BotIT2 specifically acts on Na+ channels by decreasing the peak Na+current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na+‐channel activation.
Refolding and Reassociation of Glycerol Dehydrogenase from Bacillus Stearothermophilus in the Absence and Presence of Groel
Authors: Krauss Oliver , Gore Michael G.
Published: July 2004

The refolding of the tetrameric, metalloenzyme glycerol dehydrogenase (GDH) from Bacillus steurothermophilus has been investigated using stopped‐flow fluorescence and circular dichroism spectroscopy. The effects of metal ions on the refolding of the native enzyme and the refolding of a monomeric mutant ([A208]GDH) have also been studied. The refolding process of the wild‐type enzyme is at least biphasic; 70% of the respective signal changes occur in the first 2 ms followed by a slower process with a half‐life of 3 s. The presence of the metal ion does not affect the slowest biphasic refolding rate, which is virtually the same for all three versions of the enzyme. The presence of GroEL slows down the first phase of refolding.

The reassociation of subunits was examined by measuring the regain in catalytic activity and the enhancement in the fluorescence emission from NADH on binding to the oligomeric form of the enzyme. The rate and extent of reassociation is dependent on enzyme concentration and the extent of reactivation is dependent on the presence of the metal ion. The reassociation process was more efficient in the presence of NADH particularly for the metal‐depleted enzyme (apo‐GDH). The presence of GroEL or GroEL plus ATP leads to a higher yield of reassociation and therefore catalytically active enzyme. The additional presence of Mg‐ATP does not affect the extent of reassociation, but has a small positive effect on the rate of reassociation. These data suggest that GDH is bound weakly to GroEL and that GroES is not required for release of the protein.
The Protein Conformation of Cd‐Substituted Horse Liver Alcohol Dehydrogenase and its Metal‐Site Coordination Geometry in Binary and Ternary Inhibitor Complexes
Authors: Hemmingsen Lars , Bauer Rogert , Bjerrum Morten Jannik , Adolph Hans Werner , Zeppezauer Michael , Cedergren‐Zeppezauer Eila
Published: July 2004

The coordination geometry of the metal at the active site in Cd‐substituted horse liver alcohol dehydrogenase (LADH) has been investigated for the binary complexes of LADH with imidazole, isobutyramide, decanoic acid and C1−, and for the ternary complexes of LADH with NADH and imidazole, NADH and isobutyramide, NAD+ and decanoic acid and NAD+ and Cl−, by using the method of perturbed angular correlation of γ‐rays (PAC). The spectral results are consistent with a flexible structure around the metal for the binary complexes with inhibitors. For ternary complexes, however, a rigid structure is observed. An exception is the ternary complex between LADH, NADH and imidazole, in which the metal site is still flexible. Comparing with available structures determined by X‐ray crystallography, we found a correlation between open structures and flexible metal sites, and between closed structures and rigid metal sites. This indicates that the PAC technique can be applied to distinguish the two conformations in solution.

The spectral parameters, ωo and η, of the experiments, except for the complexes with imidazole, fall into two groups: one with low ωo and one with high ωo, (η is relatively constant in all experiments). In this work it is clarified that the low ωo, values are connected with the presence of a negatively charged solvent ligand. Using an angular‐overlap approach to interpret the results, the low ωo values are found to be compatible with a coordination geometry where the S‐Cd‐S (Cys174 and Cys46 coordinate to the metal) angle is about 110° as suggested in [Hemmingsen, L., Bauer, R., Danielsen, E., Bjerrum, M. J., Zeppezauer, M., Adolph, H. W., Formicka, G. & Cedergren‐Zeppezauer, E. (1995)Biochemistry 34, 7145571531, whereas high ωo values are compatible with an S‐Cd‐S angle of 130°. The presence of a negatively charged metal ligand, therefore, might trigger the movement of the sulfur of Cys174. As it is believed that alcohols coordinate to the metal as alcoholate ions this could be important for catalysis.
Structural and Functional Characterization of Vitronectin‐Derived RGD‐Containing Peptides from Human Hemofiltrate
Authors: Ständker Ludger , Enger Andreas , Schulz‐Knappe Peter , Wohn Karl‐Dieter , Germer Matthias , Raida Manfred , Forssmann Wolf‐Georg , Preissner Klaus T.
Published: July 2004

Bioactive peptides derived from the adhesive plasma protein vitronectin are present at submicromolar concentrations in human hemofiltrate of patients with renal diseases and were isolated by a combination of high‐efficiency chromatographic steps. The structural and functional properties of these peptides were characterized. Sequencing and mass spectrometry revealed the existence of peptide isoforms (5‐6 kDa) which corresponded to the N‐terminus (residues 1 to 44‐50) of vitronectin. The isolated peptides bound directly to plasminogen‐activator inhibitor‐1 (PAI‐1) and were effective competitors of the interaction of PAI‐1 with isolated intact vitronectin or extracellular matrix. These functional properties were indistinguishable from the binding properties of a recombinant fusion protein containing residues 1‐52 of vitronectin linked to a portion of glutathione S‐transferase, expressed in Escherichia coli. Peptides containing the RGD sequence of vitronectin competed for vitronectin binding to the αvβ3 integrin. No indication for direct growth‐factor binding was noted, whereas natural peptides were found associated with PAI‐1 as the major binding protein in plasma. These data demonstrate that functionally active vitronectin‐derived peptides are released by unknown protease(s) from the mature protein and that these peptides are identical, in terms of activity, to recombinant vitronectin fragments. These natural peptides may interact with active PAI‐1 in plasma or at extravascular sites and thereby interfere with established biological functions of intact vitronectin.
α‐Crystallin Stabilizes Actin Filaments and Prevents Cytochalasin‐Induced Depolymerization in a Phosphorylation‐Dependent Manner
Authors: Wang Keyang , Spector Abraham
Published: July 2004

α‐crystallin, a major lens protein of approximately 800 kDa with subunits of about 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress‐induced damage and to share sequence similarity with small heat‐shock proteins, sHsp. It is now demonstrated that this chaperone effect extends to protection of the intracellular matrix component actin. It was found that the powerful depolymerization effect of cytochalasin D could be almost completely blocked by α‐crystallin, αA‐crystallin or αB‐crystallin. However, phosphorylation of α‐crystallin markedly decreased its protective effect. It is suggested that phosphorylation of α‐crystallin may contribute to changes in actin structure observed during cellular remodeling that occurs with the terminal differentiation of a lens epithelial cell to a fiber cell and contributes to cellular remodeling in other cell types that contain α‐crystallin species. This communication presents biochemical evidence clearly demonstrating that α‐crystallin is involved in actin polymerization‐depolymerization dynamics.

It is also shown that α‐crystallin prevented heat‐induced aggregation of actin filaments. α‐crystallin was found to stabilize actin polymers decreasing dilution‐induced depolymerization rates up to twofold while slightly decreasing the critical concentration from 0.23 μM to 0.18 μM. Similar results were found with either α‐crystallin or its purified subunits αA‐crystallin and αB‐crystallin. In contrast to the experiments with cytochalasin D, phosphorylation had no effect. There does not appear to be an interaction between α‐crystallin and actin monomers since the effect of α‐crystallin in enhancing actin polymerization does not become apparent until some polymerization has occurred. Examination of the stoichiometry of the α‐crystallin effect indicates that 2‐3 α‐crystallin monomers/actin monomer give maximum actin polymer stabilization.
Oncostatin M Differentially Regulates Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐3 Gene Expression in Human Synovial Lining Cells
Authors: Gatsios Petros , Haubeck Hans‐Dieter , Van De Leur Eddy , Frisch Wiltrud , Apte Suneel S. , Greiling Helmut , Heinrich Peter C. , Graeve Lutz
Published: July 2004

Tissue inhibitor of metalloproteinases (TIMP) 1, 2 and 3 are related proteins that can form complexes with all known matrix metalloproteinases (MMPs). They inhibit the action of MMPs on extracellular matrix components. The balance of MMPs and TIMPs is important for tissue remodeling and its disturbance is believed to play a crucial role in pathophysiological processes such as tumor metastasis, destruction of cartilage and fibrosis. Cytokines and growth factors were found to regulate TIMPs and MMPs in a complex manner. In order to better understand the role of TIMPs in inflammatory joint diseases we have studied in vitro the regulation of TIMP‐1 and TIMP‐3 by inflammatory cytokines in cultured human synovial lining cells. We found that transforming growth factor β as well as interleukin‐1β induce gene expression of both TIMP‐1 and TIMP‐3. In contrast, oncostatin M, an interleukin‐6‐type cytokine produced by activated T‐lymphocytes and monocytes, had a differential effect on TIMP mRNA levels. After oncostatin M treatment, TIMP‐1 expression was up‐regulated but basal, as well as interleukin‐1β‐induced, TIMP‐3 expression was inhibited. Interleukin‐6 itself had no effect on synovial lining cells but a complex of interleukin‐6 and the soluble interleukin‐h receptor induced activation of signal transducer and activator of transcription (STAT) factors in these cells and regulated TIMP‐1 and TIMP‐3 expression in a similar fashion as oncostatin M. Since TIMP‐3 is matrix‐associated whereas TIMP‐1 is found in many body fluids, the role of oncostatin M during inflammatory processes might be to promote ECM degradation in the local environment but to prevent it systemically.
Location of Cholesterol in Model Membranes by Magic‐Angle‐Sample‐Spinning NMR
Authors: Villalaín José
Published: July 2004

High‐resolution magic‐angle‐sample‐spinning 13C‐NMR was applied to determine the specific location of cholesterol in non‐perturbed multilamellar model membranes formed by egg yolk phosphatidylcholine. 13C spin‐lattice relaxation times of both the phospholipid and cholesterol molecules were measured in the absence and in the presence of Gd3+, a paramagnetic agent, in order to obtain information on molecular distances. The effect of Gd3+ on the spin‐lattice relaxation times of the lipid resonances has an explicit distance dependence, allowing it to be used to evaluate relative distances on a molecular scale. It has been found that cholesterol is placed in such a position that it is not readily exposed to the solvent: the hydrophobic steroid ring is oriented parallel to the membrane phospholipids, the hydroxyl group is in close vicinity to the phospholipid ester carbonyl groups and the isooctyl side chain is deeply buried in the center of the membrane. These data are consistent with an organization such that mixtures of cholesterol and phospholipids present a packing similar to that found in interdigitated lipid bilayer systems.
Complete NMR Structural Elucidation of the Capsular Polysaccharide Adjuvant from Klebsiella I‐714
Authors: Adam Olivier , Rivière Michel , Vercellone Alain , Monsan Pierre F. , Puzo Germain
Published: July 2004

The capsular polysaccharide (CPS) produced by the non‐pathogenic Klebsiella strain 1–714, selected for its immunomodulating activity, has been purified and almost completely detoxified in a previous study [Adam, O., Vercellone, A., Paul, F., Monsan, P. F. & Puzo, G. (1995)Anal. Biochem. 225, 321–3271, The present report concerns the structural elucidation of this CPS by several one‐dimensional and two‐dimensional 1H‐NMR and 13C‐NMR experiments performed on the native molecule. It was found to be a high molecular‐mass branched polymer constituted by a hexasaccharide repeating unit of following structure:

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The presence of two glycosidic substitutions on the Galp residue resulted in a strong overlapping of its proton signals, preventing direct assignment of both proton and carbon resonances. However, assignment could be achieved using the two‐bond and three‐bond 1H‐13C heteronuclear coupling observed in the 1H‐13C heteronuclear multiple‐bond correlation (HMBC) spectrum.