bluuebunny/crossref_metadata_2025 huggingface dataset:
Four Recombinant Isoforms of Cor a 1, the Major Allergen of Hazel Pollen, Show Different Reactivities with Allergen‐specific T‐lymphocyte Clones
Authors: Schenk Siegfried , Hoffmann‐Sommergruber Karin , Breiteneder Heimo , Ferreira Fatima , Fischer Gottfried , Scheiner Otto , Kraft Dietrich , Ebner Christof
Published: March 2005

Purified preparations of allergenic proteins from plants, and in particular from pollens, consist of multiple closely related isoforms. These isoforms are highly similar in their amino acid sequences, yet they display different properties with respect to antibody binding. In this study we report of differential potencies of cross‐reacting tree pollen allergens and cloned isoforms of these allergens to activate allergen‐specific T‐lymphocyte clones (T‐cell clones; TCC). Six TCC with specifity for Bet v 1, a representative tree pollen major allergen, were established from peripheral blood of five birch‐pollen‐allergic donors. All TCC displayed the helper‐cell phenotype. Five TCC reacted with distinct epitopes present on natural (n) and on recombinant (r) Bet v 1. One TCC could not be stimulated with r Bet v 1, in spite of strong reactivity with purified natural Bet v 1. The TCC were tested in proliferation assays using purified n Bet v 1, n Cor a 1 (the homologous major allergen of hazel pollen), r Bet v 1, four recombinant isoforms of Cor a 1 and peptides representing corresponding T‐cell stimulating regions (isoepitopes) on these proteins. The clones showed different patterns of reactivity in response to stimulation with the five recombinant molecules and the corresponding peptides. Certain exchanges of amino acids within stimulating peptides correlated with a lack of proliferation of the TCC tested. These findings are important with respect to the use of broadly cross‐reactive recombinant allergens or allergen‐derived peptides for immunotherapy of type I allergy.
Acyl Carrier Protein (ACP) Import into Chloroplasts
Authors: Yang Li‐Ming , Fernandez Michael D. , Lamppa Gayle K.
Published: March 2005

During the import of the precursor for the acyl carrier protein (ACP) into chloroplasts, apoACP is converted to holoACP by the attachment of a phosphopantetheine group transferred from coenzyme A (CoA) by a chloroplast holoACP synthase [Fernandez, M. and Lamppa, G. (1990) Acyl carrier protein import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holoACP synthase, Plant Cell 2, 195–‐2061. Here it is shown that exogenous addition of CoA to intact chloroplasts in the import assay stimulates the conversion of apoACP to holoACP. If adenosine 3′,5′‐bisphosphate [Ado(3′,5′)P2], the byproduct of the transfer reaction, was also included the extent of conversion was greatly reduced. CoA has its effect after ACP precursor (pre‐ACP) import and proteolytic removal of the transit peptide, thus indicating that the chloroplast holoACP synthase resides in the stroma where fatty acid synthase is found. When Ado(3′,5′)P2 was added alone to the import assay, it inhibited the synthesis of holoACP. Inhibition of the conversion of apo‐ to holoACP with Ado(3′,5′)P2 made it possible to examine whether the holoform of preACP could be imported into chloroplasts. Pre‐apoACP was synthesized in Escherichia coli and shown to be competent for import in an ATP‐ and temperature‐dependent manner. A partially purified chloroplast holoACP synthase converted 60–90% of the pre‐apoACP to pre‐holoACP. Pre‐holoACP incubated with chloroplasts in the presence of Ado(3′,5′)P2 yielded >60% holoACP, whereas the control reaction with pre‐apoACP gave primarily apoACP. Hence the phosphopantetheine prosthetic group of ACP does not block precursor movement through the translocation apparatus of the chloroplast envelope.
An intrinsic‐tryptophan‐fluorescence study of phage φ29 connector/nucleic acid interactions
Authors: Urbaneja María Angeles , Rivas Susana , Carrascosa José L. , Valpuesta José María
Published: March 2005

The protein p10 of bacteriophage φ29 assembled into connectors exhibit an intrinsic fluorescence with an emission peak centered at 335 nm, which suggests a hydrophobic environment of the three tryptohan residues that the protein contains. Upon incubation with linear DNA (but not with circular DNA), a decrease in the connector intrinsic fluorescence is measured which does not show any sequence specificity. The decrease in fluorescence is not observed when DNA is incubated with proteolyzed connectors, which lack the DNA‐binding domain, suggesting that the fluorescence quenching is related to the binding of DNA to the φ29 connectors. Acrylamide quenching studies reveal a higher accessibility of tryptophan residues to the quencher when the connector is bound to DNA. Protein denaturation by guanidine hydrochloride occurs at lower denaturant concentrations in the presence of linear DNA (but not circular DNA) than in its absence, suggesting a conformational change of φ29 connector upon binding to linear DNA. This hypothesis is supported by the fact that the proteolyzed connectors, which do not bind DNA, are denatured at the same denaturant concentration, regardless of the presence of DNA. φ29 connectors also bind RNA, but this interaction does not exert any effect on acrylamide quenching or guanidine hydrochloride denaturation. This result, together with that showing that proteolyzed connectors are able to interact with RNA, reinforces the idea that φ29 connectors have two independent domains for interaction with DNA and RNA.
Antibodies for the Immunochemistry of the Human β3‐Adrenergic Receptor
Authors: Guillaume Jean‐Luc , Petitjean Françoise , Haasemann Martina , Bianchi Cesario , Eshdat Yuval , Strosberg A. Donny
Published: March 2005

Based on the amino acid sequence deduced from the recently cloned human β3‐adrenergic receptor (huβ3AR) gene, polyclonal antibodies were prepared against synthetic peptides, corresponding to regions of huβ3AR presumed to be exposed at the outer or the inner side of the membrane on the basis of the putative three‐dimensional structure of the previously characterized β1 and β2 adrenergic receptors. Affinity‐purified antibodies directed against N‐terminal, extracellular or intracellular loops and C‐terminal peptides reacted specifically with the huβ3AR and not with either the human β1 or β2 adrenergic receptor. Using these antibodies, it was demonstrated that the receptor is present at the surface of Chinese Hamster Ovary (CHO) cells transfected with the huβ3AR gene; in addition, the presence of the receptor protein was established in a human tissue (gall bladder). Immuno‐affinity chromatography of solubilized CHO huβ3AR‐containing cell membranes allowed the isolation of huβ3AR protein with an overall yield of 30%. The degree of purity of the receptor was more than 80%, as assessed by N‐terminal sequencing of the protein eluted from the column. Sequence analysis demonstrated the absence of a methionine residue at the N‐terminal position, and suggested that the side chain of the asparagine residue at position 7 is glycosylated.
Elongation on the Amino‐terminal Part of Stefin B Decreases Inhibition of Cathepsin H
Authors: Jerala Roman , Kroon‐žitko Louisa , Popovič Tatjana , Turk Vito
Published: March 2005

Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino‐terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily. The mutant proteins were expressed in Escherichia coli and purified to homogeneity. Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H. Inhibition of both amino‐terminal elongated stefin B mutants was decreased particularly for cathepsin H.

A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed. The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases. Variable regions were selected from fragments of other proteins from the protein data base. We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes. This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H. We conclude that the amino‐terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H.
Proton Resonance Assignments and Secondary Structure of Bovine Angiogenin
Authors: Reisdorf Christine , Abergel Daniel , Bontems Francois , Lallemand Jean‐Yves , Decottignies Jean‐Pierre , Spik Genevieve
Published: March 2005

Angiogenins are 14‐kDa proteins able to induce blood vessel growth in various preparations and are thus thought to be involved in the development of solid tumors. Angiogenins have significant similarities with extracellular ribonuclease and possess a characteristic nuclease activity against large RNA molecules. These proteins are also able to induce second‐messenger pathways. We have undertaken the determination of the three‐dimensional structure of bovine angiogenin by using nuclear magnetic resonance (NMR) spectroscopy. Since this protein was directly purified from cow milk, it was not possible to enrich angiogenin with 13C or 15N isotopes. However, extensive use of two‐dimensional and three‐dimensional proton NMR experiments enabled us to identify all but four spin systems and to assign all corresponding proton resonances. Identification of most backbone‐backbone nuclear Overhauser enhancements led to the characterization of the secondary structure elements of the protein. Comparison with the structure of ribonuclease A and analysis of the location of conserved residues confirmed that the two molecules have very similar structures.
Inhibition of T7 RNA Polymerase Transcription by Phosphate and Phosphorothioate Triplex‐Forming Oligonucleotides Targeted to a R · Y Site Downstream from the Promoter
Authors: Alunni‐Fabbroni Marianna , Manfioletti Guidalberto , Manzini Giorgio , Xodo Luigi E.
Published: March 2005

The effect of triplex‐forming oligonucleotides (TFO) on the transcription activity of T7 RNA polymerase has been investigated by an in vitro assay. The TFOs, either containing only phosphate (PO2) or phosphate and phosphorothioate (POS) internucleotide linkages, were targeted to a 30‐bp homopurine · homopyrimidine (R · Y) site cloned in plasmid Bluescript KS+ about four helical turns downstream from the T7 RNA promoter. Band‐shift and ultraviolet absorption melting experiments showed that the designed pyrimidine PO2 and POS TFOs form stable triple‐helical complexes with the R · Y target duplex (the ΔGTFO values of triplex formation vary from –42 to –63 kJ/mol). The triple‐helical complexes resulting from POS oligonucleotides were less stable (by 4–12 kJ/mol) than those obtained with PO2 analogues, the magnitude of destabilization being dependent on the number of POS groups present in the third strand. The designed TFOs were shown to efficiently repress bacteriophage T7 RNA polymerase transcription under different experimental conditions. The repression depended on pH, TFO concentration and temperature. When the TFO/template ratio was fixed to 100, a strong repressive effect was observed with normal and phosphorothioate pyrimidine TFOs, also under physiological conditions. In contrast, a purine‐rich oligonucleotide containing 44% of guanine residues promoted only a weak transcription inhibition, even at a TFO/template ratio as high as 750. Both PO2‐ and POS‐containing pyrimidine TFOs produced their strong repressive effect on T7 RNA polymerase transcription even when they were added to the reaction mixture simultaneously with the polymerase. A mechanism of transcription repression is discussed. The data reported in this paper are useful for designing oligonucleotides acting as artificial repressors in the antigene strategy and indicate that the R · Y target need not to be precisely confined to the promoter.
Effects of Amino Acid Insertions on the Proteolysis of a Staphylococcal Protein A Derivative inEscherichia Coli
Authors: Yang Shaojun , Bergman Tomas , Veide Andres , Enfors Sven‐Olof
Published: March 2005

In vivoproteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated inEscherichia colibefore and after insertion of 1 – 3 multiples of the tetrapeptide Ala‐Trp‐Trp‐Pro close to the C‐terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1 – 3 units of Ala‐Trp‐Trp‐Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein. The time for 50%in vivohydrolysis of the full length protein derivative that was most susceptible to proteolysis, i.e. with three tetrapeptide units, was about 40 min when cultivated in a bioreactor and about 4 min in a shaken flask culture. Molecular masses and N‐terminal sequences of the main degradation products indicated that protein ZZT0 is cleaved at identical sites irrespective of the number of inserted tetrapeptide units and that the cleavage sites are located far from the insertion point. Insertion of another hydrophobic amino acid, isoleucine, as the tetrapeptide Ala‐Ile‐Ile‐Pro, only induced a slight proteolysis of the ZZT0 molecule under similar conditions. This indicates that the insertion of tryptophan residues, rather than of a general hydrophobic segment, plays an essential role in the induced proteolysis of the ZZT0 protein.
The Phosphotyrosyl Phosphatase Activator of Protein Phosphatase 2A
Authors: Van Hoof Christine , Cayla Xavier , Bosch Mariette , Merlevede Wilfried , Goris Jozef
Published: March 2005

A simple, improved procedure for the isolation of the phosphotyrosyl phosphatase activator (PTPA) from rabbit skeletal muscle has been developed. The majority of the protein phosphatase 2A (PP2A) was separated from PTPA at an early stage in the procedure. The procedure yields approximately 1 mg essentially pure PTPA/kg rabbit skeletal muscle; it was also applied to porcine brain and the yeast Saccharomyces cerevisiae. The physico‐chemical properties of PTPA obtained from all sources are very similar. The pure rabbit skeletal muscle protein was used to raise polyclonal goat antibodies and to affinity purify these antibodies. Immunological studies revealed the presence of PTPA in all mammalian tissues and cell lines examined with differences in tissue distribution, brain showing the highest concentration. PTPA could only be detected in cytosolic fractions. Using a semi‐quantitative immunological assay (Western blot), the in vivo concentration could be estimated to be micromolar, which is in the same range as the PP2A target. The purified Xenopus oocyte PTPA showed only a weak cross reactivity, whereas yeast PTPA was not recognised by the antibody indicating some evolutionary diversity of the protein. In a PTPA‐affinity column chromatography, the weak interaction with PP2A was independent of the presence of ATP · Mg, a necessary cofactor in the activation process. Interaction of PTPA with PP2A in a 1:1 ratio induces a low (kcat= 3 min‐1) ATPase activity that is inhibited by okadaic acid, ADP and non‐hydrolysable ATP analogues.
High‐level Expression of the Human Neurokinin‐1 Receptor in Mammalian Cell Lines using the Semliki Forest Virus Expression System
Authors: Lundström Kenneth , Mills Ann , Buell Gary , Allet Elisabeth , Adami Nadia , Liljeström Peter
Published: March 2005

Human neurokinin‐1 receptor cDNA was introduced into the pSFV1 Semliki Forest virus (SFV) vector and the in vitro transcribed RNA was electroporated into BHK cells with pSFV‐Helper RNA. This procedure resulted in the packaging of a high‐titer SFV‐NK‐1 virus stock containing approximately 5×109 infective units/ml. Infection of baby hamster kidney, COS‐7, Chinese hamster ovary and human osteosarcoma cells yielded high levels of human neurokinin‐1 receptor expression as assessed by [3H]substance P binding. The maximal receptor expression level obtained was 4×106 receptors/cell and studies of the post‐infection time indicated that a high level of receptor expression was observed 10–24 h post‐infection. The human neurokinin‐1 receptor expressed in infected baby hamster kidney, COS‐7 and Chinese hamster ovary cells was able to stimulate Ca2+ mobilization indicating functional coupling to guanine‐nucleotide‐binding proteins. The application of the Semliki Forest virus expression system will permit the rapid and efficient production of large quantities of receptor protein for both pharmacological and structural studies.
O ‐Demethylation by the Homoacetogenic Anaerobe Holophaga Foetida Studied by a New Photometric Methylation Assay Using Electrochemically Produced Cob(I)Alamin
Authors: Kreft Jan‐Ulrich , Schink Bernhard
Published: March 2005

The previously studied complete methyl transfer sequence of tetrahydrofolate‐dependent O ‐demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non‐physiological substrates: electrochemically produced cob(I)alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5‐methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isosbestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (Δɛ540= 4.40 mM‐1· cm‐1). The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of syringate‐grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme‐bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho‐hydroxyl or para‐carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol · min‐1· (mg protein)‐1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km= 1.2 mM, Vmax= 220 nmol · min‐1· (mg protein)‐1, and Vmax/Km= 180 nmol · min‐1· (mg protein)‐1· mM‐1. 3,5‐Dihydroxy‐anisole demethylation by extracts of 3,5‐dihydroxyanisole‐grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower.
ω‐Oxidation of Lipoxin B4 by Rat Liver
Authors: Mizukami Yoichi , Sumimoto Hideki , Isobe Ryuichi , Minakami Shigeki , Takeshige Koichiro
Published: March 2005

Lipoxin B4 (LXB4) is metabolized to 20‐hydroxy‐LXB4 by rat liver microsomes. The ω‐hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, indicating involvement of a cytochrome P‐450 (P‐450). This is supported by inhibition of the reaction by antibodies raised against NADPH–P‐450 reductase. The P‐450 appears to be the one responsible for leukotriene B4ω‐hydroxylation, because leukotriene B4 inhibits the formation of 20‐hydroxy‐LXB4 and LXB4 blocks the leukotriene B4ω‐hydroxylase activity in microsomes. Incubation of 20‐hydroxy‐LXB4 with both rat liver cytosol and NAD+ leads to formation of a more polar metabolite on high‐performance liquid chromatography. The metabolite is identified as 20‐carboxy‐LXB4, a novel metabolite of LXB4, based on analyses by ultraviolet spectrometry and by gas chromatography/mass spectrometry. The 20‐carboxy‐LXB4‐forming activity is localized in cytosol, with an optimal pH of 8.5. The activity is dependent on NAD+, but NADP+ can not replace NAD+. The reaction is inhibited by pyrazole and 4‐methylpyrazole, inhibitors of alcohol dehydrogenase, and by substrates of the enzyme such as ethanol and 20‐hydroxy‐leukotriene B4. Disulfiram, an inhibitor of aldehyde dehydrogenase, also blocks the 20‐carboxy‐LXB4 formation. These observations suggest that both alcohol dehydrogenase and aldehyde dehydrogenase participate in the oxidation of 20‐hydroxy‐LXB4 to 20‐carboxy‐LXB4.
Decrease in Cytosolic ATP/ADP Ratio and Activation of Pyruvate Kinase after in vitro Addition of Almitrine in Hepatocytes Isolated from Fasted Rats
Authors: Leverve Xavier M. , Fontaine Eric , Putod‐Paramelle Florence , Rigoulet Michel
Published: March 2005

Previously, we have shown in experiments with isolated mitochondria that almitrine, a drug used for patients with chronic lung disease, affects the H+/ATP stoichiometry of the F0F1‐ATPase [Rigoulet, M., Fraisse, L., Ouhabi, R., Guérin, B., Fontaine, E. & Leverve, X. M. (1990) Biochim. Biophys. Acta 1018, 91–97]. In the present study, we have investigated the effect of almitrine on gluconeogenesis and oxygen consumption in isolated hepatocytes. Almitrine decreased both the cytosolic and mitochondrial ATP/ADP ratios but had no effect on oxygen consumption in cells incubated with and without octanoate. This must have been due to a double effect. On the one hand, a decrease in the ATP/ADP ratio decreases ATP utilization; on the other hand, in the presence of almitrine more oxygen is required to synthesize ATP. Almitrine did affect gluconeogenesis from various substrates (lactate + pyruvate, glycerone or fructose), but had no effect on glycerol or glutamine metabolism. The effect on gluconeogenesis from glycerone was due to an increase in glycolytic flux. The rate of lactate + pyruvate production increased whereas there was no effect on glycerone utilization. This effect was caused by an activation of pyruvate kinase. Our data indicate that this enzyme is an extremely sensitive sensor of the cytosolic ATP/ADP ratio. Hence, under our experimental conditions, the cytosolic ATP/ADP ratio decrease affects only the balance between glucose and lactate + pyruvate productions, and not the phosphorylation of glycerone, the first and controlling step of this pathway.
Generation of Proton‐Motive Force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius
Authors: Gleissner Michael , Elferink Marieke G. L. , Driessen Arnold J. M. , Konings Wil N. , Anemüller Stefan , Schäfer Günter
Published: March 2005

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermo‐acidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N′,N′‐tetramethyl‐p‐phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50°C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady‐state transmembrane electrical potential (ΔΨ, interior negative), and transmembrane pH gradient (ΔpH, interior alkaline) at temperatures up to 70°C. The successful functional reconstitution of the cytochrome aa3‐type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.
Characterization and Expression of the Lactobacillus helveticus pepC Gene Encoding a General Aminopeptidase
Authors: Vesanto Erkki , Varmanen Pekka , Steele James L. , Palva Airi
Published: March 2005

An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8‐kb fragment, which also encoded aminopeptidase activity. This fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORF1 was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with ΔG of −49.0 kJ mol−1 was found downstream of the coding region. The deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4‐kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, respectively, thus confirming that ORF1 codes for an aminopeptidase C. mRNA size analyses revealed 1.7‐kb and 2.7‐kb transcripts in Northern blot with the pepC‐specific probe. A further analysis with the pepC‐ and ORF2‐specific probes showed that downstream ORF2 is co‐transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter were below the detection limit, and the ORF2 was expressed by its own promoter. The 5′ end mapping of the pepC transcripts with primer extension revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. helveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcription of pepC was typical to exponential growth phase expression. The level of total thiol‐aminopeptidase activity, however, remained nearly constant throughout the stationary growth phase.
The Modulation of Enzyme Reaction Rates Within Multi‐Enzyme Complexes
Authors: Gontero Brigitte , Giudici‐Orticoni Marie‐Thérèse , Ricard Jacques
Published: March 2005

Octameric ribulose bisphosphate carboxylase‐oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6‐phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase‐oxygenase is inserted in the five‐enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase‐oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein–protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase‐oxygenase. This alteration may be expressed in terms of stabilization‐destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi‐protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co‐ordinate of−1.68 kJ/mol and a constant stabilization energy of –3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi‐enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase‐oxygenase, as well as to increase the catalytic rate constant.

The binding of the substrate analog 6‐phosphogluconate to free ribulose bisphosphate carboxylase‐oxygenase is non‐co‐operative. It becomes positively co‐operative if this enzyme is inserted in the multi‐protein complex. Under these conditions, only one type of enzyme‐inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme‐inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is –1.03 kJ/mol.

Neither free nor associated ribulose bisphosphate carboxylase‐oxygenase display any co‐operativity relative to substrate binding. However, in the presence of the substrate analog 6‐phosphogluconate, this enzyme displays positive co‐operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is ≤2. As 6‐phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co‐operativity might be of functional importance in vivo.
Purification and Properties of Phosphofructokinase from Dictyostelium Discoideum
Authors: MartÍnez‐Costa Oscar H. , Estévez Antonio M. , Sanchez Valentina , Aragón Juan J.
Published: March 2005

Phosphofructokinase (PFruK) from the slime mold Dictyostelium discoideum has been purified to homogeneity over 15000‐fold with a 29% yield. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the final preparation revealed a single band of 95 kDa. The native molecular mass was determined by gel filtration to be 382 kDa, indicating that the enzyme is a homotetramer. An antibody raised in rabbits against the 95‐kDa band immunoprecipitated PFruK activity while it did not react with the enzyme from yeast and mammalian cells. The apparent PI was 6.8 and the pH optimum was 7.6. The enzyme had an activation energy (Ea) of 29.1 kJ/mol. The amino acid composition was distinctive in having high ser, Gly and Glx and low Ala, Val and Tyr compared with other eukaryotic PFruKs. Enzyme activity did not have a sigmoidal saturation curve for fructose 6‐phosphate, was only mildly inhibited by MgATP at acidic pH values, was not affected by enzyme concentration and was insensitive to any of the typical allosteric effectors of PFruKs from other sources. However, the enzyme binds fructose 2,6‐bisphosphate as indicated by protection against thermal denaturation. Treatment with cAMP‐dependent protein kinase led to phosphorylation of the enzyme without change in activity. The metabolic significance of these properties and their relationship to structure/function are discussed.
pH‐dependent Properties of a Mutant Horseradish Peroxidase Isoenzyme C in which Arg38 has been Replaced with Lysine
Authors: Sanders Stephen A. , Bray Robert C. , Smith Andrew T.
Published: March 2005

Arg38 in the active site of horseradish peroxidase isoenzyme C (HRP‐C) has been replaced with lysine by site‐directed mutagenesis. As a prelude to a detailed kinetic analysis of this variant, the present study characterizes a pH‐dependent cycle of reactions for recombinant horseradish peroxidase isoenzyme C with Arg38 replaced by lysine ([R38K]HRP‐C*), which involves time‐dependent changes in both specific activity and the electronic absorption spectrum of the enzyme. This pH‐dependent cycle resembles that previously suggested for a cytochrome‐c peroxidase variant in which Asp235 was replaced with asparagine. When the pH of a solution of resting [R38K]HRP‐C* at pH 6.6 (form AH) is raised to pH 8.6, a rapid alkaline transition occurs. This results in spectral changes characteristic of a shift from a predominantly pentacoordinate to a completely hexacoordinate high‐spin haem iron (form A−) with a pKa of 7.5. When the pH of a solution of form A− is raised from 8.7 to 12.0, no further spectral changes are observed. The reaction is reversible, but when the high‐pH form of the enzyme (A−) is allowed to stand at pH 8.6, it slowly becomes converted into a third enzyme form (form I−) at a rate which is independent of pH (k=0.56 h−1). When the pH of a sample of form I− is lowered from 8.6 to 6.6, the original low‐pH form (AH) of the enzyme is recovered. Recovery of form AH from form I− does not occur via form A−, but via at least one further intermediate, form X. Following a downward pH jump, the rate constant for the formation of form X from form I− shows a small dependence on pH, changing from 48 s−1 at pH 6.8 to 39 s−1 at pH 7.4. The rate of formation of form AH from form X is also pH dependent and biphasic in nature, with measured rate constants ranging from 11.9–2.1 h−1 The possible structures of the different forms of [R38K]HRP‐C* are discussd in the light of similar data in the literature for variants of cytochrome‐c peroxidase. The properties may be indicative of a greater degree of conformational flexibility within the active site of this mutant caused by the smaller bulk of the lysine side‐chain and the probable disruption of a part of the haem‐linked hydrogen‐bonding network in the distal haem pocket. The wild‐type enzyme undergoes no such pH induced changes.
Preliminary Crystallographic Studies of Four Crystal forms of Serum Albumin
Authors: Carter Daniel C. , Chang Brian , Ho Joseph X. , Keeling Kim , Krishnasami Ziporah
Published: March 2005

Several crystal forms of serum albumin suitable for three‐dimensional structure determination have been grown. These forms include crystals of recombinant and wild‐type human serum albumin, baboon serum albumin, and canine serum albumin. The intrinsic limits of X‐ray diffraction for these crystals are in the range 0.28–0.22 nm. Two of the crystal forms produced from human and canine albumin include incorporated long‐chain fatty acids. Molecular replacement experiments have been successfully conducted on each crystal form using the previously determined atomic coordinates of human serum albumin illustrating the conserved tertiary structure.
Covalent Modification of Catalytic Sites on Membrane‐bound Beef Heart Mitochondrial ATPase by 2‐Azido‐adenine Nucleotides
Authors: Martins Ione S. , Penefsky Harvey S.
Published: March 2005

Incubation in the dark of 32P‐labeled 2‐azido‐adenine nucleotides with submitochondrial particles from beef heart led to tight binding of the label by membrane‐bound F1. That is, the label remained with the particles following two passages through centrifuge columns. After removal of free nucleotides and ultraviolet irradiation, the radioactive label was covalently bound exclusively to the β subunit of the ATPase. Extraction of the modified enzyme from the membrane with chloroform followed by tryptic digestion and separation of peptides by reverse‐phase high‐pressure liquid chromatography indicated that the radioactive label had been inserted into a peptide fragment that included part of the catalytic site.

Covalent modification of catalytic sites by 2‐azido‐ADP was accompanied by parallel inhibition of both ATP synthesis and ATP hydrolysis by submitochondrial particles. Estimation of the likely amount of F1 participating in the reaction and extrapolation to complete inhibition suggested that modification of no more than a single site was sufficient to block both reactions. The results support suggestions of cooperative interactions between catalytic sites as well as a single catalytic pathway for both enzymic reactions.
The Complete Sequence of a 40–kDa Actin‐Modulating Protein from the Earthworm Lumbricus terrestris
Authors: Giebing Thomas , Hinssen Horst , D'haese Jochen
Published: March 2005

The complete primary structure of a 40‐kDa actin‐modulating protein from the earthworm Lumbricus terrestris is presented. A muscle‐specific cDNA library of the earthworm was constructed and screened with a specific DNA probe obtained by polymerase chain reaction considering information from peptide sequencing. A full‐length clone with a coding region of 1098 bp was isolated. The deduced polypeptide sequence of 366 amino acids (41457 Da) reveals the segmental structure typical of both the 40‐kDa and 80‐kDa actin‐modulating proteins. Prominent similarities to the 80‐kDa protein gelsolin especially exist with respect to the first segment and to the C‐terminal segment. The comparatively high nucleation efficiency of the earthworm actin modulator is probably determined by its third segment which seems to enable the earthworm actin modulator to bind a second G‐actin molecule more tightly than other previously described 40‐kDa modulators.
Active‐Site Covalent Modifications of Quinoprotein Amine Oxidases from Aspergillus niger
Authors: Frébort Ivo , Peč Pavel , Luhová Lenka , Matsushita Kazunobu , Toyama Hirohide , Adachi Osao
Published: March 2005

Interactions of two distinct quinoprotein amine oxidases from Aspergillus niger, AO‐I and AO‐II, with active‐site covalent modifiers have been investigated. Both enzymes are inhibited similarly by phenylhydrazine or p ‐nitrophenylhydrazine, forming an orange Schiff base with a carbonyl group of topaquinone cofactor. Modification of histidyl and tyrosyl residues by diethylpyrocarbonate and sulfhydryl groups by 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) and 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole have been described. A substrate analog, 1,4‐diamino‐2‐butyne, was found to function as a mechanism‐based inhibitor. It shows both substrate saturation kinetics and time‐dependent irreversible inhibition caused by formation of pynole bound to the active site. The pyrrole formation was confirmed spectrophotometrically by reaction with Ehrlich's reagent at 525 nm. Inhibition by 1,4‐diamino‐2‐butyne produces a new maximum in the absorption spectra of AO‐I and AO‐II at 310 nm and 306 nm, respectively. Inactivated AO‐I was digested by proteases; labeled peptides were purified by C18 HPLC and sequenced by Edman degradation. Data reveal the evidence that 1,4‐diamino‐ 2‐butyne reacts with the ε‐amino group of the Lys356 residue in the sequence Lys‐Met‐Pro‐Asn‐Ala of Aspergillus niger amine oxidase AO‐I.
Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast
Authors: Bellamine Aouatef , Gautier Jean‐Charles , Urban Philippe , Pompon Denis
Published: March 2005

An expression library of hybrid cDNAs was constructed in vivo by homeologous recombination in yeast between human P450 1A1 and P450 1A2 sequences. Two clones exhibiting highly enhanced monooxygenase activities in vivo were selected. Chimera S12 includes the 88 N‐terminal residues of P450 1A1 fused to the complementary part of the P450 1A2 sequence. Chimera S71 derives from P450 1A1 by the substitution of the 36 C‐terminal amino acid residues by the corresponding 38 residues of the 1A2 sequence. Biochemical analysis on microsomal fractions indicated that S12 and S71 have the same substrate specificities as 1A2 and 1A1, respectively. The observed increase in the in vivo monooxygenase activity is related to a ninefold increase in the microsomal S12 content as compared to the 1A2 content. In contrast, the expression level of S71 is slightly reduced but its turnover numbers are increased as compared to 1A1. The folding stability of chimeric P450 enzymes was evaluated by thermal and chaotropic agent denaturation. No difference was found between S12 and 1A2, but S71 appeared slightly less stable than 1Al. In vivo experiments indicated that S12 mRNA accumulation and stability are quite similar to the stability of parental 1A2 and, for both chimeras and parental enzymes, the protein half‐lives are longer than the cell doubling time. The surprising accumulation of chimera S12 in the microsomal membrane is discussed in terms of the relationship of protein folding with transport to the endoplasmic reticulum membrane and the apparent expression levels of human P450 enzymes produced in yeast.
Sodium‐Dodecyl‐Sulfate‐Resistant Complex Formation of Epimorphin Monomers and Interaction of the 150‐kDa Complex with the Cell Surface
Authors: Hirai Yohei
Published: March 2005

Epimorphin was previously identified as a mesenchymal factor essential for epithelial/mesenchymal interaction. Since a monoclonal antibody, MC‐1, inhibits the biological activity of epimorphin when added to the extracellular medium, the location of this factor is thought to be extracellular. However, sequence analysis of the isolated epimorphin cDNA revealed that its product resembled other proteins that are involved in intracellular vesicular transport. Furthermore, the molecular size of epimorphin predicted from the cloned cDNA was not consistent with the size observed by immunoblot analyses. In the present study, an attempt was made to resolve these inconsistencies in the nature and function of epimorphin. Evidence is presented for the following: (a) monomeric epimorphin forms various sodium‐dodecyl‐sulfate‐resistant complexes, each displaying a unique immunoreactivity, of which MC‐1 recognizes only a certain form; (b) the MC‐1‐reactive form of epimorphin is sensitive to extracellular trypsin whereas the other forms are not; (c) cells selectively attach to surfaces coated with recombinant epimorphin fragments containing the MC‐1 epitope in vitro. These results suggest that a certain population of epimorphin molecules is involved in cell/cell interaction through a process of complex formation, transportation to extracellular regions, and direct binding to the cell surface.
Structure/Activity Characterization of Glucagon‐Like Peptide‐1
Authors: Gallwitz Baptist , Witt Maike , Paetzold Gabriele , Morys‐Wortmann Corinna , Zimmermann Bodo , Eckart Klaus , Fölsch Ulrich R. , Schmidt Wolfgang E.
Published: March 2005

Glucagon‐like peptide‐1 is a gastrointestinal hormone that strongly stimulates insulin release via specific receptors on the pancreatic β‐cell. To characterize the side‐chain groups required for interaction of glucagon‐like peptide‐1 with its receptor, we performed binding studies with alanine‐substituted glucagon‐like peptide‐1 analogues on RINm5F insulinoma cells. The binding affinity and biological activity of glucagon‐like peptide‐1 have been found to be sensitive to alanine exchanges in the N‐terminal positions 1, 4, 6 and the C‐terminal positions 22 and 23. Alanine substitutions at positions 5, 8, 10–12, 14, 16–21 and 25–30 do not change receptor affinity. These findings could be exploited to design glucagon‐like peptide‐1 agonists and probably antagonists for further physiological studies.
The Solution Structure of a DNA Duplex Containing the cis ‐Pt(NH3)2[d(‐GTG‐)‐N 7(G), N 7(G)] Adduct, as Determined with High‐Field NMR and Molecular Mechanics/Dynamics
Authors: Van Garderen Carla J. , Van Houte Leo P. A.
Published: March 2005

The solution structure of the cis ‐Pt‐GTG adduct in the double‐stranded oligomer d(CTCTAGTG‐CTCAC) · td(GTGAGCACTAGAG) was studied with high‐resolution NMR techniques. For model building, the distance information obtained from two‐dimensional NOE experiments was used in molecular mechanic computations and molecular dynamic structure refinements.

The structural distortion upon platination appears to be restricted to the base pairs Pt‐G6 · C21 and T7 · A20; Pt‐G8 · C19 forms a normal Watson‐Crick base pair. T7 is positioned in the minor groove and stacks with the highly propeller‐twisted Pt‐G6. There is no hydrogen bonding between T7 and A20. The complementary strand is undistorted; A20 stacks with its flanking cytidines (C19 and C21) as in regular B‐DNA. The duplex is locally unwound (from base pair A5 · T22 to G8 · C19: 19°) and is slightly kinked (20°) at the platination site. The platinum coordination distorts the DNA structure at the 5′ side of the platinated ‐GTG‐ sequence and changes the minor groove face.
Overexpression of Human Cardiac Troponin‐I and Troponin‐C in Escherichia coli and Their Purification and Characterisation
Authors: Al‐Hillawi Eman , Minchin Stephen D. , Trayer Ian P.
Published: March 2005

We have overexpressed human cardiac troponin‐I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N‐terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG → GCC and (Gly4) GGG → GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin‐C. Both proteins were purified using ion‐exchange chromatography and have been proved to be biologically active. The recombinant troponin‐I was able to bind to a troponin‐C affinity column in the presence of 9 M urea in a calcium‐dependent manner. The calcium‐dependent troponin‐I–troponin‐C complex between both recombinant proteins was also demonstrated by alkaline‐urea gel electrophoresis. In addition, troponin‐I inhibited the acto‐S1 Mg‐ATPase activity; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin‐C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium‐dependent force generation to calcium‐insensitive skinned muscle fibres.
Hybrid Fo Complexes of the ATP Synthases of Spinach Chloroplasts and Escherichia coli
Authors: Burkovski Andreas , Deckers‐Hebestreit Gabriele , Altendorf Karlheinz
Published: March 2005

Hybrid Fo complexes of the ATP synthases of spinach chloroplast (CFo) and Escherichia coli (EFo) were investigated. Immunoprecipitations with polyclonal antibodies against the different Fo subunits clearly revealed that hybrid Fo complexes derived from CFo subunit III and EFo subunits a and b were formed in vivo. In addition, the ATPase activities of the hybrid ATP synthase, measured in everted cytoplasmic membranes of an atpE mutant strain transformed with the atpH gene coding for CFoIII, were comparable to activities obtained for the same mutant strain complemented with the atpE gene (EFoc). Nevertheless, CFoIII was not able to replace EFoc functionally, since the strain containing the hybrid ATP synthase was not able to grow on succinate. In order to investigate the reason for this lack of function, hybrid proteolipids of CFoIII and EFoc were constructed. Only a chimaeric protein comprising the seven N‐terminal amino acid residues from CFoIII and the remaining part of EFoc was able to replace wild‐type EFoc, whereas hybrid proteins with 13 and 33 N‐terminal amino acids of CFoIII were not functional. The results suggested that a network of interactions between the subunits essential for proton translocation and/or coupling of the F1 part exists, which was optimized for each species during evolution, although the overall structure complexes has been conserved.
Characterization of NTPase, RNA‐binding and RNA‐helicase Activities of the Cytoplasmic Inclusion Protein of Tamarillo Mosaic Potyvirus
Authors: Eagles Robin M. , Balmori‐Melián Ezequiel , Beck David L. , Gardner Richard C. , Forster Richard L. S.
Published: March 2005

The 66‐kDa cytoplasmic inclusion protein of tamarillo mosaic potyvirus was purified to near homogeneity using organic solvent clarification, differential centrifugation and sucrose density gradient centrifugation. ATPase and GTPase activities were shown to co‐purify with the 66‐kDa protein. ATPase activity was stimulated up to fivefold in the presence of 20 μM poly(A). The Km value for ATP hydrolysis (18 μM), was minimally affected upon addition of poly(A). In contrast, the Vmax value for ATP hydrolysis was increased fivefold by the addition of poly(A). Binding of RNA by the cytoplasmic inclusion protein was demonstrated by gel electrophoresis of ultraviolet cross‐linked enzyme‐RNA complexes. In the absence of added NTP, complexes between the cytoplasmic inclusion protein and single‐stranded RNA species formed rapidly in the pH range 3–7, but not at pH 8 or 9. Binding to single‐stranded RNA was markedly decreased by the addition of NaCl (10 mM), suggesting a weak association between RNA and enzyme. The cytoplasmic inclusion protein bound single‐stranded RNA or partially double‐stranded RNA duplexes with single‐stranded overhangs of 35 bases and 81 bases, respectively, but did not bind 16‐bp blunt‐ended double‐stranded RNA. RNA binding occurred in the absence of NTP (ATP, GTP, CTP or UTP), whereas dissociation of bound RNA occurred only in the presence of NTP. RNA duplex unwinding (helicase) activity of the enzyme was demonstrated in the presence of any of the above four NTPs using partially double‐stranded RNA duplexes with 3′ single‐stranded overhangs. We propose that the cytoplasmic inclusion protein of tamarillo mosaic virus is an RNA helicase, which translocates in the 3′ to 5′ direction in an energy‐dependent manner, unwinding double‐stranded regions.