bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of large nucleoprotein complexes upon binding of the high‐mobility‐group (HMG) box B‐domain of HMG1 protein to supercoiled DNA
Authors: Štros Michal , Reich Josef
Published: March 2003

High‐mobility group (HMG) 1 is a relatively highly abundant chromosomal protein with structuralrather than sequence‐specific preference for binding to DNA. HMG1 has two highly related, folded domains A and B (HMG boxes), attached by a short basic region to an acidic C‐terminal domain. We have studied binding of the B‐domain of HMG1 protein and its mutants to supercoiled DNA by a gel‐retardation assay and electron microscopy. Using a gel‐retardation assay, we have demonstrated that HMG1 or HMG1 lacking the acidic C‐terminal domain [i.e. HMG1(A+B) bi‐domain], but not the isolated B‐domain, could preferentially bind supercoiled over‐relaxed closed circular or linear DNA. Mutational analysis of the HMG1 B‐domain revealed that replacement of Lys96 of the extended N‐terminal segment (and much less the neighboring Arg97) and Lys128 of helix II to glutamic acid severely impaired binding of the HMG box domain to supercoiled DNA. The latter mutation within helix II significantly decreased the α‐helical content of the B‐domain as revealed by circular dichroism. We have also shown that mutation of several residues within helix I of the B‐domain, in particular Arg110, resulted in a diminished binding to supercoiled DNA as revealed by intensive smearing and reduced retardation of the protein/DNA complexes. These findings indicated that the extended N‐terminus, helix I and helix II of the HMG1 B‐domain are likely in contact with DNA. Electron microscopy revealed that the B‐domain could bind to supercoiled DNA at higher HMG/DNA molar ratios as oligomeric protein beads with subsequent association of the beads into large nucleoprotein complexes from which many looped DNA molecules emerged. Most of the introduced mutations within all three helices of the B‐domain (involving mainly basic and aromatic residues) abolished formation of the large nucleoprotein complexes even though the binding of the HMG box to supercoiled DNA was retained as revealed by a gel‐retardation assay. A model for the interaction of the B‐domain of HMG 1 with supercoiled DNA is presented and discussed.
Cutting at the right place
Authors: Gomis‐Rüth F. Xavier , Gómez‐Ortiz Mariola , Vendrell Josep , Ventura Salvador , Bode Wolfram , Huber Robert , Avilés Francesc X.
Published: March 2003

Proteinase E is a proteolytic enzyme which belongs to a distinct subfamily of chymotrypsin‐like serine endopeptidases. Its proform from the bovine pancreatic system has been structurally analyzed by X‐ray crystallography for the intact native form, with a 11‐residue N‐terminal activation peptide, in a ternary complex with chymotrypsinogen C and procarboxypeptidase A [Gomis‐Rüth, F. X., Gómez, M., Bode, W., Huber, R. & Avilés, F. X. (1995) The three‐dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C, EMBO J. 14, 4387−4394]. Also for a N‐terminally truncated form, lacking the first 13 residues and called subunit III, a crystal structure is available [Pignol, D., Gaboriaud, C., Michon, T., Kerfelec, B., Chapus, C. & Fontecilla‐Camps, J. C. (1994) Crystal structure of bovine procarboxypeptidase A‐S6 subunit III, a highly structured truncated zymogen E, EMBO J. 8, 1763−1771]. Both structures are well defined by electron density, except for the first 7 residues of subunit III. However, both structures present large deviations of up to 2 nm in several regions, indicating that they correspond to two quite distinct states of low free energy, influenced by very few contacts made via the N‐terminal segment. As no structure of an active proteinase E is known so far, pancreatic porcine elastase has been chosen as a model for this enzyme and an activation mechanism for this distinct serine endopeptidase subfamily is proposed.
Primary cause of mortality in the armyworm larvae simultaneously parasitized by parasitic wasp and infected with bacteria
Authors: Matsumoto Hitoshi , Noguchi Hirofumi , Hayakawa Yoichi
Published: March 2003

Parasitoid wasps never kill their hosts before the wasp larvae emerge from the host. However, almost 100 % of the host armyworm larvae Pseudaletia separata die within 2−3 days by parasitization with the wasp Cotesia kariyai or by injection of polydnavirus, the wasp symbiont virus, when they are simultaneously infected by the pathogenic bacterium Serratia marcescens. The present study was conducted to elucidate the crucial factor causing this larval mortality. An insecticidal protein has been shown to exist in the hemolymph of dying host larvae; it has been purified by procedures consisting of reverse‐phase column extraction, gel filtration and ion‐exchange column chromatography. The purified protein showed a strong insecticidal effect with a median lethal dosage (LD50) of 13 pmol/larva and was estimated to have a molecular mass of 57 kDa. The amino acid sequence of the insecticidal protein was partially characterized and used for isolation and sequencing of the genomic DNA. The deduced amino acid sequence for this protein revealed striking similarity with the metalloprotease of S. marcescens enterobacter.
Conformer selection and differential restriction of ligand mobility by a plant lectin
Authors: Gilleron Martine , Siebert Hans‐Christian , Kaltner Herbert , von der Lieth Claus‐Wilhelm , Kozár Tibor , Halkes Koen M. , Korchagina Elena Y. , Bovin Nicolai V. , Gabius Hans‐Joachim , Vliegenthart Johannes F. G.
Published: March 2003

To study conformational parameters of ligands before and after complex formation with the galactoside‐binding agglutinin of Viscum album L. (VAA) in solution, combined computer‐assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two‐dimensional rotating‐frame nuclear Overhauser effect (ROE) and two‐dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galβ1‐3GlcNAcβ1‐R and Galβ1‐3GalNAcβ1‐R as well as of a synthetic high‐affinity binding partner, i.e. the disaccharide Galβ1‐2Galβ1‐R′, were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between KY combinations of (40+/60+) and (40+/−60+). Calculations of the de‐N‐acetylated sugars revealed that presence of this group did not markedly influence the distribution of low‐energy conformers in the KY, E plot. Occupation of side minima at KY (180+/0+) or (0+/180+) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free‐state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free‐state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galβ1‐3GlcNAcβ1‐R and Galβ1‐2Galβ1‐R′. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One‐ and two‐dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin‐diffusion effects. When the NOE build‐up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galβ1‐3GalNAcβ1‐R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galβ1‐2Galβ1‐R′ in association with a galectin, namely the β‐galactoside‐binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy‐minimum conformations in the KY, E plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.
Lipocalins of boar salivary glands binding odours and pheromones
Authors: Marchese Silvana , Pes Daniela , Scaloni Andrea , Carbone Virginia , Pelosi Paolo
Published: March 2003

Large amounts of an odorant‐binding protein have been isolated from submaxillary glands of mature male pig. This polypeptide molecule is sex‐specific, being absent in females. On electrophoretic gels under denaturing conditions it migrated as a broad band with an apparent molecular mass of around 20 kDa. Electrospray mass spectrometry revealed the presence of three main components, whose mass differences are not interpretable as result of any common post‐translational modifications, indicating the presence of distinct polypeptide chains. N‐terminal Edman degradation yielded a single sequence of 29 amino acids. It includes the lipocalin signature (‐G‐X‐W‐) and shows clear homology with a subclass of odorant‐binding proteins present in mouse saliva, nasal mucus and urine. The purified protein still retained small ligands tightly bound; among them 5α‐androst‐16‐en‐3‐one and 5α‐androst‐16‐en‐3α‐ol, both known sex pheromones for the pig, were identified. The protein also binds 2‐isobutyl‐3‐methoxypyrazine, a good ligand for most odorant‐binding proteins, with a dissociation constant of 5 μM.
Ca2+ and Zn2+ bind to different sites and induce different conformational changes in human calcyclin
Authors: Kordowska Jolanta , Stafford Walter F. , Wang C.‐L. Albert
Published: March 2003

Calcyclin (CaCY) is a member of the S100 subfamily of helix‐loop‐helix (EF‐hand) calcium‐binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30 % enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8−5.8×104 M−1. Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form. The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0×107 M−1. As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence. Saturation occurs at a Zn2+/CaCY ratio of 2 : 1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol‐directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites : Ca2+ binds probably at the EF‐hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.
Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase‐like proteins
Authors: Depège Nathalie , Drevet Joël , Boyer Nicole
Published: March 2003

In Lycopersicon esculentum, two cDNAs encoding glutathione peroxidase (GPX)‐like proteins were isolated, sequenced and characterized as putative phospholipid hydroperoxide :glutathione peroxidases. After mechanical stimulation (rubbing) of the plant, each GPX tomato gene was found to behave differently in terms of inducibility and tissue distribution. The accumulation of GPX transcripts suggests that reactive oxygen species are involved in the plant response to a mechanical stress.
Thermodynamic stability of two variants of xylanase (Xys1) from Streptomyces halstedii JM8
Authors: Ruiz‐Arribas Alberto , Zhadan Galina G. , Kutyshenko Victor P. , Santamaría Ramon I. , Cortijo Manuel , Villar Enrique , Fernandez‐Abalos Jose M. , Calvete Juan J. , Shnyrov Valery L.
Published: March 2003

In a continuation of our earlier study [Ruiz‐Arribas, A., Santamaría, R.I., Zhadan, G. G., Villar, E. & Shnyrov, V. L. (1994) Differential scanning calorimetric study of the thermal stability of xylanase from Streptomyces halstedii JM8, Biochemistry 33, 13787−13791], we used high‐sensitivity differential scanning microcalorimetry, intrinsic tryptophan fluorescence and far‐ultraviolet circular dichroism to study the effect of regional sequence differences on the thermodynamic stability of xylanase (Xys1) from Streptomyces halstedii JM8 (1,4‐β‐
D‐xylanohydrolase,
EC3.2.1.8). Thermal transitions were measured for original xylanase (Xys1S) and two variants. Thermal denaturation of all the xylanases studied revealed two structural domains, each of which, despite its partial irreversibility, follows a two‐state thermal unfolding process under our experimental conditions. Both variants were found to exibit slightly decreased stability, possessing the same activity as the original. The unfolding parameters for each domain of both variants, unlike the situation with wild‐type xylanase (see our previous report), fit some correlations obtained for the most compact globular proteins. The values of enthalpy and entropy of unfolding/residue at 383 K were found to be inversely proportional to residual, well‐regulated structures in unfolded states.
The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a cellulose surface − erosion model
Authors: Väljamäe Priit , Sild Veljo , Pettersson Göran , Johansson Gunnar
Published: March 2003

Introduction of a novel method for the quantification of the cellobiose released made it possible to follow the initial stage of hydrolysis of bacterial microcrystalline cellulose (BMCC) by cellobiohydrolases 1,4‐β‐ D‐glucan‐cellobiohydrolase I (CBH I) and 1,4‐β‐
D‐glucan‐cellobiohydrolase II (CBH II) from Trichoderma reesei. A drastic retardation of the rate of the hydrolysis was observed already at a very low degree of conversion. Earlier‐suggested retardation factors, such as product inhibition by cellobiose or enzyme inactivation, could be discounted as primary causes for the pattern. A model including steric hindrance by non‐productive binding and erosion of the cellulose surface during the processive action of exoenzymes was proposed to describe the rate retardation observed. Simultaneous action of CBH I and CBH II on cellulose was not a prerequisite for synergy between them.
A domain within the tumor suppressor protein APC shows very similar biochemical properties as the microtubule‐associated protein tau
Authors: Deka Jürgen , Kuhlmann Jürgen , Müller Oliver
Published: March 2003

The tumor‐suppressor protein APC (adenomatous polyposis coli) binds to microtubules and promotes tubulin assembly. In vivo the endogenous APC protein is mainly localized at the end of microtubules that are involved in active cell migration. Since most tumor‐specific APC gene mutations lead to the loss of the microtubule binding domain this interaction is assumed to play a crucial role in tumorigenesis. In this study we show that an APC protein fragment (amino acids 2219−2580) within the C‐terminal part is enough to bind to non‐assembled tubulin with high affinity. The binding of APC to tubulin does not lead to an alteration of the intrinsic GTPase activity of the non‐assembled tubulin. The APC protein induces the tubulin assembly in a fast reaction and below the critical assembly concentration of tubulin. The APC protein induces the bundling of the assembled microtubules in a concentration‐dependent manner. Regarding its biochemical properties the analysed APC protein fragment strikingly resembles the members of the microtubule‐associated protein family tau. This analogy may help to understand the role of the APC protein in the suppression of tumorigenesis.
Methanol : coenzyme M methyltransferase from Methanosarcina barkeri
Authors: Sauer Karin , Thauer Rudolf K.
Published: March 2003

The enzyme system catalyzing the formation of methyl‐coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methylcob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+of the bound corrinoid. This active‐site His was identified by site‐directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl‐coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5 % of the activity of MtaA.
RNase L inhibitor (RLI) antisense constructions block partially the down regulation of the 2‐5A/RNase L pathway in encephalomyocarditis‐virus‐(EMCV)‐infected cells
Authors: Martinand Camille , Salehzada Tamim , Silhol Michelle , Lebleu Bernard , Bisbal Catherine
Published: March 2003

The interferon‐(IFN)‐inducible 2′,5′‐oligoadenylate (2‐5A)/endoribonuclease L (RNase L) pathway plays a major role in the antiviral and antiproliferative effects of IFN. The 2‐5A/RNase L pathway appears to be regulated by the cell‐growth status or viral infection. Viruses, and picornaviruses in particular, have evolved strategies to escape the 2‐5A/RNase L‐pathway‐associated antiviral activity.

We have recently cloned a cDNA coding for RLI, a RNase‐L‐specific protein inhibitor. Its regulated expression by viral infection could provide a new strategy to modulate the 2‐5A/RNase L pathway. Since RNase L had been shown to be down regulated upon encephalomyocarditis (EMCV) infection, we stably transfected HeLa cells with a RLI antisense cDNA expressing vector. Four independant clones named VAS1, VAS2, VAS3 and VAS4 and one clone transfected with the empty vector (VV) as control, were analyzed. The level of RLI was decreased by 20  % for VAS1, 25  % for VAS2, 75  % for VAS3 and 50  % for VAS4. The inactivation of RNase L observed during EMCV infection was decreased in these clones as compared to control HeLa cells. Here again the results vary between the four clones. The maximum inhibition of RNase L (90  %) was observed in control cells and in VAS1 while 48  % inhibition was observed in VAS4 and 25  % in VAS3. The reversal in RNase L inhibition thus reflects closely the resulting RLI level, in keeping with a major role of RLI in EMCV‐induced down regulation of 2‐5A‐binding activity of RNase L. Moreover, cells expressing a low level of RLI (VAS3 and VAS 4) are partially resistant to EMCV infection.
Structural requirements for fibromodulin binding to collagen and the control of type I collagen fibrillogenesis
Authors: Font Bernard , Eichenberger Denise , Goldschmidt Denise , Boutillon Marguerite‐Marie , Hulmes David J. S.
Published: March 2003

Fibromodulin belongs to the family of small, leucine‐rich proteoglycans which have been reported to interact with collagens and to inhibit type I collagen fibrillogenesis. Decorin and fibromodulin exhibit a noticeable degree of sequence similarity. However, as previously reported [Font, B., Eichenberger, D., Rosenberg, L. M. & van der Rest, M. (1996) Matrix Biol. 15, 341−348] the domains of these molecules implicated in the interactions with type XII and type XIV collagens are different, these being the dermatan sulphate/chondroitin sulphate chain for decorin and the core protein for fibromodulin. At the present time the fibromodulin domains implicated in the interactions with fibrillar collagens remain unknown. In experiments reported here, we have sought to identify the structural requirements for fibromodulin interaction with collagen and for the control of type I collagen fibrillogenesis. Circular dichroism spectra and fibrillogenesis inhibition studies show that fibromodulin structure and its collagen fibrillogenesis control function are strictly dependent on the presence of intact disulphide bridge(s). In addition, we show that the binding of fibromodulin (or fibromodulin‐derived fragments) to type I collagen is not necessarily correlated with fibrillogenesis inhibition. To isolate fibromodulin domains, the native proteoglycan was submitted to mild proteolysis. We have isolated an α‐chymotrypsin−resistant fragment which contains the bulk of the N‐terminal and central region of the molecule including the leucine‐rich repeats 4 and 6 reported for decorin to be involved in type I collagen binding. This fragment does not bind to type I collagen. Using enzymes with different specificities, a number of large fragments of fibromodulin were obtained, suggesting a compact structure for this molecule which is relatively resistant to proteolysis. None of these N‐glycosylated fragments were able to bind to type I collagen in co‐sedimentation experiments. Taken together these results suggest that fibromodulin‐type I collagen interactions leading to fibrillogenesis inhibition require more than one binding domain. One of these domains could be the C‐terminal end of the molecule containing the disulphide loop which is absent in the chymotrypsin‐resistant fragment.
Bacterial selenocysteine synthase
Authors: Tormay Peter , Wilting Reinhard , Lottspeich Friedrich , Mehta Perdeep K. , Christen Philipp , Böck August
Published: March 2003

Selenocysteine synthase from Escherichia coli is a pyridoxal‐5′‐phosphate‐containing enzyme which catalyses the conversion of seryl‐tRNASec into selenocysteyl‐tRNASec. Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the α/γ superfamily of pyridoxal‐5′‐phosphate‐dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass‐spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl‐tRNASec synthesis, albeit with a 330‐fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.
Phosphorylation of lens‐fiber connexins in lens organ cultures
Authors: Jiang Jean X. , Goodenough Daniel A.
Published: March 2003

Lens gap junction proteins, connexins [1], are known to be phosphorylated in vivo. Phosphorylated states of connexins were examined in lens cultures to define in vitro models for the study of the functions of lens connexin phosphorylation in lens biology. In organ and differentiated cell primary cultures, chick lens‐fiber connexins, connexin45.6 and connexin56, were labeled with [32P]orthophosphate. Pulse‐chase experiments of lens organ cultures with [35S]methionine demonstrated that connexin45.6 and connexin56 were properly processed into the phosphorylated forms observed in vivo. However, in lens cell primary cultures, both connexins had short half‐lives, and connexin56 was degraded before it was phosphorylated into the form which showed the largest mobility shift. The data suggested that the phosphorylation patterns of connexins in lens organ cultures were similar to in vivo connexin phosphorylation, while primary cultures revealed abnormal rates of protein turnover and incomplete phosphorylation. Treatment of lens organ cultures with protein kinase inhibitors indicated that protein kinase C was involved in the phosphorylation of connexin45.6 and connexin56. Comparison of the phosphopeptide patterns by two‐dimensional mapping suggested that protein kinase C was involved in the phosphorylation of connexin45.6 and that it phosphorylated the C‐terminus of connexin45.6 in vitro.
The single mutation Trp35←Ala in the 35−40 redox site of Chlamydomonas reinhardtii thioredoxin h affects its biochemical activity and the pH dependence of C36−C39 1H‐13C NMR
Authors: Krimm Isabelle , Lemaire Stéphane , Ruelland Eric , Miginiac‐Maslow Myroslawa , Jaquot Jean‐Pierre , Hirasawa Masakazu , Knaff David B. , Lancelin Jean‐Marc
Published: March 2003

The role of the invariant Trp residue at the redox site of thioredoxins was investigated by site‐directed mutagenesis of a Chlamydomonas reinhardtii thioredoxin h. Though being still redox active with NADPH−thioredoxin reductase and chemical substrates [dithiothreitol and 5,5′‐dithio‐bis(2‐nitrobenzoic acid)] the Trp35←Ala‐mutated protein completely lost the capacity to activate the thiol‐regulated NADPH‐dependent malate dehydrogenase. However, it was able to activate a mutant malate dehydrogenase where only the most exposed disulfide was retained. The pH dependence of the redox‐site Cysβ1H/13C‐NMR frequencies of the wild‐type and mutated proteins, in both the reduced and oxidised states, were compared over the pH range 5.8−10. The mutation does not affect the conserved buried Asp30, which titrates with a pKa of 7.5 in the oxidised proteins in agreement with previous studies. However, for the reduced forms of the proteins, the pH dependence of resonances of both Cys was strongly affected by the mutation. In the case of the wild‐type thioredoxin, two apparent pKa values were found around 7.0 and 9.5 and could be assigned to the titration of Cys36 and Cys39 thiol, respectively, similar to the case of Escherichia coli thioredoxin. For the mutated thioredoxin a single pKa was found around 8.3. This result can be interpreted as a single pKa of either Cys36 or Cys39 or both. While the mutation clearly affects ionisations, the measured redox potentials of the active‐site Cys pair are not significantly affected by the Trp35←Ala mutation. Possible roles of an aromatic side chain on the reactivity of the catalytic Cys residues in thioredoxins are proposed.
Purification and characterization of two enone reductases from Saccharomyces cerevisiae
Authors: Wanner Peter , Tressl Roland
Published: March 2003

Two enone reductases catalyzing irreversibly the enantioselective reduction of α,β‐unsaturated carbonyls have been purified 165‐fold and 257‐fold, respectively, from the cytosolic fraction of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G‐25 filtration, DEAE‐Sepharose CL‐6B chromatography, blue Sepharose CL‐6B chromatography and Superdex 200 preparation‐grade filtration.

One enzyme (E I) was NADPH‐dependent, showed a molecular mass of 75 kDa and decomposed under denaturing electrophoretic conditions into two subunits of 34 kDa and 37 kDa. The other enzyme (E II) was NADH linked and the molecular mass estimated by means of Superdex 200 preparation‐grade filtration, was 130 kDa. The enzyme decomposed into subunits of 56 kDa and 64 kDa on SDS/PAGE.

Both enzymes were most active at pH 4.8 and accepted 1‐octen‐3‐one, 1‐hexen‐3‐one, 3‐alken‐2‐ones, 4‐alken‐3‐ones, 2‐cyclohexen‐1‐ones, 2‐alkenals, 2,4‐alkadienals, 2‐phenyl‐2‐alkenals, and 2‐alkyl‐2‐alkenals as substrates. Both enzymes showed their highest activities with 1‐hexen‐3‐one and 1‐octen‐3‐one, respectively. The Km values for 1‐octen‐3‐one were estimated as 0.54 mM (E I) and 0.20 mM (E II), respectively. Both enzymes catalyzed the enantioselective reduction of cis‐ and trans‐2‐phenyl‐2‐butenal into (R)‐2‐phenylbutanal.
Characterization of a O‐fatty‐acylated sulfatide from equine brain
Authors: Mikami Takeshi , Tsuchihashi Keiko , Kashiwagi Motoi , Yachida Youichi , Daino Takumi , Hashi Kazuo , Akino Toyoaki , Gasa Shinsei
Published: March 2003

A sulfatide, O‐fatty‐acylated 3‐sulfogalactosylceramide at C6‐O on galactoside, was isolated from equine brain and the chemical structure was characterized by proton NMR and MS. The O‐acylation site of the acylated sulfatide was determined by the down‐field shift of protons attached to a carbon having an O‐acyl group in the NMR spectrum and by analysis of a partially methylated derivative before and after acetalization of the intact sulfatide using GC‐MS. The O‐acyl chain length was determined by GLC, revealing that it exclusively had palmitoyl and stearoyl residues as the major fatty acids. The enzymatic conversion to the O‐acyl sulfatide was further examined using equine brain microsomes as an enzyme source and different lipid substrates, resulting in O‐acylation of 3‐sulfogalactosylceramide from stearoyl CoA, while 6‐O‐acyl galactosylceramide was not O‐sulfated from phosphoadenosine phosphosulfate. The results were supported by the comparably different N‐linked fatty acid components between two lipid substrates, in which the component of 6‐O‐acyl sulfatide was mostly similar to that of sulfatide, but not to 6‐O‐acyl galactosylceramide.
Identification of a stable semiquinone intermediate in the purified and membrane bound ubiquinol oxidase‐cytochrome bd from Escherichia coli
Authors: Hastings Stuart F. , Kaysser Tamma M. , Jiang Fashun , Salerno John C. , Gennis Robert B. , Ingledew W. John
Published: March 2003

The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O2 to water and using ubiquinol‐8 or menaquinol‐8 as its immediate reductant. The oxidation of quinol is by the low‐spin ferri‐haem, cytochrome b558. This occurs at a quinol‐binding site by sequential one electron steps, requiring the stabilisation of the semiquinone intermediate. We have observed, by EPR spectroscopy, the properties of this semiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess of ubiquinone‐8 and menaquinone‐8 analogues. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a semiquinone anion of this type. The line shape is Gaussian. The semiquinone is highly stabilised with respect to free semiquinone; significant free radical can be observed at pH 7.0 and above. The pH dependence of the redox reactions indicate that the anionic form of the semiquinone and the neutral form of the quinol predominate in the pH range studied. The pH dependence of the mid‐point potentials of the one electron reactions from pH 7.0−9.0 is 120 mV/pH change for the semiquinone anion to quinol (E2) and none for the quinone to semiquinone (E1). The semiquinone radical is attenuated on titration with putative inhibitors of this quinone‐binding site. We have similarly studied the semiquinone in membrane preparations from a strain with overexpression of cytochrome bd oxidase. The data can be fitted with the assumption of a single quinone‐binding site.
Reconstitution of β2‐adrenoceptor−GTP‐binding‐protein interaction in Sf9 cells
Authors: Seifert Roland , Lee Tae Weon , Lam Van T. , Kobilka Brian K.
Published: March 2003

In most studies, coupling of the β2‐adrenoceptor (β2AR) to the stimulatory, heterotrimeric GTP‐binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which β2AR‐Gs interactions can be studied directly at the level of the G‐protein. We expressed the β2AR alone, in combination with the α‐subunit of Gs (Gsα), and as fusion protein with Gsα (β2AR‐Gsα) in Sf9 insect cells. The β2AR expressed alone couples poorly to the endogenous Gsα‐like G‐protein of Sf9 cells since no high‐affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high‐affinity GTPase and guanosine 5′‐O‐(3‐thiotriphosphate) (GTP[S]) binding were small. β2AR‐Gsα reconstituted high‐affinity agonist binding and regulated adenylyl cyclase more effectively than the β2AR co‐expressed with a large excess of Gsα. In membranes expressing β2AR‐Gsα, highly effective agonist‐ and inverse agonist regulation of high‐affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing β2AR and Gsα as separate proteins was difficult to detect. Our data show that the β2AR interacts with Gsα more efficiently when expressed as a fusion protein than when expressed with an excess of non‐fused Gsα. The β2AR‐Gsα fusion protein provides a very sensitive model system to study the regulation of Gs function by β2AR agonists and inverse agonists directly at the level of the G‐protein.
Purification and characterization of a Ca2+/calmodulin‐dependent protein kinase II from hog gastric mucosa using a protein‐protein affinity chromatographic technique
Authors: Fährmann Michael , Möhlig Matthias , Schatz Helmut , Pfeiffer Andreas
Published: March 2003

A peripheral member of the Ca2+/calmodulin‐dependent protein kinase II (CaMkinase II) group has been purified from hog gastric mucosa with the use of a novel affinity‐chromatographic step. For the well known neural isotypes of CaMkinase II, it is proposed that the subunits form holoenzymes through a specific domain at the C‐terminus called the ’association domain'. We immobilized a bacterially expressed association domain from CaMkinase II‐δ‐2 and used it as an affinity column. This matrix was used as the last step in a sequential enzyme purification procedure from hog gastric mucosa and yielded a homogeneous CaMkinase II which showed the typical physical and enzymatic properties of CaMkinase II. The enzyme activity showed a dependence on Ca2+ and calmodulin (apparent K0.5 = 2.7 μM and K0.5 = 0.02 μM, respectively). We found a subunit molecular mass of 61 kDa. An apparent native molecular mass of 310 kDa was calculated. The Stokes radius and the sedimentation coefficient were 6.7 nm and 11.2 S, respectively. Moreover, the isolated CaMkinase II was very well inhibited by the CaMkinase‐II‐specific inhibitors KN‐62 (Ki = 0.52 nM) and KT5926 (Ki = 0.79 nM). The phosphorylation of several substrates revealed its multifunctionality. The purified CaMkinase II had an apparent Km for Mg‐ATP of 24.0 μM and for autocamtide‐II of 0.62 μM. CaMkinase II is considered as a strong candidate for regulating vesicle released quanta such as acid, neurotransmitters or insulin.
Structural analysis of ASCUT‐1, a protein component of the cuticle of the parasitic nematode Ascaris lumbricoides
Authors: D'Auria Sabato , Rossi Mosè , Tanfani Fabio , Bertoli Enrico , Parise Giuliano , Bazzicalupo Paolo
Published: March 2003

CUT‐1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT‐1‐like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT‐1 from A. lumbricoides (ASCUT‐1) were investigated by Fourier‐transform infrared (FT‐IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20 +C, both as insoluble inclusion bodies or in soluble form, contains about 50 %β structure, 14 %α‐helix and 25 % turns. A tendency of A. lumbricoides CUT‐1 to form aggregates was documented by FT‐IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near‐ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near‐ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT‐IR and CD data revealed that ASCUT‐1 is very stable at high temperatures. This stability and the tendency of ASCUT‐1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle.
Identification of major wheat allergens by means of the Escherichia coli expression system
Authors: Maruyama Nobuyuki , Ichise Keiko , Katsube Tomoyuki , Kishimoto Toru , Kawase Shin‐ichiro , Matsumura Yasuki , Takeuchi Yoshihiro , Sawada Tadashi , Utsumi Shigeru
Published: March 2003

Wheat proteins were fractionated into salt‐soluble, glutenin‐rich, and gliadin‐rich fractions. Reactivities of these protein fractions with sera of patients with wheat‐associated allergies were examined under various conditions. The relative reactivity of the fractions was generally in the order glutenin‐rich > gliadin‐rich @WR salt‐soluble fractions, although their reactivities were variable among patients and among the reaction conditions, indicating that the kind, the number and the epitope of allergens were variable among patients. To identify major allergens, α‐, γ‐ and ω‐gliadin, and low‐molecular‐mass (LMM)‐ and high‐molecular‐mass (HMM)‐glutenin genes were expressed in Escherichia coli by means of a pET vector. Recombinant gliadins and glutenins were partially purfied on the basis of the solubilities of prolamin and glutelin. The partially purified recombinant proteins were reacted with the patients' sera. LMM glutenin containing many Gln‐Gln‐Gln‐Pro‐Pro motifs, which was identified to be IgE‐binding epitope [Tanabe, S., Arai, S., Yanagihara, Y., Mita, H., Takahashi, K. & Watanabe, M. (1996) Biochem. Biophys. Res. Commun. 219, 290−293], exhibited the highest reactivity. The next highest reactivities were observed on α‐gliadin and γ‐gliadin, which had not been identified as allergens.
Thrombin‐induced reversal of astrocyte stellation is mediated by activation of protein kinase C β‐1
Authors: Pindon Armelle , Festoff Barry W. , Hantaï Daniel
Published: March 2003

Exogenous or endogenous injuries of the central nervous system trigger astrogliosis characterized by proliferation of astrocytes and changes in their morphology from stellate to flat polygonal. Astrocytes in culture are very sensitive to thrombin, a serine protease, which through its proteolytically activated receptor (PAR‐1) induces proliferation and morphological changes comparable to astrogliosis. Evaluation of the thrombin signal‐transduction pathway in the reversal of astrocyte stellation might help to understand astrogliosis. For this purpose, primary cultured murine cortical astrocytes were treated with H7, a protein‐kinase inhibitor, and thrombin, which resulted in an inhibition of stellation reversal. Treatments with phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, mimicked the action of thrombin. Subsequently, direct assay of astrocyte PKC activity after thrombin or PMA treatment demonstrated involvement of PKC in thrombin signaling associated with shape change. Western blotting showed that PKC isoform β‐1 was involved in this pathway, while PKC α was only weakly activated and PKC β‐2 was not activated by thrombin. PKC β‐1 translocation was also elicited by a thrombin‐receptor active peptide (SFLLRN), demonstrating the involvement of PAR‐1 in this process. PKC δ and ε were located constitutively in the membrane fraction in stellate astrocytes. Isoforms γ, η, θ, and ζ were absent from astrocytes. These results suggest that astrogliosis in vivo might be regulated by modulating the activity of thrombin, PAR‐1, or specific PKC isoforms.
Recommendations for the presentation of NMR structures of proteins and nucleic acids
Authors: Markley John L. , Bax Ad , Arata Yoji , Hilbers C. W. , Kaptein Robert , Sykes Brian D. , Wright Peter E. , Wüthrich Kurt
Published: March 2003

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer‐accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC‐IUBMB‐IUPAB inter‐union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.
Identification of a gene selectively expressed in the brain, which encodes a putative transmembrane protein and a soluble cytoplasmic isoform
Authors: Bangsow Thorsten , Schepelmann Silke , Martin Carmen , May Monika , Oberthür Angela , Perl Sabine , Knüpfer Ellen , Zinke Holger , Gassen Hans G.
Published: March 2003

Subtractive cloning procedures led to the identification of a variety of transcripts expressed in mammalian brain. However, little is known about the encoded proteins and the regulation of gene expression. Here, we describe the isolation and characterisation of a single‐copy gene (83.5) of 21.7 kb which is specifically expressed in porcine brain. In situ hybridisation and immunohistochemistry experiments showed a distinct pattern of gene expression in neuronal cell types in different parts of the brain. The gene contains two mini exons, confirming neural‐specific expression. cDNA cloning experiments revealed two species of mRNA differing in their 5′‐regions. These transcripts are generated by two distinct transcription start sites that are under the control of different potential promoter regions as shown by primer‐extension experiments. The amino acid sequences of the deduced proteins predict that one mRNA species encodes a novel type‐I transmembrane protein, whereas the other transcript encodes only a part of its cytoplasmic domain. In Western‐blot experiments, we detected two proteins of the predicted size and cellular localisation in porcine brain. The precise function of these proteins remains to be determined. However, our findings suggest that they may be generated by alternative promoter usage, leading to the expression of a membrane protein and its truncated cytoplasmic isoform.
The bZip transcription factor vitellogenin‐binding protein is post transcriptional down regulated in chicken liver
Authors: Smidt Marten P. , Snippe Lenie , van Keulen Geertje , Ab Geert
Published: March 2003

The vitellogenin‐binding protein (VBP) is a member of the proline and acidic‐region rich (PAR) family of bZip transcription factors. PAR is located N‐terminally to the DNA‐binding domain. VBP binds to specific sites within the 300‐bp 5′‐flanking region of the chicken‐liver‐specific estrogen‐dependent very‐low‐density apolipoprotein gene (apoVLDL II). One of these binding sites (site D) resembles the albumin site D and is positioned in close proximity of the major estrogen‐responsive element. Previous studies showed that VBP can bind simultaneously with the estrogen receptor to the putative complex regulatory element E1D. To investigate whether VBP is involved in apoVLDL II gene expression, we examined its capacity to enhance apoVLDL II transcription and its presence in liver. We show that VBP is capable of enhancing transcription in transfection experiments. However, VBP could not be detected in liver by Western‐blots or immuno‐electro mobility shift assays (EMSAs) using antibodies against different moieties of the protein. We examined the possible reduction in translation efficienies due to a small upstream open reading frame in the VBP leader sequence, but did not find any. Although VBP binds to the proximal apoVLDL II promoter region and enhances transcription in co‐transfection experiments, the protein is unlikely to be involved in apoVLDL II gene transcription because of its undetectable low level in liver nuclei.
The structures of asparagine‐linked oligosaccharides of rat liver cathepsin L reflect the substrate specificity of lysosomal α‐mannosidase
Authors: Towatari Takae , Miyamura Tsuyoshi , Kondo Akihiro , Kato Ikunoshin , Inoue Masahiro , Yano Mihiro , Kido Hiroshi
Published: March 2003

We have studied the structures of asparagine‐linked oligosaccharides of cathepsin L purified from rat liver in detail. The oligosaccharides released from rat liver cathepsin L on glycopeptidase‐F treatment were tagged with 2‐aminopyridine at their reducing ends. The pyridylamino (PA) derivatives were separated into seven fractions according to molecular size by normal‐phase HPLC. The structure of each oligosaccharide thus isolated was analyzed by reversed‐phase HPLC and characterized by ion‐spray mass spectrometry and high‐resolution proton nuclear magnetic resonance (1H‐NMR) spectroscopy. Our results indicate that the asparagine‐linked oligosaccharides of rat liver cathepsin L are of the oligomannose type, having two to six mannose residues. Among them, the five major ones are Manα1−6Manβ1−4‐GlcNAcβ1−4GlcNAc, Manα1−6Manα1−6Manβ1−4GlcNAcβ1−4GlcNAc, Manα1−6(Manα1−3)‐Manα1−6Manβ1−4GlcNAcβ1−4GlcNAc, Manα1−6(Manα1−3)Manα1−6(Manα1−3)Manβ1‐4GlcNAcβ1−4GlcNAc, and Manα1−6(Manα1−3)Manα1−6(Manα1−2Manα1−3)Manβ1−4GlcNAcβ1‐4GlcNAc. Their structures are shown to be products of Man6GlcNAc2 hydrolysis with lysosomal α‐mannosidase.