bluuebunny/crossref_metadata_2025 huggingface dataset:
Inactivation of the 2‐oxo acid dehydrogenase complexes upon generation of intrinsic radical species
Authors: Bunik Victoria I. , Sievers Christian
Published: March 2003

Self‐regulation of the 2‐oxo acid dehydrogenase complexes during catalysis was studied. Radical species as side products of catalysis were detected by spin trapping, lucigenin fluorescence and ferricytochrome c reduction. Studies of the complexes after converting the bound lipoate or FAD cofactors to nonfunctional derivatives indicated that radicals are generated via FAD. In the presence of oxygen, the 2‐oxo acid, CoA‐dependent production of the superoxide anion radical was detected. In the absence of oxygen, a protein‐bound radical concluded to be the thiyl radical of the complex‐bound dihydrolipoate was trapped by α‐phenyl‐N‐tert‐butylnitrone. Another, carbon‐centered, radical was trapped in anaerobic reaction of the complex with 2‐oxoglutarate and CoA by 5,5′‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO). Generation of radical species was accompanied by the enzyme inactivation. A superoxide scavenger, superoxide dismutase, did not protect the enzyme. However, a thiyl radical scavenger, thioredoxin, prevented the inactivation. It was concluded that the thiyl radical of the complex‐bound dihydrolipoate induces the inactivation by 1e– oxidation of the 2‐oxo acid dehydrogenase catalytic intermediate. A product of this oxidation, the DMPO‐trapped radical fragment of the 2‐oxo acid substrate, inactivates the first component of the complex. The inactivation prevents transformation of the 2‐oxo acids in the absence of terminal substrate, NAD+. The self‐regulation is modulated by thioredoxin which alleviates the adverse effect of the dihydrolipoate intermediate, thus stimulating production of reactive oxygen species by the complexes. The data point to a dual pro‐oxidant action of the complex‐bound dihydrolipoate, propagated through the first and third component enzymes and controlled by thioredoxin and the (NAD+ + NADH) pool.
Inhibition of hyaluronan synthesis in Streptococcus equi FM100 by 4‐methylumbelliferone
Authors: Kakizaki Ikuko , Takagaki Keiichi , Endo Yasufumi , Kudo Daisuke , Ikeya Hitoshi , Miyoshi Teruzo , Baggenstoss Bruce A. , Tlapak‐Simmons Valarie L. , Kumari Kshama , Nakane Akio , Weigel Paul H. , Endo Masahiko
Published: March 2003

As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production was also observed in group C Streptococcus equi FM100 cells treated with 4‐methylumbelliferone (MU), although there was no effect on their growth. In this study, the inhibition mechanism of hyaluronan synthesis by MU was examined using Streptococcus equi FM100, as a model. When MU was added to a reaction mixture containing the two sugar nucleotide donors and a membrane‐rich fraction as an enzyme source in a cell‐free hyaluronan synthesis experiment, there was no change in the production of hyaluronan. On the contrary, when MU was added to the culture medium of FM100 cells, hyaluronan production in the isolated membranes was decreased in a dose‐dependent manner. However, when the effect of MU on the expression level of hyaluronan synthase was examined, MU did not decrease either the mRNA level of the has operon containing the hyaluronan synthase gene or the protein level of hyaluronan synthase. Solubilization of the enzyme from membranes of MU‐treated cells and addition of the exogenous phospholipid, cardiolipin, rescued hyaluronan synthase activity. In the mass spectrometric analysis of the membrane phospholipids from FM100 cells treated with MU, changes were observed in the distribution of only cardiolipin species but not of the other major phospholipid, PtdGro. These results suggest that MU treatment may cause a decrease in hyaluronan synthase activity by altering the lipid environment of membranes, especially the distribution of different cardiolipin species, surrounding hyaluronan synthase.
Incorporation of 3‐nitrotyrosine into the C‐terminus of α‐tubulin is reversible and not detrimental to dividing cells
Authors: Bisig C. Gastón , Purro Silvia A. , Contín María A. , Barra Héctor S. , Arce Carlos A.
Published: March 2003

The C‐terminus of the α‐chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3‐nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells. Nitrotyrosine is formed in cells by nitration of tyrosine by nitric oxide‐derived species. We studied properties of tubulin modified by in vitro nitrotyrosination at the C‐terminus of the α‐subunit, and the consequences for cell functioning. Nitrotyrosinated tubulin was a good substrate of tubulin carboxypeptidase, and showed a similar capability to assemble into microtubules in vitro to that of tyrosinated tubulin. Tubulin of C6 cells cultured in F12K medium in the presence of 500 µm nitrotyrosine became fully nitrotyrosinated. This nitrotyrosination was shown to be reversible. No changes in morphology, proliferation, or viability were observed during cycles of nitrotyrosination, denitrotyrosination, and re‐nitrotyrosination. Similar results were obtained with CHO, COS‐7, HeLa, NIH‐3T3, NIH‐3T3(TTL–), and A549 cells. C6 and A549 cells were subjected to several passages during 45 days or more in the continuous presence of 500 µm nitrotyrosine without noticeable alteration of morphology, viability, or proliferation. The microtubular networks visualized by immunofluorescence with antibodies to nitrotyrosinated and total tubulin were identical. Furthermore, nitrotyrosination of tubulin in COS cells did not alter the association of tubulin carboxypeptidase with microtubules. Our results demonstrate that substitution of C‐terminal tyrosine by 3‐nitrotyrosine has no detrimental effect on dividing cells.
Conformational analysis of opacity proteins from Neisseria meningitidis
Authors: de Jonge Marien I. , Bos Martine P. , Hamstra Hendrik J. , Jiskoot Wim , van Ulsen Peter , Tommassen Jan , van Alphen Loek , van der Ley Peter
Published: March 2003

Opacity‐associated (Opa) proteins are outer membrane proteins which play a critical role in the adhesion of pathogenic Neisseria spp. to epithelial and endothelial cells and polymorphonuclear neutrophils. The adherence is mainly mediated by the CD66‐epitope‐containing members of the carcinoembryonic‐antigen family of human cell‐adhesion molecules (CEACAM). For the analysis of the specific interactions of individual Opa proteins with their receptors, pure protein is needed in its native conformation. In this study, we describe the isolation and structural analysis of opacity proteins OpaJ129 and OpaB128 derived from Neisseria meningitidis strain H44/76. When the Opa proteins were produced with the phoE signal sequence in Escherichia coli, they were localized at the cell surface and the recombinant bacteria were found to specifically interact with CEACAM1. For refolding and purification, the proteins were overproduced without their signal sequences in E. coli, resulting in its cytoplasmic accumulation in the form of inclusion bodies. After solubilization of the inclusion bodies in urea, the proteins could be folded efficiently in vitro, under alkaline conditions by dilution in ethanolamine and the detergent n‐dodecyl‐N,N‐dimethyl‐1‐ammonio‐3‐propanesulfonate (SB12). The structure of the refolded and purified proteins, determined by circular dichroism, indicated a high content of β‐sheet conformation, which is consistent with previously proposed topology models for Opa proteins. A clear difference was found between the binding of refolded vs. denatured OpaJ protein to the N‐A1 domain of CEACAM1. Almost no binding was found with the denatured Opa protein, showing that the Opa–receptor interaction is conformation‐dependent.
Characterization of the 105‐kDa molecular chaperone
Authors: Matsumori Mika , Itoh Hideaki , Toyoshima Itaru , Komatsuda Atsushi , Sawada Ken‐ichi , Fukuda Jun , Tanaka Toshinobu , Okubo Atsuya , Kinouchi Hiroyuki , Mizoi Kazuo , Hama Tokiko , Suzuki Akira , Hamada Fumio , Otaka Michiro , Shoji Yutaka , Takada Goro
Published: March 2003

We have characterized the biochemical properties of the testis and brain‐specific 105‐kDa protein which is cross‐reacted with an anti‐bovine HSP90 antibody. The protein was induced in germ cells by heat stress, resulting in a protein which is one of the heat shock proteins [Kumagai, J., Fukuda, J., Kodama, H., Murata, M., Kawamura, K., Itoh, H. & Tanaka, T. (2000) Eur. J. Biochem.267, 3073–3078]. In the present study, we characterized the biochemical properties of the protein. The 105‐kDa protein inhibited the aggregation of citrate synthase as a molecular chaperone in vitro. ATP/MgCl2 has a slight influence of the suppression of the citrate synthase aggregation by the 105‐kDa protein. The protein possessed chaperone activity. The protein was able to bind to ATP–Sepharose like the other molecular chaperone HSP70. A partial amino‐acid sequence (24 amino‐acid residues) of the protein was determined and coincided with those of the mouse testis‐ and brain‐specific APG‐1 and osmotic stress protein 94 (OSP94). The 105‐kDa protein was detected only in the medulla of the rat kidney sections similar to OSP94 upon immunoblotting. The purified 105‐kDa protein was cross‐reacted with an antibody against APG‐1. These results suggested that APG‐1 and OSP94 are both identical to the 105‐kDa protein. There were highly homologous regions between the 105‐kDa protein/APG‐1/OSP94 and HSP90. The region of HSP90 was also an immunoreactive site. An anti‐bovine HSP90 antibody may cross‐react with the 105‐kDa protein similar to HSP90 in the rat testis and brain. We have investigated the localization and developmental induction of the protein in the rat brain. In the immunohistochemical analysis, the protein was mainly detected in the cytoplasm of the nerve and glial cells of the rat brain. Although the 105‐kDa protein was localized in all rat brain segments, the expression pattern was fast in the cerebral cortex and hippocampus and slow in the cerebellum.
Does phosphorylation of the cap‐binding protein eIF4E play a role in translation initiation?
Authors: Scheper Gert C. , Proud Christopher G.
Published: March 2003

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5′‐cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E can interact either with the scaffold protein eIF4G or with repressor proteins termed eIF4E‐binding proteins (4E‐BPs). High levels of expression can disrupt cellular growth control and are associated with human cancers. A fraction of the cellular eIF4E is found in the nucleus where it may play a role in the transport of certain mRNAs to the cytoplasm. eIF4E undergoes regulated phosphorylation (at Ser209) by members of the Mnk group of kinases, which are activated by multiple MAP kinases (hence Mnk = MAP‐kinase signal integrating kinase). The functional significance of its phosphorylation has been the subject of considerable interest. Recent genetic studies in Drosophila point to a key role for phosphorylation of eIF4E in growth and viability. Initial structural data suggested that phosphorylation of Ser209 might allow formation of a salt bridge with a basic residue (Lys159) that would clamp eIF4E onto the mRNA and increase its affinity for ligand. However, more recent structural data place Ser209 too far away from Lys159 to form such an interaction, and biophysical studies indicate that phosphorylation actually decreases the affinity of eIF4E for cap or capped RNA. The implications of these studies are discussed in the light of other, in vitro and in vivo, investigations designed to address the role of eIF4E phosphorylation in mRNA translation or its control.
A novel, inducible, citral lyase purified from spores of Penicillium digitatum
Authors: Wolken Wout A. M. , Van Loo Willem J. V. , Tramper Johannes , van der Werf Mariët J.
Published: March 2003

A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118‐fold by incubating nongerminating spores with the substrate, citral. This cofactor independent hydratase/aldolase, was purified and found to be a monomeric enzyme of 31 kDa. Citral lyase has a Km of 0.058 mm and a Vmax of 52.6 U·mg−1. Enzyme activity was optimal at 20 °C and pH 7.6. The enzyme has a strong preference for the trans isomer of citral (geranial). Citral lyase also converts other α,β‐unsaturated aldehydes (farnesal, methyl‐crotonaldehyde, decenal and cinnemaldehyde).
Role of electrostatics in the interaction between plastocyanin and photosystem I of the cyanobacterium Phormidium laminosum
Authors: Schlarb‐Ridley Beatrix G. , Navarro José A. , Spencer Matthew , Bendall Derek S. , Hervás Manuel , Howe Christopher J. , De la Rosa Miguel A.
Published: March 2003

The interactions between photosystem I and five charge mutants of plastocyanin from the cyanobacterium Phormidium laminosum were investigated in vitro. The dependence of the overall rate constant of reaction, k2, on ionic strength was investigated using laser flash photolysis. The rate constant of the wild‐type reaction increased with ionic strength, indicating repulsion between the reaction partners. Removing a negative charge on plastocyanin (D44A) accelerated the reaction and made it independent of ionic strength; removing a positive charge adjacent to D44 (K53A) had little effect. Neutralizing and inverting the charge on R93 slowed the reaction down and increased the repulsion. Specific effects of MgCl2 were observed for mutants K53A, R93Q and R93E. Thermodynamic analysis of the transition state revealed positive activation entropies, suggesting partial desolvation of the interface in the transition state. In comparison with plants, plastocyanin and photosystem I of Phormidium laminosum react slowly at low ionic strength, whereas the two systems have similar rates in the range of physiological salt concentrations. We conclude that in P. laminosum, in contrast with plants in vitro, hydrophobic interactions are more important than electrostatics for the reactions of plastocyanin, both with photosystem I (this paper) and with cytochrome f[Schlarb‐Ridley, B.G., Bendall, D.S. & Howe, C.J. (2002) Biochemistry41, 3279–3285]. We discuss the implications of this conclusion for the divergent evolution of cyanobacterial and plant plastocyanins.
Role of CCP2 of the C4b‐binding protein β‐chain in protein S binding evaluated by mutagenesis and monoclonal antibodies
Authors: Webb Joanna H. , Villoutreix Bruno O. , Dahlbäck Björn , Blom Anna M.
Published: March 2003

Complement regulator C4b‐binding protein (C4BP) and the anticoagulant vitamin K‐dependent protein S form a high affinity complex in human plasma. C4BP is composed of seven α‐chains and a unique β‐chain, each chain comprising repeating complement control protein (CCP) modules. The binding site for protein S mainly involves the first of the three β‐chain CCPs (CCP1). However, recently it has been suggested that CCP2 of the β‐chain also contributes to the binding of protein S. To elucidate the structural background for the involvement of CCP2 in the protein S binding, several recombinant β‐chain CCP1‐2 variants having mutations in CCP2 were expressed and tested for protein S binding. Mutations were chosen based on analysis of a homology model of the β‐chain and included R60A/R101A, D66A, L105A, F114A/I116A and H108A. All mutant proteins bound equally well as recombinant wild type to protein S. Several monoclonal antibodies against the β‐chain CCP2 were raised and their influence on protein S binding characterized. Taken together, the results suggest that the role of CCP2 in protein S binding is to orient and stabilize CCP1 rather than to be directly part of the binding site.
Binding of activated Factor XII to endothelial cells affects its inactivation by the C1‐esterase inhibitor
Authors: Schousboe Inger
Published: March 2003

It is well known that activated Factor XII (FXIIa) and kallikrein are rapidly inactivated in plasma as a result of reaction with endogenous inhibitors. The purpose of this may be to prevent uncontrolled deleterious spreading and activation of target zymogens. Both FXII and the complex plasma prekallikrein/high molecular mass kininogen become activated when they bind, in a Zn2+‐dependent manner, to receptors on human umbilical vein endothelial cells (HUVEC). The C1‐esterase inhibitor (C1‐INH) is by far the most efficient inhibitor of FXIIa. In the present study it has been investigated whether binding of FXIIa to HUVEC might offer protection against inactivation by C1‐INH. It appeared that the relative amidolytic activity of purified FXIIa bound to the surface of HUVEC decreased according to the concentration of C1‐INH in medium; however, the decrease was smaller than that measured for inactivation of FXIIa in solution. The secondary rate constant for the inactivation was 3–10‐fold lower for cell‐bound than for soluble FXIIa. The inactivation was found to be caused by C1‐INH binding to cell‐bound FXIIa. Accordingly, the amidolytic activity of saturated amounts of cell‐bound FXIIa was reduced in the presence of C1‐INH and was theoretically nonexistent at physiological C1‐INH concentrations. Amidolytic activity was, however, present on HUVEC incubated with plasma indicating that the endogenous C1‐INH did not completely abolish the activity of FXIIa generated during the incubation period. This supports the hypothesis that binding to endothelial cells protects the activated FXII against inactivation by its major endogenous inhibitor. Hence, the function of FXII may be localized at cellular surfaces.
Role of Acinetobacter calcoaceticus 3,4‐dihydrocoumarin hydrolase in oxidative stress defence against peroxoacids
Authors: Honda Kohsuke , Kataoka Michihiko , Sakuradani Eiji , Shimizu Sakayu
Published: March 2003

The physiological role of a bifunctional enzyme, 3,4‐dihydrocoumarin hydrolase (DCH), which is capable of both hydrolysis of ester bonds and organic acid‐assisted bromination of organic compounds, was investigated. Purified DCH from Acinetobacter calcoaceticus F46 catalysed dose‐ and time‐dependent degradation of peracetic acid. The gene (dch) was cloned from the chromosomal DNA of the bacterium. The dch ORF was 831 bp long, corresponding to a protein of 272 amino acid residues, and the deduced amino acid sequence showed high similarity to those of bacterial serine esterases and perhydrolases. The dch gene was disrupted by homologous recombination on the A. calcoaceticus genome. The dch disruptant strain was more sensitive to growth inhibition by peracetic acid than the parent strain. On the other hand, the recombinant Escherichia coli cells expressing dch were more resistant to peracetic acid. A putative catalase gene was found immediately downstream of dch, and Northern blot hybridization analysis revealed that they are transcribed as part of a polycistronic mRNA. These results suggested that in vivo DCH detoxifies peroxoacids in conjunction with the catalase, i.e. peroxoacids are first hydrolysed to the corresponding acids and hydrogen peroxide by DCH, and then the resulting hydrogen peroxide is degraded by the catalase.
Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3
Authors: Chen Hueih Min , Leung King Wong , Thakur Nagendra N. , Tan Anmin , Jack Ralph W.
Published: March 2003

Different pathways of bilayer disruption by the structurally related antimicrobial peptides cecropin B, B1 and B3, revealed by surface plasma resonance analysis of immobilized liposomes, differential scanning calorimetry of peptide–large unilamellar vesicle interactions, and light microscopic analysis of peptide‐treated giant unilamellar vesicles, have been identified in this study. Natural cecropin B (CB) has one amphipathic and one hydrophobic α‐helix, whereas cecropins B1 (CB1) and B3 (CB3), which are custom‐designed, chimaeric analogues of CB, possess either two amphipathic or two hydrophobic α‐helices, respectively. Surface plasma resonance analysis of unilamellar vesicles immobilized through a biotin–avidin interaction showed that both CB and CB1 bind to the lipid bilayers at high concentration (>10 µm); in contrast, CB3 induces disintegration of the vesicles at all concentrations tested. Differential scanning calorimetry showed the concentration‐dependent effect of bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase‐transition curves obtained. The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular‐shaped, nonlamellar structures which rapidly disintegrate into twisted, microtubule‐containing debris before being completely destroyed. On the basis of these observations, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed.
Structural and biological effects of a β2‐ or β3‐amino acid insertion in a peptide
Authors: Sagan Sandrine , Milcent Thierry , Ponsinet Rachel , Convert Odile , Tasseau Olivier , Chassaing Gérard , Lavielle Solange , Lequin Olivier
Published: March 2003

Molecular mechanics calculations on conformers of Ac‐HGly‐NHMe, Ac‐β2‐HAla‐NHMe and Ac‐β3‐HAla‐NHMe indicate that low‐energy conformations of the β‐amino acids backbone, corresponding to gauche rotamers around the Cα–Cβ bond, may overlap canonical backbone conformers observed for α‐amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)‐β2‐HPhe and of Gly9 by HGly (R)‐β2‐HAla or (S)‐β3‐HAla. [(R)‐β2‐HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)‐β3‐HAla9]SP show a 30‐ to 50‐fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a β‐amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)‐β3‐HAla9]SP and [(R)‐β2‐HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of β2‐HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a β‐amino acid could replace an α‐amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the α‐amino acid.
Promoter analysis of the human succinate dehydrogenase iron‐protein gene
Authors: Au Harry C. , Scheffler Immo E.
Published: March 2003

The iron‐sulfur subunit of succinate dehydrogenase is one of the four subunits of complex II of the mitochondrial electron transport chain. Its gene, SDH2, is one of the four nuclear‐encoded genes for this complex. Reporter gene analysis of the human SDH2 promoter indicates that it is transcriptionally regulated by the nuclear respiratory factors NRF‐1 and NRF‐2. Their binding sites reside immediately upstream (within 90 bp) of the transcription start site. Site‐directed mutagenesis of either site lowers the reporter gene activity by tenfold to a basal level. Gel shift experiments and competition experiments with the authentic NRF‐1 and NRF‐2 DNA oligomers from previously characterized nuclear respiratory genes strengthen the proposed role of these two transcriptional regulators. These experiments extend the proposed regulatory role of these two transcription factors to complex II of the respiratory chain.

The expression of three of the four genes of complex II was also examined when mouse myoblast C2C12 cells were induced to differentiate into myotubes. Up‐regulation upon differentiation in tissue culture is only modest, 2−3 fold over the myoblast cells.
Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae
Authors: Eriksson Tove Lisa Jenny , Johansson Eva , Whitley Paul , Schmidt Margit , Elsayed Said , van Hage‐Hamsten Marianne
Published: March 2003

The complete cDNA encoding a major allergen from the dust mite Tyrophagus putrescentiae, Tyr p 2, has been sequenced and expressed. A degenerate primer was designed to the N‐terminal amino acid sequence of the 16‐kDa protein. The complete cDNA sequence was achieved by using reverse transcriptase PCR, PCR+1, standard cloning and sequencing techniques. The cDNA of Tyr p 2 is 552 nucleotides in length from the start codon including 126 nucleotides after the stop codon up to the beginning of the poly(A) tail. The leader sequence consists of 15 amino acids. Regarding the predicted amino acid sequence, there are no potential N‐glycosylation sites (N‐X‐S/T). The sequence showed similarity to group II allergens from other mite species, and some regions are completely conserved. To show that the cloned cDNA sequence was coding for an allergen, Tyr p 2 was expressed in Escherichia coli and shown to react with a T. putrescentiae‐positive serum pool.
Large‐scale production, purification and refolding of the full‐length cellular prion protein from Syrian golden hamster in Escherichia coli using the glutathione S‐transferase‐fusion system
Authors: Völkel Dirk , Blankenfeldt Wulf , Schomburg Dietmar
Published: March 2003

Until quite recently, high‐level expression of full‐length cellular prion protein (Prpc) in bacterial cells was not possible. We describe here the effective purification of mature Syrian golden hamster PrPc (residues 23−231) as a C‐terminal fusion to glutathione S‐transferase (GST) from inclusion bodies expressed in Escherichia coli. Purification of the denatured fusion protein was simplified greatly by the introduction of a C‐terminal histidine anchor, leading to 255 mg pure GST‐PrPc‐His6/l bacterial broth, which could be refolded easily by dilution in 20 mM Tris, 5 mM dithiothreitol, 1 mM EDTA, pH 9.0. Refolding was monitored by following GST activity. Mature Syrian hamster PrPc (residues 23−231) was released from the refolded fusion protein by thrombin digestion, yielding 73 mg homogeneous protein/l bacterial culture after purification. The recombinant protein was identified by monoclonal antibodies, Edman sequencing and matrix‐assisted laser‐desorption/ionization MS. Correct folding was confirmed by near‐ultraviolet circular dichroism spectroscopy. Samples resulting from different purification steps were sensitive to proteinase K digestion and showed no signs of infectivity in animal experiments, demonstrating that the PrPc produced is identical with the cellular isoform. The presented purification procedure should prove useful for the production of other GST‐fusion proteins.
Binding of pyrene isothiocyanate to the E1ATP site makes the H4‐H5 cytoplasmic loop of Na+/K+‐ATPase rigid
Authors: Linnertz Holger , Miksik Ivan , Kvasnicka Peter , Bertoli Enrico , Mazzanti Laura , Schoner Wilhelm , Amler Evzen
Published: March 2003

1‐Pyreneisothiocyanate was shown to be an inhibitor of Na+/K+‐ATPase. Reverse‐phase HPLC and activity studies indicated binding of 1‐pyreneisothiocyanate at the H4‐H5 loop of the α subunit and competition with the fluorescein 5′‐isothiocyanate for the E1ATP site. While fluorescein 5′‐isothiocyanate, the fluorescent ATP pseudo‐analog, was shown to be immobilized at the E1ATP site, there was no possibility to draw any conclusion about the flexibility of the E1ATP site due to its short lifetime. Employing 1pyreneisothiocyanate as a long‐lived fluorophore and a label for the E1ATP site, we found that the ATP‐binding site of Na+/K+‐ATPase and, in fact, the whole large intracellularly exposed H4‐H5 loop of the catalytic α subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function.
Mechanism of the leakage induced on lipid model membranes by the hemolytic protein sticholysin II from the sea anemone Stichodactyla helianthus
Authors: de los Rios Vivian , Mancheño José M. , Lanio María E. , Oñaderra Mercedes , Gavilanes José G.
Published: March 2003

A potent hemolytic polypeptide, sticholysin II, has been purified to homogeneity from the sea anemone Stichodactyla helianthus. The protein produces leakage of aqueous contents of model lipid vesicles composed of either phosphatidylcholine or sphingomyelin if cholesterol is present in these membranes. The leakage has been analyzed by measuring the dequenching of the fluorescent dye 8‐aminonaphthalene‐1,3,6‐trisulfonic acid, coencapsulated with its quencher N,N′‐p‐xylenebispyridinium bromide, upon dilution of the vesicle contents into the external medium. The protein displays a maximum effect on vesicles containing 20−25 % cholesterol. Leakage is also produced in vesicles composed of mixtures of phosphatidylcholine and sphingomyelin, the maximum effect being observed for 20−30 % sphingomyelin molar content. The extent of the leakage is dependent on the molecular mass of the vesicle entrapped solutes in the range 445−960 Da. This suggests the involvement of a pore of about 1 nm in diameter based on the limiting size observed for the leakage of the different solutes. Oligomerization of the protein is apparently involved in the membrane permeabilization, based on the kinetic analysis of the leakage process which is shown to proceed through an all‐or‐none mechanism.
Three‐dimensional structure of moenomycin A
Authors: Kurz Michael , Guba Wolfgang , Vértesy László
Published: March 2003

The first three‐dimensional structure of moenomycin A in aqueous solution based on NMR‐derived distance constraints and molecular dynamics simulations is presented. The antibiotic moenomycin A was obtained from the FlavomycinR complex by ultrafiltration, chromatography on a DEAE‐cellulose ion exchanger and reverse‐phase chromatography in 98 % purity. In contrast to the previously reported behaviour, the compound gave rise to well‐resolved NMR spectra in standard solvents. Using several two‐dimensional experiments, a complete assignment of proton and carbon chemical shifts was achieved in (CD3)2SO, CD3OD and H2O/D2O (9 : 1, pH 7.3). A total of 175 interproton distances were determined from 600‐MHz rotating‐frame NOE (ROE) spectra and were used as restraints in molecular dynamics calculations. These restraints included 84 ROEs between protons of the moenocinol part leading to a very well‐defined structure of the lipid part of the molecule. The relative orientation of the subunits was determined by 66 ROEs among different sugar rings and between the sugar rings and moenocinol. As a result of the molecular dynamics calculation, rings D, E, and F as well as the moenocinol part are very well‐defined (average rms deviation over all heavy atoms 0.48 Å) whereas rings A, B and C display a higher degree of conformational flexibility which might be an artefact due to the lower number of ROEs in this part of the molecule. A three‐dimensional pharmacophore hypothesis comprising functional groups of rings E and F and the carboxyl group of glyceric acid is presented on the basis of the degradation and derivatization studies of Welzel and coworkers [Welzel, P., Kunisch, F., Kruggel, F., Stein, H., Scherkenbeck, J., Hiltmann, A., Duddeck, H., Müller, D., Maggio, J. E., Fehlhaber, H.‐W., Seibert, G., van Heijenoort, Y. & van Heijenoort, J. (1987) Moenomycin A : Minimum structural requirements for biological activity, Tetrahedron 43, 585−598; Welzel, P. (1993) Transglycosylase inhibition, in Antibiotics and antiviral compounds − Chemical synthesis and modifications (Krohn, K., Kirst, H. A. & Maag, H., eds) pp. 373−378, VCH Weinheim].
The conversion of recombinant human mast cell prochymase to enzymatically active chymase by dipeptidyl peptidase I is inhibited by heparin and histamine
Authors: McEuen Alan R. , Ashworth Doreen M. , Walls Andrew F.
Published: March 2003

Chymase, a major product of mast cell activation, is secreted as a fully active enzyme. We have prepared recombinant human prochymase and have investigated the conditions under which it may be activated by dipeptidyl peptidase I (DPP I). The gene for human chymase was cloned in a baculovirus vector and expressed in High Five insect cells, and the recombinant protein purified by heparin‐agarose and gel‐filtration chromatography. The purified prochymase was homogeneous by SDS/PAGE with the same molecular mass as native human chymase, and its identity confirmed by N‐terminal sequence analysis and Western blotting with chymase‐specific antibodies. Treatment with DPP I to remove the N‐terminal dipeptide prosequence resulted in enzymatically active chymase, with substrate and inhibitor profiles very similar to those of the native human enzyme. The activation of prochymase by DPP I was strongly inhibited by heparin (IC50 = 0.5 μg ml−1) and histamine (IC50 = 2 mM), though these mast cell products had little effect on the action of DPP I towards a low molecular‐mass substrate. The pH optimum of DPP I was also higher and narrower with prochymase. The inhibitory action of heparin was lost at NaCl or KCl concentrations sufficient to elute prochymase from a heparin agarose column. Dextran sulphate was as inhibitory as heparin, whereas chondroitin sulphate C was more than 10‐fold less effective. Our findings suggest that the activation of prochymase might be restricted to the early stages of vesicle maturation, when the pH is close to neutrality and the histamine and heparin concentrations are low.
Engineering of factors determining α‐amylase and cyclodextrin glycosyltransferase specificity in the cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1
Authors: Wind Richèle D. , Buitelaar Reinetta M. , Dijkhuizen Lubbert
Published: March 2003

The starch‐degrading enzymes α‐amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of α‐amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacteriumthermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active‐site cleft. All α‐amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(Δ′DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an α‐amylase protein in terms of the identity of residue 196, and in the domain structure containing, however, some additional Cterminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(Δ′DE) displayed a strongly reduced raw‐starch‐binding capacity, similar to the situation in most α‐amylases that lack a raw‐starch‐binding E domain. Compared to wild‐type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(Δ′DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild‐type CGTase converted starch into 35 % cyclodextrins and 11 % linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(Δ′DE) converted starch into 21 % cyclodextrins and 18 % into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to α‐amylase specificity.
Blockage of apoptotic signaling of transforming growth factor‐β in human hepatoma cells by carboxyfullerene
Authors: Huang Yu‐Lun , Shen Clifton K.‐F. , Luh Tien‐Yau , Yang Hong Chou , Hwang Kuo Chu , Chou Chen‐Kung
Published: March 2003

Transforming growth factor‐β (TGF‐β) has been shown to induce apoptosis in normal hepatocytes and hepatoma cells both in vivo and in vitro. However, the mechanism by which TGF‐β induces apoptosis is not clear. The antiapoptotic activity of antioxidants including N‐acetyl‐
L‐cysteine (Ac‐Cys), ascorbic acid and a novel free radical scavenger, carboxyfullerene (C60) on TGF‐β‐treated human hepatoma Hep3B cells was examined. Only the water‐soluble hexacarboxylic acid derivative of C60 was found to prevent TGF‐β‐induced apoptosis. Antiapoptotic activity of C60 correlated its ability to eliminate TGF‐β‐generated reactive oxygen species (ROSs). However, C60 did not interfere with TGF‐β‐activated PAI‐1 promoter activity in the Hep3B cells. These results indicate that the signaling pathway of TGF‐β‐induced apoptosis may be related to the generation of ROSs and may be uncoupled from the TGF‐β‐activated gene promoter activity. Furthermore, the regioisomer of C60 with a C3 symmetry was more potent in protecting cells from apoptosis than that with a D3 symmetry, and the C3 isomer had stronger interactions with lipid bilayers than the D3 isomer. The spectroscopic analysis revealed that the C3 isomer had stronger interactions with artificial lipid bilayers than the D3 isomer. Therefore, our study indicates that C60 may interact with membrane to eliminate TGF‐β‐induced ROSs and to prevent apoptosis occur in human hepatoma cells.
Gene cloning, biochemical characterization and physiological role of a thermostable low‐specificity L‐threonine aldolase from Escherichia coli
Authors: Liu Ji‐Quan , Dairi Tohru , Itoh Nobuya , Kataoka Michihiko , Shimizu Sakayu , Yamada Hideaki
Published: March 2003

The ltaE gene encoding for a thermostable low‐specificity
L‐threonine aldolase, which catalyzes the cleavage of
L‐threonine/
L‐allo‐threonine to glycine and acetaldehyde, was cloned from Escherichia coli GS245 by the polymerase chain reaction. Construction and expression of the plasmid pLTAE, which contained the ltaE gene under the control of the lac promoter, resulted in a 227‐fold increase in the specific activity above the level detected in E. coli cells containing the control vector. The enzyme is thermostable : it retained its full activity upon heating at 60 +C for 1 h. The enzyme was thus feasibly purified to homogeneity by heat treatment and butyl‐Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, gene disruption was performed. Knockout of the ltaE gene of wild‐type E. coli did not effect the cellular growth rate, while disruption of the ltaE gene of E. coli GS245, whose serine hydroxymethyltransferase gene was knocked out, caused a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild‐type E. coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert.
Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum
Authors: Heikoop Judith C. , Grootenhuis Peter D. J. , Blaauw Mieke , Veldema Jacob S. , Van Haastert Peter J. M. , Linskens Maarten H. K.
Published: March 2003

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non‐covalently associated subunits. We aimed for the expression of a single‐chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single‐chain hCG is indeed produced by Dictyostelium. Furthermore, this single‐chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptor‐binding affinity is comparable to single‐chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.
Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two‐dimensional nuclear magnetic resonance data
Authors: Mandard Nicolas , Sodano Patrick , Labbe Henri , Bonmatin Jean‐Marc , Bulet Philippe , Hetru Charles , Ptak Marius , Vovelle Françoise
Published: March 2003

Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad range of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two‐dimensional (2D) 1H‐NMR spectroscopy and molecular modelling have been used to determine its three‐dimensional (3D) structure in water. Thanatin adopts a well‐defined anti‐parallel β‐sheet structure from residue 8 to the C‐terminus, including the disulfide bridge. In spite of the presence of two proline residues, there is a large degree of structural variability in the N‐terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibacterial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins, which have a two‐stranded β‐sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hétru, C. & Hoffmann, J. (1996) Proc. Natl Acad. Sci. USA 93, 1221−1225], our structural study evidences the importance of the β‐sheet structure and also suggests that anti‐Gram‐negative activity involves a site formed by the Arg20 side‐chain embedded in a hydrophobic cluster.
Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase
Authors: Venning Jamie D. , Bizouarn Tania , Cotton Nick P. J. , Quirk Philip G. , Jackson J. Baz
Published: March 2003

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped‐flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP+ (k≈0.03 s−1). Phase A of the burst (k≈600 s−1) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k≈10−50 s−1), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I : III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I :III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B‐2H]NADPH, and by [4B‐1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27 535−27 538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I :III complex.
The influence of 5′ codon context on translation termination in Saccharomyces cerevisiae
Authors: Mottagui‐Tabar Salim , Tuite Mick F. , Isaksson Leif A.
Published: March 2003

Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation‐assay system. Codon changes that were made at position −2 relative to the stop codon, gave a 3.5‐fold effect on termination in a release‐factor‐defective (sup45) mutant strain, in line with the effect observed in a wild‐type strain. The influence of the −2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C‐terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing −1 (P‐site) codons in yeast gave a 10‐fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso‐codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P‐site tRNAis able to influence termination at UGAC in yeast.
Reconsidering the energetics of ribonuclease catalysed RNA hydrolysis
Authors: Loverix Stefan , Laus Georges , Martins José C. , Wyns Lode , Steyaert Jan
Published: March 2003

In principle, all biochemical reactions are reversible, though some are more reversible than others. The classical ribonuclease mechanism involves a reversible transphosphorylation step, followed by quasi irreversible hydrolysis of the cyclic intermediate. We performed isotope‐exchange and intermediate‐trapping experiments showing that the second hydrolysis step is readily reversible in the presence of RNase A or RNase T1. As a consequence, the equilibrium between a phosphodiester and a 2′,3′‐cyclophosphate accounts for all catalysed reactions, even if the leaving/attacking group is a water molecule. Therefore, ribonucleases are transferases rather than hydrolases. The equilibrium constant for the catalysed interconversion is close to 1 M. From this result, we estimate the effective concentration of the 2′‐hydroxyl nucleophile in the cyclization step to be 107 M. The high effective concentration of the vicinal hydroxyl group balances the strain‐associated and solvation‐associated instability of the pentacyclic phosphodiester.