bluuebunny/crossref_metadata_2025 huggingface dataset:
Immobilization and single‐step purification of fusion proteins using DEAE‐cellulose
Authors: SANCHEZ‐PUELLES José M. , SANZ Jesús M. , GARCIA José L. , GARCIA Ernesto
Published: March 2005

We have developed a new single‐step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline‐binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline‐binding‐domain –β‐galactosidase chimera, which can be purified by this procedure and shows a high β‐galactosidase activity when immobilized in the column. A vector plasmid, pCUZ1, containing the lppp‐5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well‐known blue/white 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactoside procedure. A chimera between the choline‐binding domain and the pneumococcal hemolysin was also constructed and purified using pCUZ1.
Crystallization and characterization of monoacylglycerol and diacylglycerol lipase from Penicillium camembertii
Authors: ISOBE Kimiyasu , NOKIHARA Kiyoshi , YAMAGUCHI Shotaro , MASE Tamio , SCHMID Rolf D.
Published: March 2005

A new lipase from Penicillium camembertii U‐150, which is specific for monoacylglycerols and diacylglycerols, but not triacylglycerols, was purified as four active components using concanavalin‐A–Sepharose column chromatography, crystallized in the form of needles, and its properties investigated. No significant difference was observed in substrate specificity, but molecular mass and other enzymatic properties, such as pH, heat stability and optimum pH and temperature, were clearly different between the unadsorbed and the three adsorbed components on concanavalinA–Sepharose; the three adsorbed components were similar to each other and more stable than the unadsorbed component. On the other hand, after enzymatic removal of carbohydrates from the three adsorbed components, their enzymatic properties became similar to those of the unadsorbed component. The carbohydrates of this lipase contribute to the stability of the enzyme, but not to its enzyme activity. The amino acid compositions of the four components did not differ from each other, and tryptic mapping of the deglycosylated components and amino acid composition of the tryptic fragments were identical. The carbohydrate compositions of four intact components were, however, different from each other. All four components have the same polypeptide backbone and multiple forms of this lipase are due to the differences in composition of the carbohydrates bound in this lipase.
Interleukin 1β and tumour necrosis factor α induce a macrophage‐type of nitric oxide synthase in rat renal mesangial cells
Authors: PFEILSCHIFTER Josef , ROB Peter , MÜLSCH Alexander , FANDREY Joachim , VOSBECK Klaus , BUSSE Rudi
Published: March 2005

Treatment of mesangial cells with interleukin 1β (IL‐1β) or tumour necrosis factor α (TNFα) has been shown to increase cGMP formation, most probably due to induction of nitric oxide synthase. Here we report that maximum stimulation of cGMP formation over a 24‐h period required the presence of IL‐1β or TNFα during the first 18 h of induction. N4‐monomethyl‐l‐arginine (L‐NMMA) was a potent inhibitor of cytokine‐induced cGMP formation while N4‐nitro‐l‐arginine (L‐NNA) was less active. Formation of nitric oxide was detected in the cytosol of cytokine‐treated mesangial cells by activation of purified soluble guanylate cyclase and was stimulated by tetrahydrobiopterin, but not by calcium calmodulin. Treatment of cells with IL‐1β or TNFα markedly attenuated the contractile response to a subsequent challenge with angiotensin II. Furthermore, conditioned medium from IL‐1β‐treated cells increased cGMP in untreated control cells.
Fluorescent CMP‐sialic acids as a tool to study the specificity of the CMP‐sialic acid carrier and the glycoconjugate sialylation in permeabilized cells
Authors: GROSS Hans Jürgen
Published: March 2005

The specificity of the Golgi carrier for CMP–sialic‐acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi‐intact CHO 15B cells) was studied with CMP‐activated fluorescent sialic acids as sensitive markers.

Semi‐intact cells represent a well‐established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane‐impermeable CMP–sialic‐acids to gain access to cellular organelles. The subcellular structures of semi‐intact cells remain morphologically intact and hence synthetic CMP–sialic‐acids can be assayed as substrates for the corresponding Golgi sugar‐nucleotide transporter.

The results prove that the CMP–sialic‐acid carrier is able to translocate fluorescent CMP‐glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic‐acid moiety; the data suggest a slightly higher affinity of the carrier for the C9‐substituted CMP‐glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP‐5‐N‐fluoresceinyl‐aminoacetylneuraminic acid (5‐FTIUNeuAc; 5‐N‐fluoresceinylaminoneuraminic acid).

Using CMP‐9‐fluoresceinylthioureido‐N‐acetylneuraminic acid (CMP‐9‐FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi‐intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N‐acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds.

The suitability of fluorescent CMP‐glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP‐N‐acetylneuraminic‐acid analogues can be produced and used for the characterization of the Golgi CMP‐sialic‐acid carrier.
Lignin peroxidase L3 from Phlebia rediata
Authors: LUNDELL Taina , WEVER Ron , FLORIS René , HARVEY Patricia , HATAKKA Annele , BRUNOW Gösta , SCHOEMAKER Hans
Published: March 2005

The catalytic cycle of lignin peroxidase (LiP, ligninase) isozyme L3 from the white‐rot fungus Phlebia radiata was investigated using stopped‐flow techniques. Veratryl (3,4‐dimethoxybenzyl) alchol and a lignin model compound, non‐phenolic β‐O‐4 dimer 1‐(3,4‐dimethoxyphenyl)‐2‐(2‐methoxyphenoxy)propane‐1,3‐diol, were used as electron donors. This is the first report on the detailed kinetic analysis of a LiP‐catalysed Cα‐Cβ bond cleavage of the dimer, representing the major depolymerisation reaction in the lignin polymer. The native enzxyme showed a typical heme peroxidase absorbance spectrum with a Soret maximum at 407 nm. Following the reaction with H2O2, the Soret band decreased in absorbance, shifted to 403 nm and then to 421 nm, demonstrating the formation of compound I followed by the formation of compound II, respectively. Similar results have been reported for the LiP from Phanerochaete chrysosporium upon reaction with H2O2. However, compound I of L3 was more stable in the absence of additional electron donors. thesecond‐order rate constant of compound I formation by H2O2was determined to be 6X105 M−1 s−1 and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound II and further to native enzyme when either veratryl alcohol or the β‐O‐4 dimer was supplied as electron donor and in both cases veratraldehyde appeared as the major product. At pH 6.0, the second‐order rate constant for compound II formation was similar with either veratryl alcohol or the β‐O‐4 dimer (6.7 × 103 and 6.5 × 103 M−1 s−1, respectively). At pH 3.0 formation of compound II with either reductant proceeded so rapidly that determination of the respective rate constants was not possible. The results point to identical catalytic cycles of L3 with veratryl alcohol or the β‐O‐4 dimer involing both compounds I and II as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound II. Chemical evidence of such a radical, formed after the initial LiP‐catalysed one‐electron oxidation of β‐O‐4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A & Brunow, G. (1993) Holzforschung, in the press]. The catalytic redox‐cycle and the oxidation mechanism presented here reconcile seemingly contradictory results obtaind in previous studies on LiP kinetics during the last decade.
Structure of the promoter region of the gene encoding cytochrome c oxidase subunit V in Dictyostelium
Authors: RIZZUTO Rosario , SANDONÀ Dorianna , BRINI Marisa , MARSCHALEK Rolf , DINGERMANN Theodor , BISSON Roberto
Published: March 2005

A cDNA for the nuclear‐encoded subunit V of Dictyostelium discoideum cytochrome‐c oxidase was used as a probe to screen a genomic library and isolate the complete gene. Primer‐extension analysis revealed two transcription start sites located 32 and 39 nucleotides upstream of the translation intiation codon. The chloramphenicol acetyltransferase assay in transient and stable Dictyostelium transformants indicated that the 400‐bp dT‐rich segment 5′ to the transcription start sites retained promoter activity. This region contains an octanucleotide sequence similar to the yeast HAP2/3/4 responsive element.
Eclosion hormone activates phosphatidylinositol hydrolysis in silkworm abdominal ganglia during adult metamorphosis
Authors: SHIBANAKA Yasuhiko , HAYASHI Hideaki , TAKAI Michihiro , FUJITA Norihisa
Published: March 2005

Eclosion hormone (EH), an insect neuropeptide, stimulated phosphatidylinositol (PtdIns) hydrolysis in abdominal ganglia isolated from Bombyx mori in a specific stage of adult development. Incubation of abdominal ganglia from silkworm pharate adults with EH led to an increase in formation of inositol 1,4,5‐trisphosphate but this increase took place transiently, maximum increase being observed 30 s after the addition of EH. PtdIns hydrolysis was stimulated by exogenous EH in a dose‐dependent fashion and was completely abolished by the phospholipase C inhibitors, neomycin and compound 48/80. The EH‐induced PtdIns hydrolysis developed in parallel to the EH‐induced eclosion behaviour during development of the adult. These results suggest that the EH‐stimulated PtdIns Hydrolysis plays an important role in EH‐mediated signal transduction during adult development of B. mori.
Hydroperoxide metabolism in rat liver
Authors: HALLBRUCKER Christian , RITTER Markus , LANG Florian , GEROK Wolfgang , HÄUSSINGER Dieter
Published: March 2005

Addition of t‐butylhydroperoxide (0.2 mM) to isolated perfused rat liver led to a net K+ release of 7.2 ± 0.2 μmol/g within 8 min and a net K+ reuptake of 6.6 ± 0.4 μmol/g following withdrawal of the hydroperoxide, in line with earlier findings by Sies et al. [Sies, H., Gerstenecker, C., Summer, K. H., Menzel, H. & Flohé, R. (1974) in Glutathione (Flohé, L., Benöhr, C., Sies, H., Waller, H. D., eds) pp. 261–276, G. Thieme Publ. Stuttgart]. Net K+ release roughly paralleled the amount of GSSG released from the liver under the influence of the hydroperoxide. The t‐butylhydroperoxide‐induced K+ efflux was inhibited by approximately 70% in the presence of Ba2+ (1 mM), by 30% in Ca2+‐free perfusions and was decreased by 50–60% when the intracellular Ca2+ stores were simultaneously depleted by repeated additions of phenylephrine. t‐Butylhydroperoxide‐induced K+ efflux was accompanied by a decrease of the intracellular water space by 58 ± 14 μl/g (n= 4), corresponding to a 10% cell shrinkage. The effect of t‐butylhydroperoxide on cell volume was inhibited by 70–80% in the presence of Ba2+.

In isolated rat hepatocytes treatment with t‐butylhydroperoxide led to a slight hyperpolarization of the membrane at concentrations of 100 nM, but marked hyperpolarization occurred at t‐butylhydroperoxide concentrations above 10 μM.

t‐Butylhydroperoxide (0.2 mM) transiently increased the portal‐perfusion pressure by 3.3 ± 0.6 cm H2O (n= 18), due to a slight stimulation of prostaglandin‐D2 release under the influence of the hydroperoxide. In the presence of Ba2+ (1 mM), t‐butylhydroperoxide increased the perfusion pressure by 12.7 ± 1.2 cm H2O (n= 9) and produced an approximately tenfold increase of prostaglandin‐D2 and thromboxane‐B2 release. Under these conditions, glucose output from the liver rose from 0.9 ± 0.03 to 2.9 ± 0.7 μmol · g−1· min−1 (n= 4) with a time course roughly resembling that of portal‐pressure increase and prostaglandin‐D2 overflow. These effects were largely abolished in the presence of ibuprofen or the thromboxane‐receptor‐antagonist BM 13.177.

The t‐butylhydroperoxide effects on perfusion pressure, glucose and eicosanoid output were also enhanced in the presence of insulin or during hypotonic exposure; i.e. conditions known to swell hepatocytes, but not during hyperosmotic exposure.

The data suggest that t‐butylhydroperoxide induces liver‐cell shrinkage and hyperpolarization of the plasma membrane due to activation of Ba2+‐sensitive K+ is apparently required for the t‐butylhydroperoxide‐induced K+ channel activation, and K+ efflux may be related to cellular thiol oxidation. t‐Butylhydroperoxide stimulates the formation of cyclooxygenase products. The data show that hydroperoxide effects on hepatic metabolism are more complex than previously thought; both, cell shrinkage and eicosanoid formation under the influence of t‐butylhydroperoxide may contribute to its known glycogenolytic effect.
CD and 1H‐NMR studies on the conformational properties of peptide fragments from the C‐terminal domain of thermolysin
Authors: JIMENEZ M. Angeles , BRUIX Marta , GONZALEZ Carlos , BLANCO Francisco J. , NIETO José L. , HERRANZ José , RICO Manuel
Published: March 2005

The propensity of the peptide fragments 233–248, 245–260, 258–276, 279–298 and 299–316 from the thermolysin C‐terminal domain to form non‐random structures has been examined by CD and two‐dimensional NMR spectroscopy. The conformational properties of these fragments have been studied in aqueous solution and in the mixed solvent trifluoroethanol/H2O (3:7 by vol.). Small but detectable populations of helical structures (up to 10–20%) in aqueous solution have been found for the fragments 233–248, 279–298 and 299–316. These populations are remarkably enhanced (50–70%) in the more hydrophobic mixed solvent, where the fragment 258–276 also forms a comparable helical population. These four fragments are helical in the native crystal structure and the spanning of the corresponding helices in the isolated peptides in solution matches very closely the ones in the native structure. In contrast, the fragment 245–260, an Ω‐loop in the crystal, remains unstructured in both solvents. Medium‐range NOE between protons in sidechains indicate the adoption of preferred sidechain coformations accompanying helix formation. Results are in agreement with the framework model of folding, in which native elements of secondary structure are formed first and folding follows from the collapse of these structural elements.
Structure/function relationships in the pyruvate dehydrogenase complex form Azotobacter vinelandii
Authors: SCHULZE Egbert , WESTPHAL Adrie H. , HANEMAAIJER Roeland , de KOK Arie
Published: March 2005

The role of the hinge region between the binding domain and the catalytic domain in dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii was addressed by deletion mutagenesis. Mutated dihydrolipoyl transacetylase proteins were constructed with a deletion of 11 amino acids in the hinge region between the binding domain and the N‐terminal part of the catalytic domain of E2p [E2p(pAPE1)] and with a further deletion of 9 amino acids into the N‐terminal sequence protrading from the globular structure of the catalytic domain [E2p(pAPE2)] and found to take part in the intratrimer interaction. Both proteins behaved as wild‐type E2p with respect to catalytic activity and quaternary structure. The interaction of the perpheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) with the mutated E2p proteins was studied. E2p‐(pAPE1) assembles to a trimeric pyruvate dehydrogenase complex (PDC) with 15% decreased complex activity. No difference in affinity towards the peripheral components was detected. Upon binding of E3, E2p(pAPE2) dissociates into trimers and monomers. At saturation, two dimers of E3 were bound/E2p monomer instead of one dimer/E2p chain in trimeric wild‐type E2p or E2p (pAPE1). The monomeric E2p species was catalytically inactive. Upon binding of excess E1p, some monomer formation of the E2p mutant took place. E1p however can prevent monomerization by E3. It is concluded that E1p is bound between two different E2p chains in the trimer. The substrates CoA and acetyl‐CoA also prevent monomerization because they are bound by amino acid residues of two different E2p chains. In the presence of CoA no difference in affinity with respect to E1p and E3 binding was observed. CoA (and acetylCoA) also prevent dissociation of the 24‐subunit core structure of wild‐type E2p when added before addition of E1p or E3. Therefore, it seems likely that in vivo A vinelandii PDC is based on a 24‐subunit E2p core, like Escherichia coli PDC. A functional difference between complexes based on a trimer or a 24‐subunit core has not been observed. A role of the hinge region as a spacer to allow binding of E1p or E3 seems unlikely. The results are discussed on the basis of the three‐dimensional structure of the catalytic domain.
Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin
Authors: WÜRFEL Martina , HÄBERLEIN Ingo , FOLLMANN Hartmut
Published: March 2005

Thioredoxins and glutaredoxins, in their oxidized form, possess a single disulfide bridge located on an edge of the small compact molecules. In contrast to most other disulfide‐containing proteins, this S‐S bridge is cleaved by millimolar concentrations of sulfite in the absence of protein denaturing agents at pH 7–8 and ambient temperature; however, the reaction is not quantitative. Sulfitolysis of Escherichia coli thioredoxin was found to be associated with an increase in fluorescence at 345 nm. A comparative study of sulfitolysis in 12 different thioredoxins and glutaredoxins of bacterial and plant origin has been made. Although they are all thought to be highly conserved in three‐dimensional structure, their reactivities towards sulfite and the effects of 6 M guanidinium chloride (not affecting, or enhancing sulfitolysis) very strongly in the series, with E. coli thioredoxin being less reactive and plant thioredoxins and E. coli glutaredoxin being more susceptible molecules. Contrary to expectation, reaction with sulfite is not generally correlated with the presence of negatively or positively charged amino acid residues near the disulfide loop but is determined by individual sequence and surface features in every single protein. These results confirm our hypothesis that thioredoxin sulfitolysis and inactivation [Würfel, M., Häberlein, I., Follmann, H. (1990) FEBS Lett. 268, 146–148] can occur in plant cells under physiological conditions and provide a biochemical rationale for the phytotoxicity of SO2.
Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds
Authors: GONZÁLEZ‐MAÑAS Juan M. , LAKEY Jeremy H. , PATTUS France
Published: March 2005

The interacion of colicin‐A thermolytic fragment with negatively charged liposmes was studied by flurescence spectroscopy. 1,2‐Dioleoyl‐sn‐glycero‐3phospho‐1‐sn‐glycerol (Ole2GroPGro) containing liposomes do not dinginficantly alter the fluorescene properties of the protein, and thus cannot ginve much ingormaion about this interacion. 1,2‐Bis(9,10‐dibromooleoyl‐sn‐glytcero‐3‐phospho‐1‐sm‐glycerol (Br4Ole2Gor) is easily synthesized by addition of bromine atoms to the double nond located at the mid‐point of the fatty‐acid acyl chain of Ole2GroPGro. The brominated phospholipid forms vesicles that strogly quench the protein fluorescence emission. The results presented here show that conversion of Ole2GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allwes for the estimation of the distribution of the quenchin gphospholipd milecules around the fluorophores [Yeager, M. D. & Feigenosn G. W. (1990) Biochemistry 29, 4380–4392]. Binding of the protein to the vesicles is an itteversible process, since inseted milecules do not dissociate from the vesicle. From steadeystate measurements, it can be concluded that in the membrane‐bound form, the tryptophans are located within quenching distance of the bormine atoms, i.e. close to the lipid head‐graup/hydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid milecules in cntact with the fluorescent domian of the protein is nine.
Reduction and mobilization of iron by a NAD(P)H: flavin oxidoreductase from Escherichia coli
Authors: COVES Jacques , Fontecave Mare
Published: March 2005

Iron is an essential element in all living cells. Solubilization, uptake and transport of iron by microorganisms is controlled by highly efficient and specific Fe3+ ‐chelating agents named siderophores. However, mechanisms of mobilization of iron from ferrisiderophores are still enigmatic. Here, we demonstrte that Escherichia coli contains a powerful enzymatic system for the reduction of ferrisederophores. Siderophores have a much lower affinity for ferrous iron, which then can be liverated. This sstem has been previously purified and characterized as a NAD(P)H: flavin oxidoreductase [Fontecave, M., Eliassor, R. and Reichard, P. (1987) J. Biol. Chem. 262 12325–12331)]. It catalyzes the reduction of free flavins, FMN, FAD or riboflavin by NADH or NADPH. Reduced flavins, in turn, transfer their electrons to physiological ferric complexes: ferrisiderophores, ferric citrateand ferritins. The reaction is inhibied by molecular oxygen and greatly stimulated by Fe2+ ‐acceptors such as ferrozine or the iron‐free form of ribonucleotide reductase subunit R2. We suggest that the reduction and the mobilization of iron from ferrisiderophores in the cell might be regulated by the presence of physiological ferrous traps such as apoproteins.
Products of enzymatic reduction of benzoyl‐CoA, a key reaction in anaerobic aromatic metabolism
Authors: KOCH Jürgen , EISENREICH Wolfgang , BACHER Adelbert , FUCHS Georg
Published: March 2005

Benzoyl‐coenzyme A is the most common central intermediate of anaerobic aromatic metabolism. Studies with whole cells of different bacteria and in vitro had shown that benzoyl‐CoA is reduced to alicyclic compounds, possibly via cyclohexadiene intermediates. This reaction is considered a ‘biological Birch reduction’. We have elucidated by NMR techniques the structures of six products of [ring‐13C6]benzoate reduction. The reaction is catalyzed by extracts from cells of a denitrifying Pseudomonas strain K172 anaerobically grown with benzoate and nitrate as sole carbon and energy sources. The assay mixture contained [ring‐13C6]benzoate plus traces of [U‐14C]benzoate, Mg2+, ATP, coenzyme A (CoA), and Ti(III) as reductant. The use of the multiply 13C‐labelled precursor increases the sensitivity of NMR detection and allows the analysis of crude product mixtures by two‐dimensional coherence transfer procedures such as total correlation 13C‐NMR spectroscopy and 13C‐filtered 1H‐NMR spectroscopy. The time course of product formation is consistent with the following order of events.

Benzoyl‐CoA is formed from benzoate via benzoate‐CoA ligase. The first ring reduction product observed is cyclohex‐1,5‐diene‐1‐carboxyl‐CoA. The next intermediate is 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA which is derived from the diene by addition of water. Part of the diene seems to be reduced to cyclohex‐1‐ene‐1‐carboxyl‐CoA which becomes hydrated to trans‐2‐hydroxycyclohexane‐1‐carboxyl‐CoA; these two intermediates may be side products in vitro. The first non‐cyclic intermediate formed by β‐oxidation is 3‐hydroxypimelyl‐CoA. This aliphatic C7 dicarboxylic acid is proposed to be oxidized via glutaryl‐CoA and crotonyl‐CoA to three molecules of acetyl‐CoA and one molecule of CO2.

A similar product pattern was observed in the benzoate‐degrading phototrophic bacterium Rhodopseudomonas palustris. This indicates that the enzymatic reduction of benzoyl‐CoA may be mechanistically similar in different anaerobes.
Localisation of a receptor‐recognition domain on the S3 subunit of pertussis toxin by peptide mapping
Authors: TALLETT April , SEABROOK Richard N. , IRONS Laurence I. , ROBINSON Andrew , VAN HEYNINGEN Simon , ATKINSON Tony
Published: March 2005

Overlapping 10‐amino‐acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46–55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44–58) was also found to bind α‐1‐acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide vielded a fluorescence‐emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L‐α‐lysolecithin, but not N‐acetylneuramin‐lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue‐shifted to 340 nm by ganglioside, or to 345 nm by L‐α‐lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence‐titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 ± 2.8 μM. These findings demonstrate that region 44–58 of the pertussis‐toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target‐cell membranes.
Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene
Authors: LINDQUIST Lennart , KAISER Rudolf , REEVES Peter R. , LINDBERG Alf A.
Published: March 2005

We here report on the purification and characterization of glucose‐1‐phosphate thymidylyl‐transferase, the first of four enzymes commited to biosynthesis of dTDP‐l‐rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the rfbA gene encoding this enzyme. Pure enzyme was obtained by 109‐fold enrichment in three chromatography steps.

The glucose‐1‐phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a ‘ping‐pong’ mechanism. The Km values for dTTP and α‐d‐glucose 1‐phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the Km values for dTDP‐d‐glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP‐d‐glucose and α‐d‐glucosamine 1‐phosphate is accepted equally as well as α‐d‐glucose 1‐phosphate.

The NH2‐terminal sequence of glucose‐1‐phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the orf6.1 gene of the rfb gene cluster. The SDS/PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the orf6.1 gene (32453 Da).
Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli
Authors: SAKAI Hiroshi , OHTA Takahisa
Published: March 2005

Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5‐phosphate but not by fructose 1,6‐bisphosphate. The gene for the enzyme was cloned in Escherichia coli and its entire nucleotide sequence was determined. The deduced amino acid sequence consisted of 587 residues and the molecular mass was calculated to be 62 317 Da. The sequence was highly similar to other pyruvate kinases, indicating that they have the same evolutional origin. Similarly to the E. coli enzymes, the enzyme does not contain an N‐terminal domain, in contrast to the eukaryotic pyruvate kinases. However, the Bacillus stearothermophilus enzyme had an extra C‐terminal sequence consisting of about 110 amino acid residues. A phosphoenolpyruvatebinding motif, which is observed in pyruvate phosphate dikinase, phosphoenolpyruvate: sugar phosphotransferase system enzyme I and phosphoenolpyruvate synthase, was present in the extra C‐terminal sequence. There was an open reading frame upstream of the pyruvate kinase gene. The homology of the sequence showed that the gene encodes phosphofructokinase. Both phosphofructokinase and pyruvate kinase were expressed in E. coli cells, and the evidence suggesting that both genes consitute an operon is presented.
RNA polymerase III
Authors: WILLIS Ian M.
Published: March 2005

Recent studies on RNA polymerase III (pol III) gene transcription have provided a new awareness of the molecular complexity of this process. Fortunately, while the number of transcription components has been increasing, fundamental similarities have emerged regarding the function of eukaryotic promoter elements and the factors that bind them to form preinitiation complexes. Among these, the ability of transcription factor IIIB (TFIIIB) and pol III to transcribe the Saccharomyces cerevisiae U6 gene suggests that the concept of a minimal pol II promoter comprising a TATA box and an initiator region has a parallel in the pol III system. Furthermore, for each of the three classes of eukaryotic RNA polymerase, the assembly of transcription preinitiation complexes and, to some extent, the nature of these complexes appears to be more similar than was previously anticipated.

This work highlights the novel functions and transcriptional properties of newly identified pol III genes, discusses the diversity of pol III promoter structures and presents the notion that the exclusive use of extragenic promoters by some pol III genes (so‐called type‐3 genes) may have evolved since the divergence of yeast and higher eukaryotes. Additionally, recent progress is reviewed on the identification and cloning of subunits for TFIIIC and TFIIIB. Particular emphasis is given to two components of TFIIIB, the TATA‐binding protein and a protein with TFIIB homology (PCF4), since the properties of these molecules suggest a model whereby the polymerase specificity of transcription complexes is determined.
Isolation and identification of N‐terminally extended forms of 5‐oxoprolylglutamylprolinamide (Glp‐Glu‐Pro‐NH2), a thyrotropin‐releasing‐hormone(TRH)‐like peptide present in human semen
Authors: KHAN Zeena , SMYTH Derek G.
Published: March 2005

N‐terminally extended forms of 5‐oxoprolylglutamylprolinamide (Glp‐Glu‐Pro‐NH2), a thyrotropin‐releasing‐hormone(TRH)‐like peptide associated with the male reproductive system, were isolated from human semen by gel exclusion on Sephadex G50, ion‐exchange chromatography on SP‐Sephade C25 and QAE‐Sephadex A25, and by HPLC. The peptides were located by trypsin‐catalysed release of their C‐terminal fragments which were detected by RIA with a TRH‐specific antibody. A series of overlapping peptides containing 16,18, 22 and 25 residues was obtained in homogeneous form and their sequences were determined by automatic Edman degradation. The peptides all terminated in ‐Lys‐Gln‐Glu‐Pro‐NH2 and were found to correspond to sequences occurring between residues 350–374 of semenogelin, a protein present in human semen. In semenogelin, however, the Gln‐Glu‐Pro sequence is followed by tryptophan and not glycine which is normally essential for formation of the C‐terminal amide group. Model experiments with the synthetic peptide Glp‐Glu‐Pro‐Trp showed that under a range of experimental conditions the tetrapeptide did not undergo conversion to Glp‐Glu‐Pro‐NH2. This would indicate that the tripeptide and its extended forms are generated from a precursor that is related to semenogelin but in which Trp375 is replaced by glycine.
Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein
Authors: PIERIK Antonio J. , WASSINK Hans , HAAKER Huub , HAGEN Wilfred R.
Published: March 2005

In Azotobacter vinelandii MoFe protein the oxidation of the P clusters to the S= 7/2 state is associated with a redox reaction with Em,7.5=+90 ± 10 mV (vs the normal hydrogen electrode), n= 1. A concomitant redox process is observed for a rhombic S= 1/2 EPR signal with g= 1.97, 1.88 and 1.68. This indicates that both S= 1/2 and S= 7/2 signals are associated with oxidized P clusters occurring as a physical mixture of spin states. The maximal intensity of the S= 1/2 and S= 7/2 signals in the mediated equilibrium redox titration is similar if not identical to that of solid‐thionine‐treated samples. Summation of the spin concentration of the S= 1/2 spin state (0.25 ± 0.03 spin/α2β2) and the S= 7/2 spin state (1.3 ± 0.2 spin/α2β2) confirms that the MoFe protein has absolutely no more than two P clusters. In spectra of enzyme fixed at potentials around −100 mV a very low‐intensity g= 12 EPR signal was discovered. In parallel‐mode EPR the signal sharpened and increased > 10‐fold in intensity which allowed us to assign the g= 12 signal to a non‐Kramers system (presumably S= 3). In contrast with the non‐Kramers EPR signals of various metalloproteins and inorganic compounds, the sharp absorption‐shaped g= 12 signal is not significantly broadened into zero field, implying that the zero field splitting of the non‐Kramers doublet is smaller than the X‐band microwave quantum. The temperature dependence of this g= 12 EPR signal indicates that it is from an excited state within the integer spin multiplet. A bell‐shaped titration curve with Em,7.5=−307 ± 30 mV and +81 ± 30 mV midpoint potentials is found for the g= 12 EPR signal. We propose that this signal represents an intermediate redox state of the P clusters between the diamagnetic, dithionite‐reduced and the fully oxidized S= 7/2 and S= 1/2 state. Redox transitions of two electrons (−307 ± 30 mV) and one electron (+90 ± 10 mV) link the sequence S= 0 ⇌S= 3 ⇌ (S = 7/2 and S= 1/2). We propose to name the latter paramagnetic oxidation states of the P clusters in nitrogenase POX1 and POX2, and to retain PN for the diamagnetic native redox state. The magnetic circular dichroism and Mössbauer data on thionine‐oxidized MoFe protein have to be re‐evaluated bearing in mind that the oxidized P clusters can exist in two redox‐states. Finally, an account is given of the EPR spectroscopic properties of S= 9/2 and other systems obtained upon superoxidation of the MoFe protein.
The role of the Crabtree effect and an endogenous fuel in the energy metabolism of resting and proliferating thymocytes
Authors: GUPPY Michael , GREINER Erich , BRAND Karl
Published: March 2005

Rat thymocytes have been used to characterize the changes in energy metabolism that occur as cells undergo a resting/proliferation transition. In the resting state, anaerobic ATP production accounts for only 4% of ATP turnover. The remainder is fueled by the oxidation of a mixture of an unidentified endogenous fuel (62%), glucose (18%) and glutamine (16%). 48 h after mitogen stimulation, the ATP turnover has increased twofold. In these proliferating cells, glucose inhibits oxygen consumption by 58%, indicating a profound Crabtree effect which is not present in resting cells. Consequently, proliferating cells, in the presence of glucose and glutamine, fuel the majority (61%) of ATP turnover anaerobically, producing lactate from glucose. The development of a Crabtree effect may be the result of the 8–10‐fold increase in glycolytic enzyme activities which occurs with proliferation. Possible advantages of such a proliferative metabolism are a sparing of endogenous fuel, and a minimizing of oxidative metabolism, with its concurrent production of free radicals.
Specificity of Streptomyces griseus aminopeptidase and modulation of activity by divalent metal ion binding and substitution
Authors: BEN‐MEIR Daniella , SPUNGIN Anya , ASHKENAZI Ruth , BLUMBERG Shmaryahu
Published: March 2005

Streptomyces griseus aminopeptidase is a calcium‐activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass [Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471–477]. A study of the specificity of S. griseus aminopeptidase using amino acid 4‐nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4‐nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa‐proline N‐terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate‐dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1,10‐phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4‐nitroanilide at a 10‐fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4‐nitroanilide at a more than 20‐fold faster rate than the zinc or manganese enzymes.
An upstream region of the rat spermatogenesis‐specific heat‐shock‐like Hst70 gene confers testis‐specific expression in transgenic mice
Authors: WISNIEWSKI Jan , MALEZEWSKI Marek , KRAWCZYK Zdzislaw , GEDAMU Lashitew
Published: March 2005

In order to study the temporal and spatial regulation of a rat testis‐specific heat‐shock‐like hst70 gene, an 0.8‐kb fragment of its upstream DNA was fused to the lacZ gene and microinjected into one‐cell murine embryos. Independent tgHST1 and tgHST2 trangsgenic mice strains were established, containing about 5–7 and 40–60 transgene copies/haploid genome, respectively. Enzyme assays in various tissues showed that transgene‐encoded β‐galactosidase accumulates exclusively in testes of transgenic animals and cannot be detected until 16–17 days after birth. In‐situ assays revealed that the enzyme accumulates mainly in pachytene primary spermatocytes. Our data complement previous studies on the endogenous rat hst70 and suggest that its 0.8‐kb upstream region contains sufficient information to function as an active spermatogenesis‐specific promoter.
Immunologically unrelated Heliothis sp. and Spodoptera sp. midgut membrane‐proteins bind Bacillus thuringiensis CryIA(b) δ‐endotoxin
Authors: ODDOU Pascale , HARTMANN Herbert , RADECKE Franck , GEISER Martin
Published: March 2005

The Bacillus thuringiensis toxins, CryIA(a), CryIA(b), CryIA(c) and CryIC were used in a ligand‐blot assay to detect specific toxin‐binding proteins in the brush‐border membranes of Heliothis virescens, Helicoverpa zea, Spodoptera littoralis, Spodoptera exigua and Spodoptera litura. While CryIA(a) and CryIA(b) always recognize the same protein(s) in a given species, CryIA(c) and CryIC were found to bind to other proteins. Polyclonal antibodies directed against the CryIA(b) binding protein of H. virescens and polyclonal anti‐idiotype antibodies recognizing some determinants on the CryIA(b)‐binding protein involved in the interaction with the toxin, were used to analyse immunological relationships among the toxin‐binding proteins. The results showed that the 170‐kDa toxin‐binding proteins from the H. virescens and H. zea are immunologically related. However, the toxin‐binding proteins from the Spodoptera species did not cross‐react with either type of antibodies. Therefore, we conclude that the CryIA(b) toxin has different binding determinants on the toxin molecule itself which can interact with specific binding sites on the toxin‐binding proteins from Heliothis sp. and Spodoptera sp.
An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis
Authors: ZHAO Genshi , XIA Tianhui , INGRAM Lonnie O. , JENSEN Roy A.
Published: March 2005

The key enzyme of tyrosine biosynthesis in many Gram‐negative prokaryotes is cyclohexadienyl dehydrogenase. The Zymomonas mobilis gene (tyrC) coding for this enzyme was cloned in Escherichia coli by complementation of a tyrosine auxotroph. The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da. The Z. mobilis cyclohexadienyl dehydrogenase expressed in E. coli was purified to electrophoretic homogeneity. The subunit molecular mass of the purified enzyme was 32 kDa as determined by SDS/PAGE. The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3: 1) remained constant throughout purification, and the two activities were therefore inseparable. The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions. Km values of 0.25 mM and 0.18 mM were obtained for prephenate and L‐arogenate, respectively. The Km value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehdrogenase or as prephenate dehydrogenase. Unlike the corresponding enzyme of Pseudomonas aeruginosa or E. coli, the cyclohexadienyl dehydrogenase of Z. mobilis lacks sensitivity to feedback inhibition by L‐tyrosine. A typical NAD+‐binding domain was found to be located at the N‐terminus of the protein.

Although the deduced amino‐acid sequence of the Z. mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19–32%) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T‐proteins of E. coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules.

Immediately upstream of tyrC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino‐acid sequence with the B. subtilis hisH gene product. Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tyrosine‐pathway dehydrogenase and a histidine‐pathway aminotransferase in B. subtilis.
Conformation of deltorphin‐II in membrane environment studied by two‐dimensional NMR spectroscopy and molecular dynamics calculations
Authors: OHNO Yasunori , SEGAWA Motozumi , OHISHI Hirofumi , DOI Mitsunobu , KITAMURA Kunihiro , ISHIDA Toshimasa , INOUE Masatoshi , IWASHITA Takashi
Published: March 2005

Two‐dimensional homonuclear Hartmann‐Hahn spectroscopy and NOESY (nuclear Overhauser effect and exchange spectroscopy) 1H‐NMR techniques have been used to obtain complete proton resonance assignments and to perform a conformational investigation of deltorphin‐II (Tyr‐D‐Ala‐Phe‐Glu‐Val‐Val‐Gly‐NH2), a naturally occurring δ‐selective opioid peptide, in the membrane‐mi‐metic micelles of perdeuterated dodecylphosphocholine. This was done in order to examine conformational characteristics that would be closely related to the selectivity towards the δ‐opioid receptor. With the use of the proton‐proton distances derived from NOESY measurements, 50 possible three‐dimensional structures were generated by means of distance‐geometry calculations, and 25 of them were subjected to the molecular‐dynamics simulations of 10 ps, which were energetically constrained for the NOE interproton distances. Most of the possible conformers simulated showed a common feature such that the conformation of deltorphin‐II is characterized by the S‐shaped back‐bone structure in which the turn conformation of the Val‐Val‐Gly‐NH2 sequence is located under the helically folded conformation of the N‐terminal Tyr‐D‐Ala‐Phe‐Glu sequence. The possible relationship between this conformational characteristic and the δ‐opioid‐receptor selectivity has been discussed.
Fructose‐1,6‐bisphosphatase of the yeast Kluyveromyces lactis
Authors: ZAROR Isabel , MARCUS Frank , MOYER Donna L. , TUNG James , SHUSTER Jeffrey R.
Published: March 2005

The fructose‐1,6‐bisphosphatase [Fru(1,6)P2ase] gene of the budding yeast, Kluyveromyces lactis, was cloned and sequenced. The gene encodes one open reading frame predicting a 354‐amino‐acid polypeptide. The polypeptide is different from other Fru(1,6)P2ases in that it contains a short amino‐acid‐insert region close to a basic residue located at the binding site for the allosteric inhibitor AMP. Comparison of the biochemical properties of the K. lactis enzyme with its closest homolog, the Saccharomyces cerevisiae Fru(1,6)P2ase (74% amino acid identity), reveals that the K. lactis enzyme is significantly less sensitive to AMP (Ki= 540 μM) than the S. cerevisiae enzyme (Ki= 190 μM). However, studies with a K. lactis Fru(1,6)P2ase mutant, in which the insert region (amino acids 152–160) was deleted by site‐directed mutagenesis [(des‐152–160)Fru(1,6)P2ase], showed that the mutant enzyme had higher sensitivity to AMP inhibition (Ki= 280 μM) than the control K. lactis enzyme. Thus, the nine‐amino‐acid insert region appears to be responsible for the decreased AMP inhibition shown by the K. lactis wild‐type enzyme. Catabolite‐repression and catabolite‐inactivation studies show that, unlike the complete repression of FBP1 mRNA and inactivation of enzyme activity by glucose seen in S. cerevisiae, mRNA levels and enzyme activity of K. lactis Fru(1,6)P2ase decreased only about 2–4‐fold due to the presence of glucose in the cell‐culture medium.
Alternative pre‐rRNA processing pathways in human cells and their alteration by cycloheximide inhibition of protein synthesis
Authors: HADJIOLOVA Krassimira V. , NICOLOSO Monique , MAZAN Sylvie , HADJIOLOV Asen A. , BACHELLERIE Jean‐Pierre
Published: March 2005

rRNA processing pathways in humans have been reinverstigated through systematic Northernblot hybridizations of HeLa cell nuclear RNA with a collection of digoxigenine‐lebeled rDNA probes from different regions of the human rDNA transcriptional unit. In addition to the known 45S, 41S, 32S and 21S pre‐rRNA, two major pre‐rRNA fractions were identified; a ‘30S’ (about 5800 nucleotides) precursor to 18S rRNA containing an external transcribed spacer at the 5′ end (ETS) and internal trnscribed spacer (ITS) 1 sequences and a ‘12S’ (about 950 nucleotides) precursor to 5.8S rRNA containing ITS 2 sequences. These pre‐rRNA species do not react with probes located near the 3′‐terminal segments of ITS 1 or ITS 2, thus suggesting that processive endonuclease cuts occur within ITS spacer sequences. The simultaneous occurrence of at least two alternative 45S pre‐rRNA processing pathways is deduced, which correspond to a different temporal order of endonuclease attack at the sites located near the 5′ end of 18S rRNA and within ITS 1. In‐vivo labeling experiments with [14C] uridine revealed that inhibition of protein synthesis with cycloheximide abolishes the endonuclease cut at the 5′ end of 18S rRNA and the formation of 41S pre‐rRNA, while the cut within ITS 1 and the processing to 32S and ‘30S’ pre‐rRNA remains relatively unaltered.