bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterisation of the Nucleic‐Acid‐Binding Activity of KH Domains Different Properties of Different Domains
Authors: Dejgaard Kurt , Leffers Henrik
Published: July 2004

The KH module is a sequence motif recently identified in a number of diversified RNA‐binding proteins and suggested to be the functional element responsible for RNA binding. So far, however, this hypothesis has not received direct experimental support. We have expressed the three KH‐domains from heterogeneous nuclear ribonucleoprotein K (hnRNP‐K), the poly(C)‐binding proteins PCBP‐1 and PCBP‐2, the first three to four domains from the high‐density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile‐X protein FMR1 in Escherichia coli and analysed their nucleic‐acid‐binding properties in vitro. The results showed that the in vitro poly(rC)‐binding activity of hnRNP‐K can be assigned to KH‐domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC). In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level. The first KH domain from the FMR1 protein binds poly(rG) and single‐stranded and double‐stranded DNA. The N‐terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single‐stranded and double‐stranded DNA. Thus, single KH domains are discrete and independent nucleic‐acid‐binding units. Moreover, different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic‐acid‐binding, structure that is fine tuned, by sequence variation, resulting in sequence‐specific nucleic‐acid‐binding entities.
Cloning, Sequencing and Functional Overexpression of the Streptococcus equisimilis H46A gapC Gene Encoding a Glyceraldehyde‐3‐phosphate Dehydrogenase that also Functions as a Plasmin(ogen)‐Binding Protein
Authors: Gase Klaus , Gase Ariane , Schirmer Heike , Malke Horst
Published: July 2004

We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms that control the balance between the plasminogen activating and plasmin(ogen) binding capacities of H46A, we describe here its gapC gene encoding glyceraldehyde‐3‐phosphate dehydrogenase (GraP ‐DH, EC 1.2.1.12), a glycolytic enzyme apparently transported to the cell surface where it functions as a plasmin(ogen) binding protein. The gapC gene was cloned and sequenced and found to code for a 336‐amino‐acid polypeptide (≈35.9 kDa) exhibiting 94.9% sequence identity to the Plr protein from Streptococcus pyogenes shown by others to be capable of plasmin binding [Lottenberg, R., Broder, C. C., Boyle, M. D., Kain, S. J., Schroeder, B. L. & Curtiss, R. III (1992) J. Bacteriol. 174, 5204–5210. To study the properties of the GapC protein, its gene was inducibly overexpressed in Escherichia coli from QIAexpress expression plasmids to yield the authentic GapC or (His)6 GapC carrying a hexahistidyl N‐terminus to permit affinity purification. Both proteins were functionally active, exhibiting specific GraP ‐DH activities of about 80 kat/mol (≈130 U/mg) after purification. Their binding parameters [association (ka) and dissociation (kd) rate constants, and equilibrium dissociation constants (Kd= kd/ka] for the interaction with human Glu‐plasminogen and plasmin were determined by real‐time biospecific interaction analysis using the Pharmacia BIAcore instrument. For comparative purposes, the commercial GraP ‐DH from Bacillus stearothermophilus (BstGraP ‐DH), a nonpathogenic organism, was included in these experiments. The Kd, values for binding of plasminogen to GapC, (His)6 GapC and BstGraP ‐DH were 220 nM, 260 nM and 520 nM, respectively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the binding to plasmin. These data show that both the zymogen and active enzyme posse low‐affinity binding sites for the gapC gene product and that the hexahistidyl terminus does not affect its function. Prior limited treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP ‐DHs, presumably by the exposure of new C‐terminal lysine residues for binding to the zymogen.
The Solution Structure Refinement of the Paramagnetic Reduced High‐Potential Iron‐Sulfur Protein I from Ectothiorhodospira Halophila by Using Stable Isotope Labeling and Nuclear Relaxation
Authors: Bertini Ivano , Couture Manon M. J. , Donaire Antonio , Eltis Lindsay D. , Felli Isabella C. , Luchinat Claudio , Piccioli Mario , Rosato Antonio
Published: July 2004

The reduced high‐potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe‐4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994)Eur. J. Biochem. 225, 715–7251. We have performed classical HNHA, HNCA soft‐COSY, soft‐HCCH E. COSY and 15N‐1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single‐quantum experiments preceded by a 180° pulse and variable delay times, the non‐selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (pppara) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and φpara data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the φpara constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one‐dimensional NOEs,39 3JHNHα and 37 3JHNC′ (providing 45 φ dihedral angle constraints) 54 3JHαHβ and 31 3JNHβ (providing 26 χ1 dihedral angle constraints), 4 χ2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the βCH protons, and 58 φpara constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.
X‐ray Structure of Native Full‐Length Human Fibroblast‐Growth Factor at 0.25‐nm Resolution
Authors: Romero Antonio , Pineda‐Lucena Antonio , Giménez‐Gallego Guillermo
Published: July 2004

Acidic fibroblast‐growth factor (aFGF) is one of the typical members of a group of nine polypeptides of relatively similar amino acid sequence known as the fibroblast‐growth‐factor family of proteins. Widely distributed throughout the organism, fibroblast‐growth factors seem to be involved in numerous physiological processes ranging from control of cell proliferation and differentiation to modulation of animal behaviour and arterial blood pressure. This wide assortment of biological activities explains their involvement in numerous pathologies. Instability and low yields of the purified protein have precluded high‐resolution structural studies of the physiological form of aFGF. Nevertheless, modifications introduced recently into the synthesis and purification procedures of this protein have allowed preparations of samples that, as shown here, are reliable substrates to obtain crystals suitable for X‐ray‐diffraction studies. These analyses have allowed us to elucidate the three‐dimensional structure of the physiological form of human aFGF by molecular‐replacement methods, from the previously reported structure of a shortened form of bovine aFGF that was stabilized by point‐directed mutagenesis. The structure was refined at a resolution of 0.25 nm to an R factor of 20.4% for 13109 reflections between 0.6 nm and 0.25 nm, with rmsd of 1.1 pm and 1.9° from ideal bond lengths and bond angles, respectively. Human aFGF folds according to a β‐trefoil topology. This fold consist of six β‐strand pairs. Three of them form a six‐stranded β‐barrel structure that is capped at one end by the other three pairs arranged in a triangular array. The N‐terminus of aFGF up to residue Pro19 appears flexible in the structure and does not specifically interact with the rest of the molecule.
Structural Alterations of Horseradish Peroxidase in the Presence of low Concentrations of Guanidinium Chloride
Authors: Chakrabarti Abhijit , Basak Soumen
Published: July 2004

The presence of very low concentrations of guanidinium chloride (GdmCl) alters the tertiary structure of the monomeric heme‐containing enzyme, horseradish peroxidase (HRP). The change in tertiary structure of the protein was reflected in the mean fluorescence lifetime of its single tryptophan residue, which increased from 2.3 ± 0.1 ns in the native enzyme to 2.7 ± 0.2 ns in the presence of 100 mM GdmCI. More convincing evidence in support of such alterations came from quenching study of tryptophan fluorescence using the most widely used quencher, acrylamide. It revealed significant differences between the Stern‐Volmer quenching constants observed in the absence and in the presence of GdmCl concentrations below 100 mM. The fluorescence emission maximum of 6‐propionyl‐2‐(dimethylamino)naphthalene (PRODAN), used as an extrinsic fluorophore to probe any changes in the tertiary structure of the enzyme, was blue‐shifted from 522 nm in aqueous buffer to 509 nm in the presence of 27 μM native HRP. However, this emission maximum appeared at 519 nm when the PRODAN was incorporated in HRP previously incubated with 100 mM GdmCl. The fluorescence lifetime of PRODAN incorporated in HRP was also different from that of PRODAN in buffer, but much more so in absence of GdmCl than in its presence. Taken together, these results indicate partial unfolding of HRP leading to a conformation with native‐like secondary structure and unaltered enzymatic activity, in presence of millimolar concentrations of GdmCI.
Chemically and Conformationally Authentic Active Domain of Human Tissue Inhibitor of Metalloproteinases‐2 Refolded from Bacterial Inclusion Bodies
Authors: Williamson Richard A. , Natalia Dessy , Gee Christopher K. , Murphy Gillian , Carr Mark D. , Freedman Robert B.
Published: July 2004

The aggregation of recombinant proteins into inclusion bodies is a major problem for expression in bacterial systems. The inclusion bodies must be solubilized and the denatured protein renatured if an active molecule is to be recovered. We have developed such a procedure for the active N‐terminal domain of tissue inhibitor of metalloproteinases‐2 [TIMP‐2‐(1‐127)], a small mammalian protein containing three disulfide bonds. Conditions for its renaturation were determined by studying the refolding behaviour of reduced and denatured mammalian‐cell‐expressed TIMP‐(1‐127) by intrinsic fluorescence. This strategy allows the development of a refolding protocol before generation of a bacterial expression system, and allows rapid and systematic optimization of each refolding variable by assessing its effect on the rate and extent of the refolding reaction. TIMP‐(1‐127) was expressed at high levels inEscherichia coli, and refolded from TIMP‐2‐(1‐127) inclusion bodies, by means of the method developed with mammalian‐cell‐expressed protein, to give a refolding efficiency of 30‐40% and a final yield of 11‐14 mg purified protein/l culture. The chemical structure and conformation of this material was characterized by electrospray mass spectrometry and two‐dimensional1H‐NMR; no significant differences were found between it and the native protein. Mass analysis of uniformly13C‐labeled and15N‐labeled protein was used to help identify a mistranslated TIMP‐(1‐127) contaminant in the purified refolded sample. This technique provides additional information on the nature of the modification and allows a distinction to be made between those modifications that are cell derived, and those that arise from subsequent handling of the protein.
Induction of Programmed Cell Death and Immunosuppression by Exogenous Sphingolipids are Separate Processes
Authors: Olshefski Randal , Taylor Barbara , Heitger Andreas , Hasegawa Akira , Ladisch Stephan
Published: July 2004

Gangliosides are highly immunosuppressive molecules but the mechanism(s) by which they act upon cells remains to be fully defined. Several metabolic products of exogenous gangliosides, including ceramide, have recently been suggested as second messengers in programmed cell death (PCD). Therefore, we have probed the role of gangliosides and ceramides in the induction of PCD and in the inhibition of in vitro lymphoproliferation. PCD was caused only by exogenous ceramides with short fatty acyl groups‐ d18:1‐C2:O (C2‐ceramide, where d18:1 is sphingosine and C2:O is an acetyl group) and d18:1–C6:0 (C6‐ceramide, where C6:O is a hexanoyl group). None of the gangliosides studied induced PCD, including naturally occurring GM3 synthetic d18:1‐C18:0 GM3 (C18‐Cer GM3, where C18:0 is a stearoyl group), or even d18:1‐C2:0 GM3 (C2‐Cer GM3), which itself contains a PCD‐causing ceramide. However, these gangliosides were highly immunosuppressive, inhibiting antigen‐induced lymphoproliferation at micromolar concentrations. We conclude that exogenous sphingolipids cause inhibition of lymphoproliferation and PCD by two separate and distinct mechanisms of action.
Determination of a Binding Site for a Non‐Substrate Ligand in Mammalian Cytosolic Glutathione S‐Transferases by means of Fluorescence‐Resonance Energy Transfer
Authors: Sluis‐Cremer Nicolas , Naidoo Nirinjini N. , Kaplan Warren H. , Manoharan T. Herbert , Fahl William E. , Dirr Heini W.
Published: July 2004

To determine the location of the non‐substrate‐ligand‐binding region in mammalian glutathione S‐transferases, fluorescence‐resonance energy transfer was used to calculate distances between tryptophan residues and protein‐bound 8‐anilinonaphthalene 1‐sulphonate (an anionic ligand) in the human class‐α glutathione S‐transferase, and in a human Trp28→Phe mutant class‐π glutathione S‐transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class‐α and class‐π enzymes, respectively. Since glutathione S‐transferases bind one non‐substrate ligandprotein dimer, the ligand‐binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S‐transferase binds praziquantel, a non‐substrate drug used to treat schistosomiasis [McTigue, M. A., Williams, D. R. & Tainer, J. A. (1995)J. Mol. Biol. 246, 21–271. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S‐transferases bind non‐substrate ligands in the amphipathic groove at the dimer interface.
Two Distinct Ca2+ Influx Pathways Activated by the Bradykinin B2 Receptor
Authors: Abdalla Said , Müller‐Esterl Werner , Quitterer Ursula
Published: July 2004

The hormone‐induced depletion of cellular Ca stores provides a signal for the Ca2+ influx into electrically non‐excitable cells; however, the underlying molecular mechanisms remain elusive. Therefore, we analyzed bradykinin‐activated Ca2+ influx into human foreskin fibroblast cells, HF‐15, by fura‐2 and 45Ca labeling to discriminate between Ca2+ influx into the fura‐sensitive compartment and Ca uptake into fura‐insensitive Ca stores. Bradykinin‐activated CaZt influx into the fura‐sensitive compartment was blocked by inhibitors of NO synthases. These inhibitors also suppressed bradykinin‐activated increases in cGMP, indicating that the NO‐dependent increase in cGMP is involved in the activation of the Ca2+ influx into the fura‐sensitive compartment. The cGMP‐dependent kinase inhibitors KT5823 and Rp‐8‐bara‐chlorophenylthio)‐cGMP (Rp‐8‐pCPT‐cGMPS) blocked bradykinin‐activated Ca2+ influx into the fura‐sensitive compartment, suggesting that a cGMP‐dependent kinase step participates in the activation of this Ca2+ influx pathway. In addition to the NO/cGMP‐mediated Ca2+ influx into the fura‐sensitive compartment, bradykinin enhanced 45Ca uptake into Ca stores that were not accessible to fura‐2. This enhanced 45Ca uptake was insensitive to blockers of the NO/cGMP pathway, indicating that the 45Ca uptake pathway is distinct from the NO‐dependent Ca2+ influx into the fura‐sensitive compartment. Furthermore, bradykinin enhanced 45Ca uptake into proliferating but not into quiescent HF‐15 fibroblasts. Hence, bradykinin stimulates two distinct Ca2+ influx pathways in HF‐15 cells, (a) Ca2+ influx into the fura‐sensitive compartment which is NO/cGMP‐dependent and (b) Ca uptake into Ca stores which bypasses the cytoplasm, which is NO insensitive and which is linked to cell proliferation.
Follitropin Action on the Transferrin Gene in Sertoli Cells is Mediated by cAMP‐Responsive‐Element‐Binding‐Protein and Antagonized by Chicken Ovalbumin‐Upstream‐Promoter‐Transcription Factor
Authors: Suire Sabine , Maurel Marie‐Christine , Guillou Florian
Published: July 2004

The transcription of the transferrin (Tf) gene is induced by follitropin via cAMP in rat Sertoli cells. We previously demonstrated that the cAMP‐responsive‐element‐binding protein (CREB) interacts on the proximal region II (PRII) of the human Tf promoter (Suire et al., 1995). The PRII region is identified as essential for cAMP inducibility of the Tf promoter and contains a CCAAT box. This unexpected result led us to study the relation that exists between CREB and the PRII site.

In the liver, CCAAT/enhancer‐binding (C/EBP) proteins act at the PRII site. Although these factors are absent in Sertoli cells, their overexpression in Sertoli cells disturbs basal and induced transcription. C/EBP α and δ were able to stimulate the basal transcription driven by the–100 to +39 region, placed upstream of the chloramphenicol acetyltransferase (CAT) gene. However, only C/EBPα allowed the cAMP‐inducible expression.

The Ka of CREB bZIP (254–327), a deleted form of CREB, for the CRE site (3.92 × 108M−1) and for the PRII site (1.38 × 108M−1) were determined using the surface plasmon resonance (SPR) method. The Ka values were similar, although the derived kinetics were different: higher ka and kd of CREB for the PRII site were found compared with the CRE site.

Since we observed important dissociation kinetics, we hypothesized that the binding of CREB to the PRII site is stabilized by CREB‐binding protein (CBP) or by chicken‐ovalbumin‐upstream‐promoter transcription factor (COUP‐TF) binding to PRI site near to PRII. However, we observed that the overexpression of CBP in Sertoli cells did not potentiate the basal and cAMP‐stimulated activity of CREB of the–100 to +39Tf‐CAT construct. In basal and cAMP‐stimulated conditions, COUP‐TF appeared to repress the transcription driven by the–100 to +39 region in a specific manner.

These results demonstrate a direct action of CREB on hTf promoter, which is antagonized by COUPTF and may explain the transcriptional regulation of Tf by follitropin, via cAMP.
Direct Electrochemistry of the Hydroxylase of Soluble Methane Monooxygenase from Methylococcus Capsulatus (Bath)
Authors: Kazlauskaite Jurate , Hill H. Allen O. , Wilkins Patricia C. , Dalton Howard
Published: July 2004

The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus cupsulutus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulutus (Bath) at a modified gold electrode giving rise to two waves at 4(± 10) mV and‐386(± 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B′ on the redox reactions were determined. The redox potentials of the complex with protein B are‐25(± 14)mV and 433(± 8) mV versus SCE whereas protein B′ had no effect though it did alter the effect of protein B on the redox potentials.
Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease
Authors: Friedhoff Peter , Meiss Gregor , Kolmes Bettina , Pieper Uwe , Gimadutdinow Oleg , Urbanke Claus , Pingoud Alfred
Published: July 2004

The extracellular nuclease from Serratia marcescens is a non‐specific endonuclease that hydrolyzes double‐stranded and single‐stranded DNA and RNA with high specific activity. Steady‐state and pre‐steady‐state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serrutia nuclease. Most of the natural substrates are cleaved with similar kcat and Km values, the kcat/Km ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA) · poly(dT) or poly(dG) · poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower Km, respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligo‐deoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxy‐nucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro‐Rp‐oxygen of the phosphate group 3′ adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp‐phosphorothioate supports cleavage at the 5′ adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp‐diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereo‐chemical outcome of cleavage. Pre‐steady‐state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate‐limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3′,5′‐bis(p‐nitrophenyl)phosphate as a substrate and cleaves it at its 5′‐end to produce nitrophenol and thymidine 3′‐(p‐nitrophenylphosphate) 5‐phosphate. The rate of cleavage of this artificial substrate, however, is 6‐7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.
The Lurlenes, a New Class of Plastoquinone‐Related Mating Pheromones from Chlamydomonas Allensworth (Chlorophyceae)
Authors: Jaenicke Lothar , Starr Richard C.
Published: July 2004

Chlamydomonas allensworth is a recently described species, which differs phenotypically and in its reproductive behaviour from other members of this genus of flagellated green protists. Its female cells temporarily or constantly excrete a luring signal into the medium to attract the male cells for mating. The sperm is susceptible to the pheromone either all the time or only after a maturation process. Maturation in this species is accompanied with a colour shift from green to khaki (strain from Lemon Cove CA) or to olive (strain from Catarina TX), apparently due to degradation of chloroplast material. In the bioassay with polyacrylamide beads (plain or DEAE‐substituted) to which the lures are bound, acting as mock females, Catarina‐strain males are only attracted to Catarina strain lures, whereas Lemon‐Cove‐strain males are attracted to Lemon‐Cove‐strain lures and to a lesser extent, to Catarina‐strain lures. Being amphiphilic acid, Lemon‐Cove‐strain lure is more tightly bound to DEAE beads. Catarina‐strain lure is an uncharged amphiphile. The two signal compounds have been isolated and identified by chemical analysis and mass and NMR spectroscopies as 0(5)‐β‐D‐xylosylated degradation products of the chloroplast electron transporter hydroplastoquinone, its polyprenoid side chain being oxidatively cleaved at the fourth double bond. Lurlenic acid (lurlene L) is the resulting acid and lurlenol (lurlene C) the corresponding alcohol. The pheromonal activities of both compounds have a threshold at 1 pM, and are destroyed by deglycosylation and subsequent oxidation to the quinonoid aglycon.

This class of amphiphilic lures, having the same basic glycosylated hydroquinone structure, is named lurlenes.
Pore‐Forming Properties of the Plasmid‐Encoded Hemolysin of Enterohemorrhagic Escherichia Coli 0157: H7
Authors: Schmidt Herbert , Maier Elke , Karch Helge , Benz Roland
Published: July 2004

Lipid bilayer experiments were performed with the plasmid‐encoded hemolysin of enterohemorrhagic Escherichiu coli (EHEC) 0157:H7 strain EDL933. EHEC‐hemolysin caused the formation of transient ion‐permeable channels by integration in lipid bilayer membranes composed of asolectin, dioleoylglycero‐phosphoethanolamine and phosphoserine but not of diphytanoylglycerophosphocholine. Channel formation showed the same characteristics when culture supernatants of E. coli strains EDL 933 or HB101/pE040, precipitated or purified EHEC‐hemolysin were used for these experiments. The EHEC‐hemolysin channels had two different states at small transmembrane potential (20 mV): a prestate that represented the first step of channel formation (single‐channel conductance 40 pS in 0.15 M KCI) and an open state (550 pS in 0.15 M KCI at pH 6.0). Experiments with different salts suggested that the EHEC‐hemolysin‐induced channels were cation‐selective at neutral pH. The mobility sequence of the cations within the channels resembles their mobility sequence in the aqueous phase. The single‐channel data were consistent with the formation of wide, water‐filled channels by the EHEC hemolysin. The single channel conductance was strongly pH dependent and increased over 2.5‐fold in the pH range 5–8. The analysis of the single‐channel data using the Renkin correction factor suggested that the EHEC‐hemolysin formed channels with an average diameter of 2.6 nm. This size could be confirmed by the results of osmotic‐protection experiments. Neither sucrose nor raffinose inhibited toxin‐dependent hemolysis, whereas hemolysis did not occur in the presence of dextran 4 (molecular mass, 4 kDa). Our results demonstrate that EHEC‐hemolysin can be considered to be a highly active repeats‐in‐toxin (RTX)‐toxin with a similar but not identical pore‐forming capacity as the chromosomal encoded E. coliα‐hemolysin.
The Role of Conserved Histidine Residues in the Pyridine Nucleotide Transhydrogenase of Escherichia Coli
Authors: Bragg Philip D. , Hou Cynthia
Published: July 2004

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+, coupled to translocation of protons across the cyto‐plasmic membrane. The role of histidine residues in catalysis was investigated by chemical modification with diethylpyrocarbonate and by site‐directed mutagenesis. Diethylpyrocarbonate inhibited both hydride ion transfer and coupled proton translocation. Histidine residues were modified as shown spectroscopically and by the ability of hydroxylamine to cause reversal of inhibition. Complete inhibition of hydride ion transfer occurred following modification of 10 residuedenzyme molecule. Site‐directed mutagenesis of single conserved histidine residues or the presence of substrates did not provide resistance to inhibition by diethylpyrocarbonate. It is concluded that diethylpyrocarbonate inhibition was a consequence of the structural changes brought about by modification of many histidine residues. With the exception of β‐subunit residue His91 (βHis91), in which mutation can result in specific loss of proton translocation activity [Glavas, N. A., Hou, C. & Bragg, P. D. (1995)Biochemistry 34, 7694–77021, site‐directed mutation of the remaining conserved residues αHis450, βHisl61, βHis345 and βHis354 did not demonstrate a direct role for these residues in catalysis. Mutation of βHis161 had relatively little effect on the properties of the enzyme. By contrast, mutation of αHis450, βHis345 and βHis354 caused major loss of enzyme activities which was probably due to alterations in the structure of the enzyme. These alterations were reflected in changes in the Km values for transhydrogenation.
X‐ray Absorption Spectroscopy Study of Native and Phenylphosphorodiamidate‐Inhibited Bacillus pasteurii Urease
Authors: Benini Stefano , Ciurli Stefano , Nolting Hans F. , Mangani Stefano
Published: July 2004

X‐ray absorption spectroscopy (XAS) has been applied to urease from Bacillus pasteurii, a highly ureolytic soil bacterium, with the aim of elucidating the structural details of the nickel‐containing active site. The results indicate the presence of octahedrally coordinated Ni2+, in a sphere of six N/O donors at an average distance of 0.203 nm. An average of two histidine residues are bound to nickel. The experimental evidence suggests direct binding of the urease inhibitor phenylphosphorodiamidate to Ni2+. These spectroscopic results are in agreement with previous findings on both plant and microbial ureases, but differ in some respect from the results obtained by X‐ray crystallography analysis of Klebsiella aerogenes urease.
A Defined Epitope on the Human Choriogonadotropin α‐Subunit Interacts with the Second Extracellular Loop of the Transmembrane Domain of the Lutropidchoriogonadotropin Receptor
Authors: Couture Laurence , Remy Jean‐Jacques , Rabesona Hanitra , Troalen Frédéric , Pajot‐Augy Edith , Bozon Véronique , Haertle Thomas , Bidart Jean‐Michel , Salesse Roland
Published: July 2004

The monoclonal antibody, HT13 recognizes human choriogonadotropin (CG) bound to the extracellular domain of its receptor, but not to the fullolength receptor. The HT13 epitope is located in the regions of residues 15–17 and 73–75 of the human CG α‐subunit. Only one synthetic peptide, lutropin (LH)/CG‐receptor‐(481–497)‐peptide (EL2 peptide), which spans the second putative extracellular loop of the LWCG‐receptor endodomain, prevents recognition of human CG by HT13 mAb. EL2 peptide decreases hormone‐induced cAMP production, but not high‐affinity binding. An anti‐EL2 serum also displays the capacity to inhibit human CG‐stimulated cAMP production. These results suggest that the second extracellular loop of the receptor is in contact with the HT13 epitope of human CG a‐subunit and is involved in signal transduction. A relative orientation of the hormone versus the endodomain is proposed.
Ferritin mRNAs in Schistosoma Mansoni do not have Iron‐Responsive Elements for Post‐Transcriptional Regulation
Authors: Schüssler Peter , Pötters Elke , Winnen Ralf , Michel Anja , Bottke Werner , Kunz Werner
Published: July 2004

Schistosoma mansoni possesses two isoforms of ferritin, soma and yolk ferritin. The soma ferritin occurs at a low level in most cells of both genders, whereas the yolk ferritin is a female‐specific gene product that is expressed at high level in the vitellarium. In higher animals, ferritin mRNA is regulated by iron via the interaction of cytoplasmic binding proteins (IRPs) with a specific sequence element in the 5′ untranslated region (UTR) referred to as the iron‐responsive element (IRE). Sequence studies of the 5′ UTRs, gel retardation assays, and hybridization experiments show that neither ferritin mRNAs of S. mansoni is regulated by an IRE/IRP mechanism. It is suggested that ferritins in schistosomes are controlled only at the transcriptional level.
A Zyxin‐Related Protein whose Synthesis is Reduced in Virally Transformed Fibroblasts
Authors: Zumbrunn Jürg , Trueb Beat
Published: July 2004

We have cloned the gene for a novel LIM‐domain protein from human fibroblasts whose expression is substantially decreased in simian‐virus‐40‐(SV40)‐transformed cells. This protein has a calculated molecular mass of 61 kDa and comprises a prolinerich domain followed by three LIM motifs. It appears to be identical to the focal adhesion protein p83 that has recently been isolated and characterized from porcine and human platelets. Hybridization experiments demonstrate a very low degree of evolutionary conservation of its sequence between mammals and birds. It is therefore possible that the novel protein represents the human equivalent of the chicken protein zyxin as the two proteins display a very similar overall structure, although their amino acid sequences diverge markedly from each other. The repression of this zyxin‐related protein in virally transformed fibroblasts may explain, at least in part, the dramatic morphological changes that occur at the cell surface and in the cytoskeleton of transformed cells.
Expression, Characterisation and Mutagenesis of the Aspartic Proteinase from Equine Infectious Anaemia Virus
Authors: Powell David J. , Bur Daniel , Wlodawer Alexander , Gustchina Alla , Payne Susan L. , Dunn Ben M. , Kay John
Published: July 2004

The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli. The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase. EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV‐1 proteinase, including those which have been licenced as anti‐AIDS drugs. In this respect, EIAV proteinase behaves like an extreme case of a drug‐resistant mutant of HIV‐1 proteinase that has arisen under selective drug pressure. Only one potent inhibitor (HBY‐793) of HIV‐1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A‐77003 and A‐76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY‐793 from residues P2 to P2′[nomenclature of Schechter, I. & Berger, A. (1967)Biochem. Biophys. Res. Commun. 27. 157–1621, were not effective inhibitors of EIAV proteinase. Mutant forms of EIAV proteinase (Thr30→Asp and IIe54→ Gly) were generated and their ability to interact with substrates and inhibitors was characterised. HBY‐793 inhibited [Gly54]proteinase as effectively as the wild‐type proteinase but was tenfold less potent against [Asp30]proteinase. Data interpretations are presented, based on the structure solved for the complex between HBY‐793 and EIAV [Gly54]proteinase [Gustchina A., Kervinen, J., Powell, D. J., Zdanov, A., Kay, J. & Wlodawer, A. (1996)Protein Sci. 5, 1453–14651.
Inhibition by Iodoacetamide and Acetylene of the H‐D‐Exchange Reaction Catalyzed by Thiocapsa Roseopersicina Hydrogenase
Authors: Zorin Nikolay A. , Dimon Bernard , Gagnon Jean , Gaillard Jacques , Carrier Patrick , Vignais Paulette M.
Published: July 2004

The kinetics of H‐D isotope exchange catalyzed by the thermostable hydrogenase from Thiocapsa roseopersicina have been studied by analysis of the exchange between D1 and H2O. The pH dependence of the exchange reaction was examined between pH 2.5 and pH 11. Over the whole pH range, HD was produced at a higher initial velocity than H2, with a marked optimum at pH 5.5; a second peak in the pH profile was observed at around pH 8.5. The rapid formation of H2 with respect to HD in the D2/H2O system is consistent with a heterolytic cleavage of D2, into D+ and an enzyme hydride that can both exchange with the solvent. The H‐D‐exchange activity was lower in the H2/D2O system than in the D2/H2O system. The other reactions catalyzed by the hydrogenase, H2 oxidation and H2 evolution, are pH dependent; the optimal pH were 9.5 for H2 uptake and 4.0 for H2 production. Treatment of the active form of hydrogenase by iodoacetamide led to a slow and irreversible inhibition of the H‐D exchange. When iodo[1‐14C]acetamide was incubated with hydrogenase, the radioactive labeling of the large subunit was higher for the enzyme activated under H, than for the inactive oxidized form. Cysteine residues were identified as the alkylated derivative by amino acid analysis. Acetylene, which inhibits H‐D exchange and abolishes the Ni‐C EPR signal, protected the enzyme from irreversible inhibition by iodoacetamide. These data indicate that iodoacetamide can reach the active site of the H2‐activated hydrogenase from T: roseopersicina. This was not found to be the case with the seleno hydrogenase from Desulfovibrio baculatus (now Desulfomicrobium baculatus). Cysteine modification by iodoacetamide upon activation of the enzyme concomitant with loss of H‐D exchange indicates that reductive activation makes at least one Cys residue of the active site available for alkylation.
Selective Interaction Between Parathymosin and Histone H1
Authors: Kondili Katerina , Tsolas Orestes , Papamarcaki Thomais
Published: July 2004

We have studied the molecular associations of parathymosin, an acidic polypeptide with a wide tissue distribution, by means of three approaches; ligand blotting; native electrophoresis; and immunoprecipitation. We report here that parathymosin binds specifically to the linker histone H1. This binding is enhanced by Zn2+ and is dependent on the concentration of parathymosin. Poly(glutamic acid) is able to compete fully with parathymosin for binding to histone H1, suggesting that this interaction is mediated by the acidic domain of the protein. Moreover, we demonstrate that parathymosin interacts with the globular domain of histone H1 under native conditions. Based on these data, we postulate that parathymosin may belong to a group of nuclear acidic proteins that affect histone H1 function.
A Unique Phenylalanine‐Containing Lipopeptide Isolated from a Rough‐Colony Variant of Mycobacterium Avium
Authors: Rivière Michel , Puzo Germain , Wright Esther L. , Barrow William W.
Published: July 2004

Previous investigations have suggested that the biosynthesis of the Mycobacterium avium serovar‐specific glycopeptidolipid antigens involves initial steps that include the participation of lipopeptides. The prevailing assumption is that subsequent glycosylation of those lipopeptides results in the fully glycosylated form of the glycopeptidolipid components. In an effort to identify potential precursors in the biosynthetic pathway of glycopeptidolipid components, we have identified a unique lipopeptide from an M. avium rough variant (MAC702) that was isolated from a patient suffering from a chronic M. avium lung infection. Upon examination it was revealed that although the total lipid extract from MAC702 lacked serovar‐specific glycopeptidolipid antigens, it did contain a unique lipopeptide, possessing some amino acids identical to those found in the serovar‐specific glycopeptidolipid antigens. Initial examination of acid‐hydrolyzed samples of the lipopeptide (lipopeptide‐I) revealed the presence of phenylalanine, alanine, and isoleucine, but no carbohydrate. Subsequent mass spectrometric and 1H‐NMR and 1H‐CNMR correlation spectroscopy analysis confirmed the initial results and also revealed the presence of N‐methyl‐isoleucine. The following structure for lipopeptide‐I was proposed: fatty acyl (C19 or C17)‐Phe‐N‐methyl‐Ile‐Ile‐Phe‐Ala‐Ile‐Ala‐Phe. Lipopeptide‐I is unlike any heretofore identified compound, however, it does have similar features to lipopeptides previously reported in mycobacteria and fungi. Although its structure does not verify that it is a direct precursor in glycopeptidolipid biosynthesis, the presence of certain components in lipopeptide‐I indicate that it may share at least some pathways associated with the biosynthesis of the M. avium serovar‐specific glycopeptidolipids.
Molecular Cloning and Characterization of an Insect Aquaporin
Authors: Le Cahérec Françoise , Deschamps Stéphane , Delamarche Christian , Pellerin Isabelle , Bonnec Georgette , Guillam Marie‐Thérèse , Thomas Daniel , Gouranton Jean , Hubert Jean‐François
Published: July 2004

We previously described the structural organization of P25, a member of the major‐intrinsic‐protein family found in the digestive tract of homopteran sap‐sucking insects [Beuron, F., Le Cahérec, F., Guillam, M. T., Cavalier, A., Garret, A., Tassan, J. P., Delamarche, C., Schultz, P., Mallouh, V., Rolland, J. P., Hubert, J.‐F., Gouranton, J. & Thomas, D. (1995)J. Biol. Chem. 270, 17414–17422]. We demonstrated, by means of introducing P25 tetramers into the membranes of Xenopus oocytes, that this protein exhibits functional properties similar to those of aquaporin 1, the archetypal water channel [Le Cahérec, F., Bron, P., Verbavatz, J. M., Garret, A., Morel, G., Cavalier, A., Bonnec, G., Thomas, D., Gouranton, J. & Hubert, J.‐F. (1996)J. Cell Sci. 109, 1285–1295]. In the present work, we cloned a full‐length cDNA from a Cicadella viridis library with an open reading frame of 765 bp that encoded a 26‐kDa protein whose sequence was 43, 40, 36 and 36% identical to aquaporins 1, 2, z and tonoplast intrinsic protein γ, respectively. Translation of the corresponding RNA in Xenopus oocytes generated a polypeptide that was specifically recognized by polyclonal antibodies raised against native P25. Expression of the protein in Xenopus oocyte membranes was assessed by immunocytochemistry and led to a 15‐fold increase of osmotic membrane water permeability. This increase was inhibited by HgCl2. The permeability had an Arrhenius activation energy of 11.7 kJ/mol. We called this protein Cicadella aquaporin (AQPcic). The oocytes expressing Cicadella aquaporin were less sensitive to HgCl2 than oocytes expressing aquaporin 1. In the Xenopus oocyte system, Cicadella aquaporin failed to transport glycerol, urea and ions. It exhibited permeabilities to ethylene glycol and formamide similar to those measured for aquaporin 1 under the same conditions.
Structure and Biological Activity of Chemically Modified Nisin A Species
Authors: Rollema Harry S. , Metzger Jörg W. , Both Paula , Kuipers Oscar P. , Siezen Roland J.
Published: July 2004

Nisin, a 34‐residue peptide bacteriocin, contains the less common amino acids lanthionine, β‐methyl‐lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb). Several chemically modified nisin A species were purified by reverse‐phase HPLC and characterized by two‐dimensional NMR and electrospray mass spectrometry. Five constituents, [2‐hydroxy‐Ala5]nisin, [Ile4‐amide,pyruvyl‐Leu6]des‐Dha5‐nisin, [Met(O)21]nisin, [Ser33]nisin, and nisin‐(1–32)‐peptide amide, were found in a commercial nisin sample. A further species, [2‐hydroxy‐Ala5]nisin‐(1–32)‐peptide amide, was obtained by freeze drying an acidic nisin solution. These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues.

The 2‐hydroxyalanine‐containing products have a limited stability; they are spontaneously converted into the corresponding des‐dehydroalanine derivatives. The growth‐inhibiting activity of the modified nisins towards different bacteria was determined. The 2‐hydroxyalanine‐containing species and the des‐dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin. [Met(O)21]nisin and [Ser33]nisin show moderate or no reduction in biological activity.
Characterization of a 3α‐Hydroxysteroid Dehydrogenase/Carbonyl Reductase from the Gram‐Negative Bacterium Comamonas testosteroni
Authors: Oppermann Udo C. T. , Maser Edmund
Published: July 2004

A new form of the NAD(P)‐dependent 3α‐hydroxysteroid dehydrogenases (3α‐HSDs), present in the gram‐negative bacterium Comamonas testosteroni ATCC 11996, was isolated from a testosterone‐induced bacterial extract and characterized. The enzyme (HSD 28) has a monomeric molecular mass of 28 kDa. It belongs to the protein superfamily of short‐chain dehydrogenases/reductases (SDR) as established by N‐terminal sequence analysis. Along with the 3α‐hydroxysteroid dehydrogenase and 3‐oxo‐reductase activities towards a variety of cis or trans fused A/B ring steroids, it also reduces several xenobiotic carbonyl compounds, including a metyrapone‐based class of insecticides, to the respective alcohol metabolites. No dihydrodiol dehydrogenase activity towards trans‐ or cis‐benzene‐dihydrodiols could be detected, thus distinguishing it from the indomethacine‐sensitive, mammalian liver type 3α‐HSDs. Subcellular fractionation revealed that the enzyme is localized in the cytoplasm of the bacterial cell. Proteins similar to the 3α‐HSD were detected and characterized from Comamonas testosteroni strain ATCC 17454 and from a commercially available steroid‐induced extract of a patent Pseudomonas strain. The N‐terminal amino acid sequence of the 3α‐HSD from the latter strain (HSD 29) is highly similar (94% identity over 15 residues) to a previously determined primary structure of a Pseudomonas species 3α‐HSD. However, no similarities could be detected between HSD 28 and a recently determined 3α‐HSD sequence from the ATCC 11996 Comamonas strain.

The specific crossreaction of antibodies directed against mammalian liver type I 11β‐hydroxysteroid dehydrogenase (11β‐HSD I) with the isolated 3α‐HSDs suggests the existence of a functionally and structurally related subgroup within the SDR superfamily. The broad substrate specificities of the characterized 3α‐HSD enzymes lead to the conclusion that they might participate in the intestinal bioactivation or inactivation of hormones, bile acids and xenobiotics since Comamonas testosteroni and related species are found in the intestinal tract of vertebrates including man.