bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of the Amino Terminus in Maintenance of Oligomeric Structure of Sheep Liver Cytosolic Serine Hydroxymethyltransferase
Authors: Jagath Junutula Reddy , Sharma Balasubramanya , Bhaskar Brahatheeswaran , Datta Asis , Rao Naropantul Appaji , Savithri Handanahal S.
Published: July 2004

The role of the amino and carboxyl‐terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino‐terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl‐terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR‐based methods and overexpressed in Escherichia coli. The overexpressed proteins, des‐(A1‐K6)‐SHMT and des‐(A1‐ W14)‐SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des‐(A1–V30)‐SHMT and des‐(A1–L49)‐SHMT were expressed at very low levels, whereas des‐(A1–R58)‐SHMT, des‐(A1–G75)‐SHMT, des‐(Q435–F483)‐SHMT and des‐(L299‐F483)‐SHMT mutant proteins were not soluble and formed inclusion bodies. Des‐(A1–K6)‐SHMT and des‐(A1–W14)‐SHMT catalyzed both the tetrahydrofolate‐dependent and tetrahydrofolate‐independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 °C, the des‐(A1–W14)‐SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des‐(A1–K6)‐SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des‐(A1–W14)‐SHMT had a lower apparent melting temperature (52°C) compared to rSHMT (56°C) and des‐(A1–K6)‐SHMT (55 °C), suggesting that N‐terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2 ± 0.1 M) in the case of des‐(A1–W14)‐SHMT compared to rSHMT (1.8 ±0.1 M) and des‐(A1–K6)‐SHMT (1.7 ±0.1 M). The apoenzyme of des‐(A1‐ W14)‐SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des‐(A1–K6)‐SHMT were a mixture of tetramers (≈75% and ≈65%, respectively) and dimers. While, rSHMT and des‐(A1–K6)‐SHMT apoenzymes could be reconstituted upon the addition of pyridoxal‐5′‐phosphate to 96% and 94% enzyme activity, respectively, des‐(A1–W14)‐SHMT apoenzyme could be reconstituted only upto 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N‐terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.
Viscoelasticity of Actin‐Gelsolin Networks in the Presence of Filamin
Authors: Goldmann Wolfgang H. , Tempel Markus , Sprenger Irene , Isenberg Gerhard , Ezzell Robert M.
Published: July 2004

Cross‐linking of actin filaments by filamin by means of frequency‐dependent rheology yields an increase in the filament's elasticity and stiffness. Higher cross‐linker (filamin) ratios are required for mean actin‐filament lengths of 5–6 μm than for random‐length distribution of actin filaments. The loss modulus (i.e. the viscous portion) in the region of the internal‐chain dynamics [G′′(ω) ≈ωα] is influenced by the cross‐linking of filaments, and with an increasing molar ratio of filamin/actin a reduction of α is observed. Rheological measurements reveal that actin networks are already formed at the polymerizing stage at a molar ratio of filamin/actin of less than 1:100, and electron micrographs show phase separation of actin/filament networks of low density and of actin/filament bundles.
Study of Fatty Acid Specificity of Sunflower Phospholipase D using Detergent/Phospholipid Micelles
Authors: Abousalham Abdelkarim , Nari Joannès , Teissère Marcel , Ferté Nathalie , Noat Georges , Verger Robert
Published: July 2004

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water‐insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D, the maximum hydrolysis rate Vm and the kinetic constant KmB were determined with the micellar substrate. The specificity of various substrates was examined by comparing the Vm/KmB values, and it was noted that sunflower phospholipase D is most active on medium‐chain fatty PtdCho compounds. With long‐chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present in mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme.
Molecular Chaperones Protect Catalase Against Thermal Stress
Authors: Hook Darren W. A. , Harding John J.
Published: July 2004

Lenticular α‐crystallin is generally thought of as having limited chaperone functions. It can efficiently suppress the aggregation of proteins but is unable to promote the functional refolding of proteins after denaturation in many systems unlike other molecular chaperones. However, it has been reported that α‐crystallin, along with the small heat‐shock proteins, is able to promote the functional refolding of some enzymes after thermal and chemical denaturation. These chaperones are also able to confer protection against the thermal inactivation of these enzymes. In results presented here, we demonstrate that α‐crystallin, along with chaperonin 60 (GroEL), was able to provide statistically significant and specific protection against catalase thermal inactivation at stoichiometrical concentrations. The small heat‐shock protein, heat‐shock protein 25 (Hsp25), was unable to confer any such protection. α‐Crystallin however was unable to promote the functional refolding of thermally inactivated catalase. α‐Crystallin and Hsp25 both efficiently suppressed the thermal aggregation of catalase. A high‐molecular‐mass (HMM) complex was only observed to develop in solutions containing catalase and α‐crystallin after solutions were 80‐fold more concentrated relative to thermal inactivation assay conditions prior to incubation. SDS/PAGE analysis confirmed that α‐crystallin had formed a soluble complex with catalase after a period of thermal stress.
Probing Substrate Backbone Function in Prolyl Oligopeptidase Catalysis
Authors: Schutkowski Mike , Jakob Mario , Landgraf Grit , Born Ilona , Neubert Klaus , Fischer Gunter
Published: July 2004

Site‐specific effects on the catalytic activity of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond. Two series of tetrapeptide‐4‐nitroanilides, Ala‐Gly‐Pro‐Phe‐NH‐Np and Ala‐Ala‐Pro‐Phe‐NH‐Np, along with all possible monothioxylated derivatives, were synthesised andkcatandKmvalues were determined for proteolytic cleavage at the Pro‐Phe bond. Regardless of either Gly or Ala in the P2subsite, tetrapeptides were rendered uncleavable by thioxylation at the Pro‐Phe linkage. As a result, Ala‐Xaa‐Pro‐φ[CS‐NH]‐Phe‐NH‐Np (Xaa = Gly or Ala) displayed competitive inhibition withKi‐values of 12 μM and 44 μM, respectively. Furthermore, in controlling proteolytic susceptibility of the substrates, cooperation of the P3‐P2thioxylation site and the side chain at the P2subsite was obtained. Thioxylation at this position enhancedKcat/Kmfivefold in the Gly series, but led to a 1.7‐fold decrease in the Ala series of substrates. With respect to the Xaa‐Pro peptide bond, all of the substrates underwentcisltransisomerisation, thus presenting two stable conformers to the protease. However, the magnitudes of the isomerisation constants suggested that neither isomerisation rates norcisltransequilibria can explain the effect of thioxylation on the steady‐state constants of proteolysis.
NMR Assignments, Secondary Structure and Hydration of Oxidized Escherichia coli Flavodoxin
Authors: Ponstingl Hannes , Otting Gottfried
Published: July 2004

Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three‐dimensional NMR spectroscopy. Nearly complete 1H, 15N and 13C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and 3JHNHα coupling constant data. Like homologous long‐chain flavodoxins, E. coli flavodoxin contains a five‐stranded parallel β‐sheet and five helices. The β‐strands were found to comprise the residues 3–8, 29–34, 48–56, 80–89, 114–116 and 141–145. The helices comprise residues 12–25, 40–45, 62–73, 98–108 and 152–166. The FMN‐binding site was determined by intermolecular NOEs and low‐field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin [Havel, T. F. (1993)Mol. Sim. 10, 175–210]. The analysis of of water‐flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.
Isolation and Characterization of D‐Threonine Aldolase, A Pyridoxal‐5′‐Phosphate‐Dependentenzyme from Arthrobacter sp. DK‐38
Authors: Kataoka Michihiko , Ikemi Masahisa , Morikawa Tadashi , Miyoshi Teruzo , Nishi Ken‐ichi , Wada Masura , Yamada Hideaki , Shimizu Sakayu
Published: July 2004

d‐Threonine aldolase is an enzyme that catalyzes the cleavage of d‐threonine into glycine and acetaldehyde. Its activity was found in several genera of bacteria such as Arthrobacter, Alcaligenes, Xanthomonus, and Pseudonzonas, but not in yeasts or fungi. The enzyme was purified to homogeneity from one strain, Arthrobacter sp. DK‐38. The enzyme appeared to consist of a single polypeptide chain with an apparent molecular mass of 51 kDa. This enzyme, as well as L‐threonine aldolase, requires pyridoxal 5′‐phosphate (pyridoxal‐P) as a coenzyme. Unlike other pyridoxal‐P enzymes, d‐threonine aldolase also requires a divalent cation such as Co2+, Ni2+, Mn2+, or Mg2+ for its catalytic activity. The enzyme completely lost its activity in the absence of either pyridoxal‐P or a divalent cation. A divalent cation was also essential for the thermal stability of the enzyme. The metal‐free enzyme tends to become thermally unstable, resulting in the irreversible loss of its catalytic activity. The enzyme is strictly d‐specific for the α‐position, whereas it cannot distinguish between threo and erythro forms at the β‐position. Thus, d‐threonine and d‐allothreonine act as substrates of the enzyme, but their kinetic parameters are different: the Km and Vmax values are 3.81 mM and 38.8 μMol. min−1 mg−1 toward d‐threonine, and 14.0 mM and 102 μMol min−1. Mg−1 toward d‐allothreonine, respectively. The aldolase reaction is reversible, and the enzyme is therefore able to produce nearly equimolar amounts of d‐threonine and d‐allothreonine through C‐C bond formation between glycine and acetaldehyde. The enzyme also acts, in the same manner, on several other d‐β‐hydroxy‐α‐amino acids, including d‐β‐phenylserine, d‐β‐hydroxy‐α‐aminovaleric acid, d‐β‐3,4‐dihydroxyphenylserine, and d‐β‐3,4‐methylenedioxyphenylserine.
NMR Investigations of the Role of the Sugar Moiety in Glycosylated Recombinant Human Granulocyte‐Colony‐Stimulating Factor
Authors: Gervais Virginie , Zertal Aurelio , Oschkinat Hartmut
Published: July 2004

Human granulocyte‐colony‐stimulating factor (G‐CSF) is a hematopoietic growth factor that plays a major role in the stimulation of the proliferation and maturation of granulocyte neutrophil cells. With the recent increased understanding of its biological properties in vivo together with available preparations of recombinant human G‐CSF, this growth factor has become an essential agent for clinical applications. The presence of an O‐linked carbohydrate chain at position 133 greatly improves the physical stability of the protein. To clarify the molecular basis for the stabilisation effect of saccharide moieties on human G‐CSF, the whole glycoprotein expressed in CHO cells has been investigated by means of two 1H‐NMR‐spectroscopy and two 1H‐detected‐heteronuclear 1H‐13C experiments at natural abundance, and compared with the non‐glycosylated form. The present NMR study reports assignments of 1H and 13C resonances of the bound saccharidic chain NeuNAc(α2‐3)Gal(β1‐3)[NeuNAc(α2‐6)]GalNAc, where NeuNAc represents N‐acetylneuraminic acid, and demonstrates the α‐anomeric configuration of the N‐acetylgalactosamine‐threonine linkage. It also provides results suggesting that the carbohydrate moiety reduces the local mobility around the glycosylation site, which could be responsible for the stabilising effect observed on the glycoprotein.
An ADP‐Ribosylation‐Factor(ARF)‐Like Protein involved in Regulated Secretion
Authors: Icard‐Liepkalns Christine , Ravassard Philippe , Liepkalns Vis. A. , Chatail Fabienne , Mallet Jacques
Published: July 2004

A rat ADP‐ribosylation factor(ARF)‐like protein named ARL184 was identified by cDNA cloning. The corresponding recombinant protein had an apparent molecular mass of 22000. The deduced amino acid sequence had 55% identity with the human ARL1 and four functional GTP‐binding sites. Immuno‐fluorescent confocal microscopy studies showed that ARL184 was present in the cytosol as well as in the Golgi apparatus, raising the possibility that it has a role in a secretory pathway. The involvement of this ARF‐like protein in secretion was confirmed by demonstrating that ARL184 potentiated acetylcholine release in stably transfected PC12 cells. Collectively these results suggest that this ARL protein is a component of a regulated secretory pathway involved in Ca2+‐dependent release of acetylcholine.
Sphingosylphosphorylcholine Activates an Amiloride‐Nsensitive Na+‐H+‐Exchange Mechanism in GH4C1 Cells
Authors: Törnquist Kid , Woodside Michael , Grinstetn Sergio
Published: July 2004

The effect of sphingosylphosphorylcholine (SphPCho) on the intracellular pH (pHi) in GH4C1 cells was investigated. SphPCho evoked a very slow increase in basal pHi. In cells acidified with nigericin, SphPCho induced a rapid alkalinization of the cells. The effect was inhibited in a Na+‐free buffer solution, but was insensitive to ethylisopropyl amiloride, a potent inhibitor of Na+‐H+ exchangers (NHE). Reverse transcription and PCR showed that the predominant isoform of the antiport expressed in GH4C1, cells is NHE‐1. The rate of alkalinization after stimulation with propionate, and after addition of Na1 to cells acidified with NH4Cl, was enhanced in cells treated with SphPCho. The initial rate of alkalinization after addition of Na+ to acidified cells treated with SphPCho gave an apparent Km value of 15 ± 2 mM for Na+. The Vmax value was 9 ± 22 mM H+/min. The effect was insensitive to ouabain, staurosporine and bafilomycin A. However, the SphPCho‐evoked alkalinization was abolished in cells treated with 2‐deoxy‐D‐glucose. The effect was not due to the charge of the molecule, as stearylamine increased pHi in Na+‐containing and Na+‐free buffer. The results show that SphPCho may activate Na+‐H+ exchange, and that this effect is mediated via an amiloride‐insensitive exchange mechanism.
Intermolecular Cleavage by the Newt Ribozyme
Authors: Marusic Lidija , Luzi Ettore , Barsacchi Giuseppina , Eckstein Fritz
Published: July 2004

To analyse the trans‐cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop II. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and III to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondary‐structure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and III did. These results suggest that the sequence of the stems I and III impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.
Kinetic Investigations with Inhibitors that Mimic the Postomolysis Intermediate in the Reactions of Coenzyme‐B12‐Dependent Glycerol Dehydratase and Diol Dehydratase
Authors: Poppe László , Rétey János
Published: July 2004

Kinetic investigations were performed on the coenzyme‐B12‐dependent glycerol dehydratase and diol dehydratase reactions using 1,2‐propanediol as substrate and [ω‐(adenosin‐5′‐O‐yl)alkyl]cobalamins as mimics of the posthomolysis intermediate state of the coenzyme. All the coenzyme‐B12 analogues with oligomethylene chains (C3–C7) inserted between the central Co atom and the 5′ O of the adenosine moiety were competitive inhibitors with respect to coenzyme B12. The apparent inhibition constants (Ki) of the shorter‐chain inhibitors, especially the C5 inhibitor, were smaller for both enzymes than those of the longer‐chain (C6, C7) compounds. These results are in agreement with the expected (0.6–0.9 nm) distance between the Co and 5′‐methylene paramagnetic centers in the posthomolysis intermediate state of coenzyme B12 in these reactions.
Metabolism of 14C‐Labelled 5‐Nitro‐1,2,4‐Triazol‐3‐One by Rat Liver Microsomes Evidence for the Participation of Cytochrome P‐450
Authors: Le Campion Laurence , Delaforge Marcel , Noel J. Pierre , Ouazzani Jamal
Published: July 2004

In the present study, we synthesized 14C‐labelled 5‐nitro‐1,2,4‐triazol‐3‐one (NTO) and investigated its hepatic metabolism by dexamethasone‐induced murine hepatic microsomes. Under the nitrogen atmosphere, 5‐amino‐1,2,4‐triazol‐3‐one was the only detected metabolite of NTO. The microsomal nitro‐reductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5‐hydroxy‐triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductase activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole‐related inhibitors: miconazole and methimazole, and to a less extent by N‐octyl‐amine. The microsomal denitrification was induced by the treatment of rats with dexamethasone and phenobarbital. The microsomal reductare activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P‐450 in the metabolism of NTO, supported by its transformation with reconstituted cytochrome P‐450 systems.
Structural Studies on the Short‐Chain Lipopolysaccharide of Vibrio Cholerae O139 Bengal
Authors: Knirel Yuriy A. , Widmalm Göran , Senchenkova Sof'ya N. , Jansson Per‐Erik , Weintraub Andrej
Published: July 2004

A Vibrio cholerae O139 strain, MO10‐T4, lacking capsular polysaccharide, produces a short‐chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high‐performance anion‐exchange chromatography, one‐ and two‐dimensional 1H‐, 13C‐, and 31P‐NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS:
image

where PEtn stands for 2‐aminoethyl phosphate, Fru for fructose, Hep for l‐glycero‐d‐manno‐heptose, and Kdo for 3‐deoxy‐d‐manno‐octulosonic acid; unless otherwise stated, the monosaccharide residues are d and present in the pyranose form. An O‐acetyl group is present on a secondary position, tentatively O4 of the α‐linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown.

An O139‐negative mutant strain, Bengal‐2R, derived from V. cholerae O139, has also been investigated and shown to produce an O‐antigen‐lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10‐T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2‐aminoethyl phosphate, the O‐acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10‐T4 LPS, a short O‐side chain is attached at position 3 of the 7‐substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.‐R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43–58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59–65.]
Purification of Bovine Lysosomal α‐Mannosidase, Characterization of its Gene and Determination of two Mutations that Cause α‐Mannosidosis
Authors: Tollersrud Ole K. , Berg Thomas , Healy Peter , Evjen Gry , Ramachandran Umayal , Nilssen Øivind
Published: July 2004

Bovine kidney lysosomal α‐mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16kb and its corresponding cDNA contained an open reading frame of 2997 bp beginning from a putative ATG start codon. The deduced amino acid sequence contained a signal peptide of 50 amino acids adjactent to a protein sequence of 949 amino acids that was cleaved into five peptides in the mature enzyme; starting with the peptide derived from the N‐terminal part of this precursor, their molecular masses were 35/38 (peptide a), 11/13 (peptide b), 22 (peptide c), 38 (peptide d) and 13/15 kDa (peptide e). Variation in the degree of N‐glycosylation accounts for molecular mass heterogeneities of peptides a, b and e. Peptides a, b and c were disulphide‐linked. A T961°C transition, resulting in Phe321→Leu substitution, was identified in the cDNA of α‐mannosidosis‐affected Angus cattle. In affected Galloway cattle, a G662→A transition that causes Arg221→His substitution was identified. Phe321 and Arg221 are conserved among the α‐mannosidase class‐2 family, indicating that the substitutions resulted from disease‐causing mutations in these breeds.
Inhibition of Translation by mRNA Encoding NIPP‐1, a Nuclear Inhibitor of Protein Phosphatase‐1
Authors: Wera Stefaan , Van Eynde Aleyde , Stalmans Willy , Bollen Mathieu
Published: July 2004

Transient transfection of COS‐1 cells with an expression vector for NIPP‐1, a nuclear subunit of protein phosphatase‐1, did not result in an overexpression of NIPP‐1 protein, although the levels of mRNA encoding NIPP‐1 increased dramatically. Moreover, high concentrations of NIPP‐1 mRNA inhibited the translation in reticulocyte lysates of various unrelated mRNAs. This inhibition of translation was caused by the NIPP‐1 messenger and not by the translation product, since mutation of the start codon abolished NIPP‐1 protein production, but had no influence on the translational inhibition. Analysis of deletion mutants showed that the inhibition was mediated by a 0.5‐kb fragment in the 5′‐end of the NIPP‐1 mRNA. This region, when inserted in the 5′‐untranslated region of the β‐galactosidase messenger, inhibited the translation of β‐galactosidase mRNA in COS‐1 cells. A predicted highly stable secondary structure G= ‐239.5 kJ/mol) is present between residues 300 and 500 of NIPP‐1 mRNA. The possible importance of this structure in the translational inhibition is discussed.
Differential Expression of the Vegetative and Spore‐Bound Hydrophobins of Trichoderma Reesei Cloning and Characterization of the Hfb2 Gene
Authors: Nakari‐Setälä Tiina , Aro Nina , IlméN Marja , Muñoz Gaston , Kalkkinen Nisse , Penttilä Merja
Published: July 2004

The hfb2 gene encoding the hydrophobin HFBII of the filamentous fungus Trichoderma reesei was isolated by heterologous hybridization using the vegetative hydrophobin I, hfb1, gene of T. reesei as a probe. The hfb2 gene codes for a typical fungal secreted hydrophobin of 71 amino acids containing eight cysteine residues. The amino acid similarity towards HFBI is 69%. The HFBII protein was isolated from the fungal spores by extraction with trifluoroacetic acid/acetonitrile solution, and by bubbling from the lactose‐based culture medium. Expression of the hfb1 and hfb2 genes is divergent, hfb1 expression was only observed in vegetative cultures on glucose‐containing and sorbitol‐containing media. It was not expressed on media containing complex plant polysaccharides, cellulose, xylan, cellobiose or lactose, whereas hfb2 was highly expressed in vegetative cultures on these media. Expression of hfb2 was also strongly induced by N and C starvation, by light and in conidiating cultures.
Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen
Authors: Mcdevitt Damien , Nanavaty Tamanna , House‐Pompeo Karen , Bell Ewen , Turner Nancy , Mcintire Larry , Foster Timothy , HööK Magnus
Published: July 2004

The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of biomaterial and wound‐associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen‐binding protein clumping factor (ClfA) is within the C‐terminus of the fibrinogen γ chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C‐terminal residues of the γ chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C‐terminal amino acids of the fibrinogen γ chain effectively inhibited adherence of ClfA‐expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221–550) recognized intact recombinant fibrinogen γ chains, but failed to recognize recombinant fibrinogen γ chains where the 4 C‐terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C‐terminal domain of fibrinogen γ chains contains a binding site for the integrin αIIbβ3 (glycoprotein gpIIb/IIIa) receptor on platelets [Kloczewiak, M., Timmons, S., Bednarek, M. A., Sakon, M. & Hawiger, J. (1989) Biochemistry 28, 2915–1919; Farrell, D. H., Thiagarajan, P., Chung, D. W. & Davie, E. W. (1992) Proc. Natl Acad. Sci. USA 89, 10729–10732; Hettasch, J. M., Bolyard, M. G. & Lord, S. T. (1992) Thromb. Haemostasis 68, 701–706]. We now show that Clf33 inhibits ADP‐induced, fibrinogen‐dependent platelet aggregation in a concentration‐dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 ± 0.19 μM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C‐terminal γ‐chain peptide effectively inhibited this interaction.
Affinity Labeling of Escherichia Coli Glucosamine‐6‐Phosphate Synthase with a Fructose 6‐Phosphate Analog
Authors: Leriche Caroline , Badet‐Denisot Marie‐Ange , Badet Bernard
Published: July 2004

Glucosamine‐6‐phosphate synthase (GlcNP‐synthase) catalyzes the formation of glucosamine 6‐phosphate from fructose 6‐phosphate using the γ‐amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP‐synthase was recently found to catalyze the formation of glucose 6‐phosphate corresponding to a phosphoglucoisomerase‐like activity. Here we report active‐site directed, irreversible inhibition of Escherichia coli GlcNP‐synthase (Kinact= 0.60 ± 0.05 min−1, Kirr= 1.40 ± 0.20 mM) by anhydro‐1,2‐hexitol 6‐phosphates previously known as irreversible inhibitors of phospho‐glucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of side‐chain carboxylates of Glu255 and Glu258 and thiol of Cys300 of the fructose‐6‐phosphate‐binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N‐terminal cysteine from the glutamine‐binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose‐6‐phosphate‐binding sites recently suggested by Bearne
Structure/Function Relationship Study of Tyr14 and Arg22 in Trichosanthin, a Ribosome‐Inactivating Protein
Authors: Shaw Pang‐Chui , Mulot Sandrine , Ma Sun‐Kai , Xu Qiong‐Fang , Yao Hong‐Bing , Wu Shen , Lu Xiao‐Hui , Dong Yi‐Cheng
Published: July 2004

Amino acids Tyr14 and Arg22 in trichosanthin are residues on helix A1 close to the active‐site cleft. They are invariant in various type‐I and type‐II ribosome‐inactivating proteins. In this study, Tyr14 was changed to Phe and Arg22 to Lys and Leu. Modified proteins were purified, and activities compared by assaying their median inhibitory concentration (ID50) on a rabbit‐reticulocyte‐lysate protein‐synthesis system. While the ID50 of wild‐type trichosanthin was 0.02 nM, those for [Phe14], [Lys22], [Leu22] and [Phe14, Leu22]trichosanthin were 0.10, 0.03, 0.25 and 0.15 nM, respectively. Therefore, compared with Tyr14, Arg22 appears to play a more important role in trichosanthin. Structural studies on [Leu22]tricho‐santhin showed that two water molecules occupy the space left by the side chain of Arg22, and hydrogen bonds exist between these water molecules and nearby residues to retain the conformation. The use of intermolecular rather than intramolecular hydrogen bonds may have an adverse effect on stability or folding of the protein and results in a mild decrease in activity.
A Kinetic Study of Triple‐Helix Formation at a Critical R · Y Sequence of the Murine c‐Ki‐ras Promoter by (A,G)‐ and (G,T) Oligonucleotides
Authors: Xodo Luigi E. , Pirulli Doroti , Quadrifoglio Franco
Published: July 2004

The kinetics of triplex formation between the oligonucleotides d(AGGGAGG‐GAGGAAGGGAGGG) (20AG), d(TGGGTGGGTGGTTGGGTGGG) (20GT) and a 29‐bp polypurine‐polypyrimidine sequence located in the c‐Ki‐ras promoter (D) was studied by electrophoretic experiments in 50 mM Tris/acetate, pH 7.4, 50 mM NaCl, 5 mM MgCl2. Rates of triplex formation were determined at three different temperatures (20°C, 37°C and 45°C), under pseudo‐first order conditions obtained by using the triplex‐forming oligonucleotide (TFO) 500‐fold in excess over the target duplex (5 nM). Measurements at TFO/target ratios of 20 and 100 were also carried out. At 37°C the pseudo first‐order constants, kobs, were 18.9×10−5s−1 for 20AG and 13.0×10−5s−1 for 20GT, yielding association half‐lives of 1 h and 1.5 h, respectively. Second‐order association constants were found to be in the order of 102 M−1 s−1: these are slightly lower if compared with those measured for triplex formation by polypyrimidine (C,T) oligonucleotides (103 M−1 s−1) [Maher, L. J., Dervan, P. B. & Wolf, B. J. (1990)Biochemistry 29, 8820–8826; Xodo, L. E. (1995)Eur. J. Biochem. 228, 918–926; Bates, P. J., Dosanjh, H. S., Jenkins, T. C., Laughton, C. A. & Neidle, S. (1995)Nucleic Acids Res. 23, 3627–3632] but dramatically lower when compared with duplex recombination from complementary strands (106 M−1 s−1) [Craig, M. E., Crothers, D. M. & Doty, P. (1971)J. Mol Biol. 62, 383–401; Pörschke, D. & Eigen, M. (1971)J. Mol. Biol. 62, 361–381]. Dissociation rate constants, k−1, were indirectly obtained from equilibrium constants (Kd) and found to be, at 37°C, 6.7×10×7 s−1 and 5.4×10−6 s−1 for 20AG and 20GT, respectively. From the rate constants obtained at 20°C, 37°C and 45°C we estimated activation energies of triplex formation between D plus 20AG and D plus 20GT of respectively 134±29 and 88±21kJ/mol. Moreover, the activation energies for the reaction of triplex dissociation were 385 ± 50 kJ/mol for 20AG and 330 ± 42 kJ/mol for 20GT. Decreasing the TFO/target ratio from 500 to 100 or 20, we observed a concomitant decrease of the association rate, in keeping with the finding that triplex formation occurs through a bimolecular process. We found that the effect of salt on triplex formation is rather complex, as, the addition of 2 mM spermidine boosted the binding rate of 20GT, but slightly reduced that of 20AG; the increase of NaCl from 50 mM to 100 mM or 150mM decreased the rate of triplex formation. Finally, the biological implications of the kinetic behaviour exhibited by the two triplex‐forming oligonucleotides specific for the c‐Ki‐ras promoter are discussed.
Properties of an NAD(H)‐Containing Methanol Dehydrogenase and its Activator Protein from Bacillus methanolicus
Authors: Arfman Nico , Hektor Harm J. , Bystrykh Leonid V. , Govorukhina Natalya I. , Dijkhuizen Lubbert , Frank Johannes
Published: July 2004

Oxidation of C1–C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD‐dependent methanol dehydrogenase (MDH), composed of ten identical 43000‐Mr subunits. Each MDH subunit contains a tightly, but non‐covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1–2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH‐catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping‐pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH‐bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50000‐Mr activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.
Elongation of N‐Acetyllactosamine Repeats in Diantennary Oligosaccharides
Authors: Hummel Mathias , Hedrich Hans C. , Hasilik Andrej
Published: July 2004

Glycosylated [Asn22]lysozyme has been shown to contain N‐acetyllactosamine repeats when expressed in Chinese hamster ovary (CHO) cells. We find that the major portion of N‐acetyllactosamine repeats are associated with diantennary oligosaccharides. In Lec2 CHO cells, which are deficient in sialylation, glycosylated lysozyme is synthesized with increased contents of N‐acetyllactosamine repeats terminating in β‐galactosyl residues. In the Lec2 cells and the parental CHO cell line, Pro−5, only a minor portion of the oligosaccharides in lysozyme are of the triantennary type. Previously, it has been shown that the synthesis of N‐acetyllactosamine repeats in Asn‐linked oligosaccharides is enhanced by an increase in the activity of the elongating β‐N‐acetylglucosaminyl transferase and by the synthesis of β‐1,6‐linked antennae. The results with glycosylated lysozyme suggest that glycoproteins bearing diantennary oligosaccharides can contain several N‐acetyllactosamine repeats and that the number of the latter can be increased by decreasing the activity of the capping sialyl transferases.
Electrostatic Effects of Smooth Muscle Calponin on Actin Assembly
Authors: Tang Jay X. , Szymanski Pawel T. , Janmey Paul A. , Tao Terence
Published: July 2004

The contribution of electrostatic interactions to the effects of chicken gizzard calponin on the kinetics of actin polymerization and the bundling of F‐actin were characterized by a combination of fluorescence, light‐scattering, co‐sedimentation, and electron‐microscopic methods. Stoichiometric amounts of calponin accelerate actin polymerization in low‐ionic‐strength solutions, but this effect is diminished at [KCl] = 150 mM. At low ionic strengths, micromolar concentrations of calponin induce the formation of large bundles of actin filaments, and lower concentrations of calponin quench the fluorescence of pyrene‐labeled F‐actin. The latter effect is related to binding of calponin to F‐actin rather than to bundling of the filaments. The concentration of calponin required to bundle a fixed concentration of actin filaments increases with increasing ionic strength, as the average diameter of the bundles decreases. Millimolar concentrations of ATP, GTP or ITP are equally efficient at dispersing actin bundles to single filaments or smaller aggregates, even though a significant fraction of calponin remains bound to F‐actin. Our findings show that the binding of calponin to actin is determined at least in part by electrostatic interactions, and that the polycationic nature of calponin is primarily responsible for the formation of F‐actin bundles via its ability to reduce the electrostatic repulsion between the negatively charged actin filaments.
Transcriptional and Translational Adjustments of Psba Gene Expression in Mature Chloroplasts During Photoinhibition and Subsequent Repair of Photosystem II
Authors: Kettunen Reetta , Pursiheimo Saijaliisa , Rintamäki Eevi , VAN Wijk Klaas‐Jan , Aro Eva‐Mari
Published: July 2004

The D1 reaction centre protein of photosystem II (PSII), encoded by the plastid psbA gene, has the highest turnover rate of all thylakoid proteins, due to light‐induced damage to D1. The expression of the psbA gene was studied in chloroplasts of fully developed pea (Pisum sativum L.) leaves during high‐light photoinhibitory treatment and subsequent restoration of PSII function at low light. psbA transcript levels were determined and the translational activity was followed by in vivo pulse‐labelling, by in vitro translations with intact chloroplasts, and by run‐off translations on isolated thylakoid membranes. PSII photochemical efficiency was determined in vivo by monitoring the ratio of variable fluorescence to maximal fluorescence (FV/FM). Enhanced D1 synthesis in pea leaves, upon a shift first from darkness to growth light and subsequently to high light, was accompanied by a substantial increase in the total number of psbA transcripts and by the accumulation of psbA mRNA · initiation complexes on thylakoid membrane. This suggested that high‐light illumination increased the transcriptional activity of the psbA gene in mature leaves, and that enhanced translational initiation of psbA mRNA was followed by docking of the initiation complexes to the thylakoid membrane. The high‐light‐induced increase in the number of thylakoid‐associated psbA mRNA · initiation complexes, occurred in parallel with enhanced in vivo D1 synthesis. This, however, did not result in an enhanced accumulation of D1 translation products in in vitro run‐off translations when pea leaves were shifted from growth light to high light. This may suggest that at high light only a portion of thylakoid‐associated psbA mRNA can be under translational elongation at a given moment. When the leaves were shifted from high light to low light to allow repair of PSII, thylakoid‐associated psbA mRNA was rapidly released from the membrane and the high‐light‐induced pool of psbA transcripts was degraded. The synthesis of the D1 protein decreased on the same time scale. However, the restoration of PSII photochemical function, measured as FV/FM, took a substantially longer time. It is concluded that during changing light conditions, mature leaves are able to adjust psbA gene expression both at the transcriptional and at the translational level.
The Primary Structure of the Split‐Soret Cytochrome c from Desulfovibrio Desulfuricans ATCC 27774 Reveals an Unusual Type of Diheme Cytochrome C
Authors: Devreese Bart , Costa Cristina , Demol Hans , Papaefthymiou Vasilios , Moura Isabelle , Moura Jose J. R. , Van Beeumen Jozef
Published: July 2004

The complete amino acid sequence of the unusual diheme split‐Soret cytochrome c from the sulphate‐reducing Desulfovibrio desulfuricans strain ATCC 27774 has been determined using classical chemical sequencing techniques and mass spectrometry. The 247‐residue sequence shows almost no similarity with any other known diheme cytochrome c, but the heme‐binding site of the protein is similar to that of the cytochromes c3 from the sulphate reducers. The cytochrome‐c‐like domain of the protein covers only the C‐terminal part of the molecule, and there is evidence for at least one more domain containing four cysteine residues, which might bind another cofactor, possibly a non‐heme iron‐containing cluster. This domain is similar to a sequence fragment of the genome of Archaeoglobus fulgidus, which confirms the high conservation of the genes involved in sulfate reduction.
Distinct Repression of Translation by Wortmannin and Rapamycin
Authors: Pedersen Susanne , Celis Julio E. , Nielsen Jacob , Christiansen Jan , Nielsen Finn C.
Published: July 2004

The role of phosphatidylinositol 3‐kinase and FK506‐binding protein · rapamycin‐associated protein (FRAP) in translational control has been examined by treating RD‐rhabdomyosarcoma cells with wortmannin and rapamycin and studying the effects on cell‐growth, translation initiation, and protein synthesis. Whereas wortmannin and rapamycin exhibit subtle effects on global translation, examination of individual mRNAs in sucrose gradients and of individual proteins in two‐dimensional polyacrylamide gels reveals that wortmannin and rapamycin exhibit distinct effects on the translation of individual mRNAs. Wortmannin represses the synthesis of a third of cellular proteins, whereas rapamycin affects a subset of these proteins. Since ribosomal protein S6 was rapidly dephosphorylated following wortmannin and rapamycin treatment, and the phosphorylation status of the eukaryotic initiation factor 4E was unchanged, our data imply that the p70 signalling pathway has at least one branch‐point upstream of FRAP leading to an additional route of translational control.
Redox Titration of Two [4Fe‐4S] Clusters in the Photosynthetic Reaction Center from the Anaerobic Green Sulfur Bacterium Chlorobium vibrioforme
Authors: Scott M. Paul , Kjœr Bodil , Scheller Henrik Vibe , Golbeck John H.
Published: July 2004

Anaerobic green sulfur bacteria contain photosynthetic reaction centers analogous to photosystem I (PS I) of plants and cyanobacteria. These reaction centers, termed type I, are characterized by the presence of bound iron‐sulfur clusters as the terminal electron acceptors. In this work, the iron‐sulfur clusters in Chlorobium vibrioforme were studied using selective light‐induced reduction protocols, spin quantifications, and chemical redox titrations coupled with EPR detection. Illumination of a dark‐frozen sample at 12 K results in the appearance of a spectrum termed signal I. Chemical reduction in darkness at solution potentials between –414 mV and –492 mV results in the appearance of a different spectrum termed signal II. Illumination of these chemically poised samples at 12 K results in the appearance of signal I such that the sum of the intensity of signal I + signal II is nearly constant for every ratio of signal I/signal II. As the solution potential is lowered to –545 mV, the spectrum shifts to yet a third set of resonances, termed signal III. Concomitant with this shift is a loss of low temperature light‐induced reduction of signal I. Photoaccumulation of a sample poised at a solution potential of–50 mV results also in the appearance of signal III at nearly the same spin concentration as the chemically reduced sample. Spin quantifications imply that signals I and II are both derived from the reduction of one iron‐sulfur cluster, termed center I; signal III is derived from simultaneous reduction of two iron‐sulfur clusters, centers I and II. By measuring the EPR signal intensities over a range of solution potentials, centers I and II were shown to have Em (pH 10.0) values of –446 mV and –501 mV, respectively. The observations are consistent with a structural and functional analogy of centers I and II with FA and FB of PS I.
The Role of the Cap Structure in RNA Processing and Nuclear Export
Authors: Lewis Joe D. , Izaurflde Elisa
Published: July 2004

The cap structure that is characteristic of all polymerase‐II‐transcribed RNAs has been shown to play an important role in many aspects of RNA metabolism including RNA processing, RNA nuclear transport, and translation initiation. The effects of the cap structure on these different processes is mediated by proteins that recognise and bind to it, and are therefore generically called cap‐binding proteins. For example, the cap‐binding protein eIF4E, in a complex with other proteins, mediates the effect of the cap on the initiation of translation. EIF‐4E is predominantly localised in the cytoplasm. In the last five years, it has been demonstrated that a second cap‐binding protein complex, which is mainly localised in the nucleus, mediates the stimulatory effects of the cap in nuclear processes such as pre‐mRNA splicing, RNA 3′‐end formation, and RNA nuclear export. The purpose of this review is to summarise our current knowledge on the role of the cap structure and of the cap‐binding protein complex in nuclear RNA metabolism and present evidence that at least some processes may be coupled in vivo.