bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of nucleophosmin/B23 oligomers requires both the amino‐and the carboxyl‐terminal domains of the protein
Authors: LIU Qing‐Rong , CHAN Pui K.
Published: March 2005

Nucleophosmin/B23 is a nucleolar phosphoprotein which forms oligomers. To determine the domain essential for oligomer formation, various deletion and point mutation clones of nucleophosmin/B23 were constructed. Nucleophosmin/B23 and the mutant proteins were produced by (a) coupled in vitro transcription and translation and (b) expression in Escherichia coli with T7 RNA polymerase expression vector (pET‐8c). Nucleophosmin/B23 synthesized in vitro has the same peptide map as that synthesized in HeLa cells. Similarly, it formed oligomers which could be detected in SDS/PAGE and were cross‐linked with nitrogen mustard in vivo. Substitution of Met5, Met7, and Met9 with Leu or deletion of five amino acids at the C‐terminus abolished the oligomerization. Deletion of portions of amino acids in the middle of the molecule (amino acid residues 83–152, 117–186 and 185–240) had little effect on the oligomerization. Co‐expression of the N‐ and C‐terminal mutant clones in vitro did not produce oligomers. These results indicate that intra‐molecular interactions with both the N‐ and C‐terminal domains are essential for oligomer formation.
Proteolytic cleavage sites in smooth muscle myosin‐light‐chain kinase and their relation to structural and regulatory domains
Authors: PEARSON Richard B. , ITO Masaaki , MORRICE Nick A. , SMITH Alan J. , CONDRON Rosemary , WETTENHALL Richard E. H. , KEMP Bruce E. , HARTSHORNE David J.
Published: March 2005

Proteolysis of the smooth muscle myosin‐light‐chain kinase with either thermolysin or endoproteinase Lys‐C cleaves the enzyme towards the amino‐terminus between the first and second unc domains, unc‐II‐1 and unc‐II‐2, and in the calmodulin‐binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys‐C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys‐C endoproteinase results in a constitutively active 474‐residue fragment with the same amino‐terminus, but a carboxyl‐terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic‐residue‐rich connecting peptide between the amino‐terminus of the catalytic domain and the unc‐I domain, nor within the unc‐II or unc‐I domains or between the adjacent unc‐II‐2 and unc‐I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin‐light‐chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787–807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777–793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967–6971] to be important in autoregulation of the myosin‐light‐chain kinase.
NMR studies on p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida
Authors: VERVOORT Jacques , VAN BERKEL Willem J. H. , MÜLLER Franz , MOONEN Chrit T. W.
Published: March 2005

p‐Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C‐ and 15N‐enriched FAD. The protein preparations were studied by 13C‐NMR, 15N‐NMR and 31P‐NMR techniques in the oxidized and in the two‐electron‐reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the π electron distribution in oxidized free p‐hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e. g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an α‐helix microdipole.

In the reduced state, the flavin in p‐hydroxybenzoate hydroxylase is bound in the anionic form, i. e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state.

The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4α. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2‐hybridized N5 atom and a slightly sp3‐hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.
Pterocarpan phytoalexin biosynthesis in elicitor‐challenged chickpea (Cicer arietinum L.) cell cultures
Authors: TIEMANN Karin , INZÉ Dirk , VAN MONTAGU Marc , BARZ Wolfgang
Published: March 2005

NADPH:isoflavone oxidoreductase (IFR) is the first soluble enzyme of the pterocarpan‐specific part of phytoalexin biosynthesis in chickpea (Cicer arietinum L.). The enzyme was purified to apparent homogeneity by a five‐step procedure from chickpea cell cultures treated with yeast extract as elicitor. Analysis by gel filtration and SDS/PAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa. Km values for the substrates 2′‐hydroxyformononetin, 2′‐hydroxypseudobaptigenin and NADPH were 6, 6 and 20 μM, respectively. The IFR showed pronounced specificity for the substitution pattern of isoflavones. We found a 2′‐hydroxy group and a 4′,5′‐methylenedioxy or 4′‐methoxy function to be essential for acceptance as substrate. The isoelectric point of the protein was determined as 6.3 by IEF and there is no evidence for the existence of isoenzymes.

Partial amino acid sequences of IFR were determined from internal peptides obtained by tryptic digestion of the protein and corresponding oligonucleotides were synthesized. A λgt10 cDNA library was constructed using poly(A)‐rich RNA isolated from chickpea cell cultures treated with Ascochyta rabiei elicitor. 150 positive clones were obtained by screening 2 × 105 clones with an IFR‐specific oligonucleotide. The identity of sequenced clones was confirmed by comparison of the deduced amino acid sequence with the internal peptide sequences of purified IFR. The sequence of a 1183‐bp clone contained a continuous open reading frame of 954 bases encoding a polypeptide of 318 amino acids with a calculated molecular mass of 35.4 kDa, indicating that a full‐length cDNA coding for IFR was isolated.
Preparation and kinetic properties of 5‐ethylphenazine‐glucose‐dehydrogenase‐NAD+ conjugate, a semisynthetic glucose oxidase
Authors: YOMO Tetsuya , URABE Itaru , OKADA Hirosuke
Published: March 2005

5‐Ethylphenazine‐glucose‐dehydrogenase‐NAD+ conjugate (EP+ ‐GlcDH‐NAD+) was prepared by linking both poly(ethylene glycol)‐bound 5‐ethylphenazine and poly(ethylene glycol)‐bound NAD+ to glucose dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.8, and that of the NAD+ moieties was 1.2. This conjugate is a semisynthetic enzyme having glucose oxidase activity using oxygen or 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) as an electron acceptor. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis‐Menten equation with the following values of the kinetic constants: for the system with oxygen, the turnover number per subunit is 0.40 s‐1 and Km for oxygen is 1.57 mM; and for the system with MTT, the turnover number is 0.11 s‐1 and Km for MTT is 0.072 mM. The catalytic cycle of the semisynthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety. The apparent intramolecular rate constants of these steps were estimated, and the values are as follows: 0.39 s‐1 for the reductions of the NAD+ moiety, 2.2 s‐1 and 0.12 s‐1 for the oxidation of the NADH moiety in the systems with oxygen and with MTT, respectively, and 3.2 s‐1 and 0.18 s‐1 for the reduction of the ethylphenazine moiety in the systems with oxygen and with MTT, respectively. On the bases of these results, the following three rate‐acceleration mechanisms of the semisynthetic glucose oxidase are discussed: high effective concentration, intramolecular coupling of successive catalytic reactions, and multiple connection between the two kinds of the catalytic sites.
Characterization of two pterin derivatives isolated from Methanoculleus thermophilicum
Authors: RAEMAKERS‐FRANKEN Petronella C. , BONGAERTS Roy , FOKKENS Roel , VAN DER DRIFT Chris , VOGELS Godfried D.
Published: March 2005

Methanoculleus thermophilicum was shown to contain two pterin derivatives. The structures of these pterin derivatives were established from amino acid analysis, 1H‐NMR and fast‐atom bombardment mass spectrometry data. One of the pterins was identified as tatiopterin‐O, an aspartyl derivative of methanopterin with a proton at position 7 of the pterin moiety. The other pterin, which we named thermopterin, differed in the structure of the aniline group, containing two additional hydroxyl residues. The IUPAC name of thermopterin is N‐[‐1′‐(2"‐amino‐4"‐hydroxy‐6"‐pteridinyl)ethy]‐4‐[2′,3′,4′,5′,‐tetrahydroxypent‐1′‐yl(5′→ 1")O‐α‐ribofuranosyl‐5"‐phosphoric acid]‐2,5‐dihydroxyaniline, in which the phosphate group is esterified with α‐hydroxyglutarylaspartic acid.
When beef‐heart mitochondrial F1‐ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites
Authors: MILGROM Yakov M.
Published: March 2005

Inactivation of the isolated ATPase portion of ATP synthase from beef‐heart mitochondria (F1) by its natural inhibitor protein (IP) during steady‐state ATP hydrolysis is accompanied by a trapping of 1 mol nucleotide/mol F1 in one of the catalytic sites. The trapped nucleotide is not released during incubation of IP‐inhibited F1 in the presence of MgATP at pH 8.0 for at least 20 min, indicating a very low turnover rate of the IP · F1 complex. The ATP/ADP ratio of the trapped nucleotides is higher than that found for transitorily bound nucleotides under the same conditions but in the absence of IP. The IP impairs the acceleration of ATP hydrolysis and product release steps that results from the binding of ATP to an alternate catalytic site. It also inhibits ATP hydrolysis by a single catalytic site or shifts the equilibrium toward ATP formation from bound ADP and Pi. At high pH, an active acidic form of the free IP is transformed to the inactive basic one with a half‐time of 3—4 s. This process seems to be prevented by IP binding to F1. The inactive basic form of IP does not compete with the active acidic IP for the binding to F1. The data do not favor the existence of a long‐lived catalytically active IP · F1 intermediate during IP action on F1. The reactivation of IP‐inhibited membrane‐bound F1 by energization may be due to a conformational change in the IP · F1 complex allowing the transformation of IP into an inactive basic state that rapidly dissociates.
Mathematical analysis of enzymic reaction systems using optimization principles
Authors: HEINRICH Reinhart , SCHUSTER Stefan , HOLZHÜTTER Hermann‐Georg
Published: March 2005

Contents

Introduction

Optimization of the catalytic properties of single enzymes

Irreversible two‐step kinetic mechanism

Reversible three‐step kinetic mechanism

Cooperative enzymes

Optimization of multienzyme systems

Basic equations for unbranched reaction chains

Maximization ofsteady‐state fluxes

Transient times

Sensitivity to external signals

Implications of the solvent capacity and osmotic balance

Metabolic channelling

Thermodynamic principles

Optimal stoichiometries

Time hierarchy in biochemical networks

Final remarks
Human and rodent sequence analogs of Alzheimer's amyloid βA4 share similar properties and can be solubilized in buffers of pH 7.4
Authors: HILBICH Caroline , KISTERS‐WOIKE Brigitte , REED Jennifer , MASTERS Colin L. , BEYREUTHER Konrad
Published: March 2005

The filamentous amyloid protein aggregates found in the brain of patients affected with Alzheimer's disease principally consist of a peptide termed βA4, according to its secondary structure of β‐pleated sheets and its molecular mass of about 4 kDa. It has a length of up to 42 or 43 residues. By chemical means, we have synthesized peptide analogs corresponding to the human and rodent βA4 sequences. We describe structural and functional properties of peptides spanning residues 1–43, 10–23, 1–27 and 4–27 of βA4. The peptides have been tested for their ability to form filaments in vitro. Their solubilities and secondary structures in solution and in the solid state have been used to detect differences between the properties of human and rodent βA4 sequences. We show that mouse and rat βA4 homologs are as amyloidogenic as the human sequence. The absence of amyloid deposits in the brain of aged rats and mice is therefore not due to the three amino acid substitutions identified within the sequence which is homologous to βA4 of humans. Moreover, peptides corresponding to residues 1–27 of human and rodent βA4 are solubilized under physiological conditions; thus they are very unlikely to form stable filaments in vivo.
Latent fibronectin‐degrading serine proteinase activity in N‐terminal heparin‐binding domain of human plasma fibronectin
Authors: LAMBERT VIDMAR Smilja , LOTTSPEICH Friedrich , EMOD Istvan , PLANCHENAULT Thierry , KEIL‐DLOUHA Vera
Published: March 2005

The N‐terminal 70‐kDa fragment of human plasma fibronectin, purified from a cathepsin D digest, is characterized by lack of stability. It is processed proteolytically during incubation in the presence of Ca2+ into 27‐kDa N‐terminal heparin‐binding and 45‐kDa collagen‐binding domains. The N‐terminal residue in the 27‐kDa fragment was blocked as in native fibronectin. The 45‐kDa fragments began with the sequences AAVYQP, AVYQP and VYQP (residues 260, 261, 262–265 of fibronectin) that correspond to the beginning of the collagen‐binding domain. In the presence of Ca2+ the purified 27‐kDa fragment underwent further processing finally leading to the cleavage of the bond K85‐D86 and to the simultaneous appearance of a specific proteolytic activity. Inhibition studies suggests that the newly generated enzyme is a Ca2+ ‐dependent serine proteinase. Among all assayed matrix proteins, the newly generated enzyme cleaves native fibronectin and its fragments. It is proposed that this fibronectinase may originate from the N‐terminal domain of fibronectin.
Effect of calcium ions on the processing of pro‐opiomelanocortin by bovine intermediate lobe pro‐opiomelanocortin‐converting enzyme
Authors: BIRCH Nigel P. , BENNETT Hugh P. J. , ESTIVARIZ Fernando E. , LOH Y. Peng
Published: March 2005

The effect of Ca2+ on the extent and pattern of processing of pro‐opiomelanocortin and an N‐terminal fragment by a purified pituitary secretory vesicle, soluble aspartic endoprotease, was studied. Ca2+ stimulated the first cleavage of pro‐opiomelanocortin by pro‐opiomelanocortin‐converting enzyme to yield 21–23 kDa adrenocorticotropin and β‐lipotropin, but its effect was minimal. The production of adrenocorticotropin from the 21–23 kDa intermediate was stimulated approximately 2.3‐fold in the presence of 10 mM Ca2+, and processing of β‐lipotropin to β‐endorphin was stimulated about 1.3–1.4‐fold by 5–10 mM Ca2+, The production of γ‐melanotropin‐immunoreactive material from bovine N‐pro‐opiomelanocortin(1–77) was stimulated approximately 1.3‐fold at both 100 μM and 1.5–2.0 mM Ca2+. Further characterization of the γ‐melanotropin‐immunoreactive material by HPLC demonstrated that the major products were γ3‐[Lys]melanotropin and γ3‐melanotropin at both Ca2+ concentrations. These results indicate that pro‐opiomelanocortin‐converting enzyme is stimulated by Ca2+.
Transcriptional activation of early‐response genes by hydrogen peroxide in a mouse osteoblastic cell line
Authors: NOSE Kiyoshi , SHIBANUMA Motoko , KIKUCHI Keishi , KAGEYAMA Hajime , SAKIYAMA Shigeru , KUROKI Toshio
Published: March 2005

H2O2, like other oxidants, is known to act as a mitogen at low concentrations in resting Balb/3T3 or mouse epidermal JB6 cells. We described previously that H2O2 induces some early response genes in Balb/3T3 cells. We extended these observations using another cell line, MC3T3 (mouse osteoblastic) cells by examination of transcriptional activity of these genes and by using inhibitors of protein kinases. H2O2 increased the expressions of c‐fos, c‐jun, egr‐1 and JE genes which are known to be early response genes and are induced by mitogenic stimuli in many types of cells. Exogenous addition of H2O2 increased the mRNA levels of these genes, the kinetics of increase being similar to those of their inductions by a phorbol ester or serum. Nuclear run‐on transcription showed that this induction occurred at the transcriptional level. H2O2 at 0.1–0.2 mM induced maximal expressions of c‐fos and c‐jun, whereas 0.3 mM H2O2 was required for induction of stress‐induced heme oxygenase mRNA. The inductions of c‐fos and were inhibited by 50 μM H7, a protein kinase inhibitor that is relatively specific for protein kinase C, but were not affected by H9, relatively specific for cAMP‐dependent protein kinase. In cells pretreated with 12‐O‐tetradecanoylphorbol 13‐acetate, however, in which protein kinase was supposed to be downregulated, H2O2 induced c‐fos and heme oxygenase as efficiently as in untreated cells. H2O2 did not increase the phosphorylation of p80 protein, which is known to be a substrate for protein kinase C. Thus, H2O2 seemed to induce c‐fos and c‐jun by activating protein kinases distinct from protein kinase C. Activity of the chloramphenicol acetyltransferase gene under control of the serum‐response element of human c‐fos genes was increased by H2O2 treatment, whereas that under control of cAMP‐response element was not affected. These results indicate that the inductions by H2O2 of c‐fos and possibly other early response genes are mediated through activation of the serum‐response element in their enhancer.
Histone hyperacetylation is accompanied by changes in DNA topology in vivo
Authors: THOMSEN Bo , BENDIXEN Christian , WESTERGAARD Ole
Published: March 2005

The effect of histone acetylation on the topology of plasmids transfected into COS7 cells was examined. Parallel determinations of histone profiles and DNA topology showed that with increasing levels of acetylation the minichromosomal DNA is gradually relaxed. This effect could not be attributed to the increased transcriptional activity accompanying butyrate treatment since plasmids with different promoter strengths exhibited similar superhelical densities. Considering that the number of nucleosomes/minichromosome were constant under these conditions, the data suggest that in vivo histone hyperacetylation reduces the linking number change/nucleosome.
The polymerase domain of Streptococcus pneumoniae DNA polymerase I
Authors: PONS María Elena , DÍAZ Asunción , LACKS Sanford A. , LÓPEZ Paloma
Published: March 2005

The 3′‐terminal two‐thirds of the Streptococcus pneumoniae polA gene was cloned in an Escherichia coli genefusion vector with inducible expression. The resulting recombinant plasmid (pSM10) directs the hyperproduction of a polypeptide of 70.6 kDa corresponding to the C‐terminal fragment of pneumococcal DNA polymerase I. Induced cells synthesized catalytically active protein to the extent of 7% of the total soluble protein in the cells. The polymerase fragment was purified to greater than 90% homogeneity with a yield of 1.5 mg pure protein/l culture. The protein has DNA polymerase activity, but no exonuclease activity. The enzyme requires a divalent cation (MgCl2 or MnCl2) for polymerization of DNA. Comparison of the mutant and wild‐type pneumococcal polymerases shows that the construction did not affect the enzymatic affinity for the various substrates. The mutant protein, like its parent DNA polymerase I, exhibited an intermediate level of activity with primed single‐stranded DNA. At high molar ratio of enzyme/DNA substrate, the polymerase fragment catalyzes strand displacement and switching after completing the replication of a primed single‐stranded M13 DNA molecule.
The lux gene of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish
Authors: LEE Chan Yong , SZITTNER Rose B. , MEIGHEN Edward A.
Published: March 2005

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5‐kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty‐acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured.

Determination of the 7.3‐kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty‐acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem‐loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.
Microcalorimetric investigation of the interactions between the subunits of the bovine pancreatic procarboxypeptidase A‐S6 complex
Authors: MICHON Thierry , SARI Jean‐Claude , GRANON Simone , KERFELEC Brigitte , CHAPUS Catherine
Published: March 2005

A complete microcalorimetric investigation of the interactions between the native subunits of the bovine pancreatic procarboxypeptidase A‐S6 ternary complex has been performed. All the association constants and thermodynamic parameters associated with the reactions forming the various complexes have been determined.

The influence of pH and ionic strength on the binding reactions has been investigated. Interestingly, the affinity between the subunits is not significantly modified by varying the ionic strength. In this respect, an enthalpy/ entropy compensatory effect is observed for the binding of subunit III to subunit I when the ionic strength is increased, suggesting a physiological function for the association.

The various pathways for formation of the ternary complex have been studied. Binding of subunit II (or III) to subunit I, the central element of the ternary complex, does not significantly modify the affinity of the other subunit for subunit I. From a thermodynamic point of view, the same final state is obtained whatever the pathway of ternary complex formation.

This study is the first step of a kinetic investigation of the associated subunits.
Non‐random conformation of a mouse IgG2a monoclonal antibody at low pH
Authors: JISKOOT Wim , BLOEMENDAL Michael , van HAERINGEN Bart , van GRONDELLE Rienk , BEUVERY E. Coen , HERRON James N. , CROMMELIN Daan J. A.
Published: March 2005

The pH dependence of the conformation of a mouse IgG2a,K monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near‐ultraviolet and far‐ultraviolet CD, and electric‐field‐induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid‐induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16–0.18 μs) was observed for MN12 by electric‐field‐induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 μs; a further increase to 0.30 μs was observed after 24 h. The combined results suggest an acid‐induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.
Purification and characterization of a new k‐carrageenase from a marine Cytophaga‐like bacterium
Authors: POTIN Philippe , SANSEAU Alain , LE GALL Yvan , ROCHAS Cyrille , KLOAREG Bernard
Published: March 2005

A bacterial strain able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. From the cell‐free supernantant of cultures grown on crude λ‐carrageenan, a K‐carrageenase was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S 200 HR and ionexchange chromatography on DEAE–Sepharose‐CL6B. The purified k‐carrageenase was detected as a single protein upon SDS/PAGE. Its molecular mass was estimated at 40 kDa. Activity was observed against k‐carrageena over the pH range 5.0–8.5 and was optimal at pH 7.2 in Tris buffer or 7.0 in Mops buffer. The enzyme activity remained stable at 30°C, but only for up to 1h at 40°C. Analysis of the degradation products of the k‐carrageenase by gel filtration and 13C‐NMR spectroscopy indicated that the enzyme degrades K‐carrageenan down to the level of the k‐neocarratetaose sulfate. The properties of this new enzyme are compared with those of previously characterized carrageenases.
Purification and chemical modification of a phosphatidylinositol kinase from sheep brain
Authors: SCHOLZ Glen , BARRITT Greg J. , KWOK Francis
Published: March 2005

A membrane‐bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500‐fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X‐100, ammonium sulphate fractionation and a number of ion‐exchange and gel‐filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol · min−1· mg−1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 ± 10 kDa by HPLC gel filtration in the presence of Triton X‐100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 μM and for ATP 67 μM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2′(3′)‐O‐(2,4,6‐trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 μM. Enzymatic activity was found to be stimulated by Triton X‐100 but inhibited by deoxycholate.
Cloning and expression of human ciliary neurotrophic factor
Authors: NEGRO Alessandro , TOLOSANO Emanuela , SKAPER Stephen D. , MARTINI Irene , CALLEGARO Lanfranco , SILENGO Lorenzo , FIORINI Ferdinando , ALTRUDA Fiorella
Published: March 2005

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure.

Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N‐terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.
Malic enzyme in human liver
Authors: ŻELEWSKI Mateusz , ŚWIERCZYŃSKI Julian
Published: March 2005

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction.

Extramitochondrial malic enzyme has been isolated from the post‐mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography of DEAE‐cellulose, ADP–Sepharose‐4B and Sephacryl S‐300 to apparent homogenity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 μmol · min−1· mg protein−1, which corresponds to about 10000‐fold purification.

The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is tetramer composed of identical‐molecular‐mass subunits.

The isoelectric point of the isolated enzyme was 5.65.

The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction.

The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP‐linked decarboxylation reaction varied with malate concentration.

TheKm values determined at pH 7.2 for malate and NADP were 120 μM and 9.2 μM, respectively. TheKm values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 μM and 27.9 mM, respectively.

The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. TheKm values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD‐dependent decarboxylation reaction was not reversible.

The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetete and NADPH‐linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP‐linked oxidative decarboxylation of malate, respectively.

The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.
Isolation and structural characterization of an insulin‐related molecule, a predominant neuropeptide from Locusta migratoria
Authors: HETRU Charles , LI Ka Wan , BULET Philippe , LAGUEUX Marie , HOFFMANN Jules A.
Published: March 2005

Neurohaemal lobes of corpora cardiaca of Locusta migratoria are an established storage site for neurohormones produced by the neurosecretory cells of the brain. As previously reported [Hietter, H., Van Dorsselaer, A., Green, B., Denoroy, L., Hoffmann, J. A. & Luu, B. (1990) Eur. J. Biochem. 187, 241–247], the isolation and characterization of a novel 5‐kDA peptide from these lobes served as the basis for oligonucleotide screening of cDNA libraries prepared from poly(A)RNA from neurosecretory cells of the central nervous system. From subsequent cDNA cloning studies [Lagueux, M., Lwoff, L., Meister, M., Goltzené, F. & Hoffmann, J. A. (1991) Eur. J. Biochem. 187, 249–254], the existence of a 145‐residue precursor protein was deduced, which contained, in addition to the 5‐kDa peptide, amino‐acid sequences with homology to the A and B chains of an insulinrelated peptide. In the present study we have isolated the native molecule from corpora cardiaca of Locusta and characterized, by Edman degradation and plasma‐desorption mass spectrometry, the two chains as follows: A chain, Gly‐Val‐Phe‐Asp‐Glu‐Cys‐Cys‐Arg‐Lys‐Ser‐Cys‐Ser‐Ile‐Ser‐Glu‐Leu‐Gln‐Thr‐Tyr‐Cys‐Gly (Ile, isoleucine); B chain, Ser‐Gly‐Ala‐Pro‐Gln‐Pro‐Val‐Ala‐Alg‐Tyr‐Cys‐Gly‐Glu‐Lys‐Leu‐Ser‐Asn‐Ala‐Leu‐Lys‐Leu‐Val‐Cys‐Arg‐Gly‐Asn‐Tyr‐Asn‐Thr‐Met‐Phe. Taken in conjunction with the previous cloning studies, our data lead to a clear picture of the processing of Locusta preproinsulin. They indicate that locusta corpora cardiaca contain remarkably large amounts of one single insulin form, in contrast to multiple insulin isoforms of Bombyx mori, the only other insect species from which insulin‐related peptides have been isolated and characterized [Nagasawa, H., Kataoka, H., Isogai, A., Tamura, S., Suzuki, A., Mizoguchi, A., Fujiwara, Y., Suzuki, A., Takahashi, S. & Ishizaki, H. (1986) Proc. Natl Acad. Sci. USA 83, 5840–5843].
The catalytic domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex From Azotobacter vinelandii and Escherichia coli
Authors: SCHULZE Egbert , WESTPHAL Adrie H. , OBMOLOVA Galya , MATTEVI Andrea , HOL Wim G. J. , de KOK Arie
Published: March 2005

Partial sequences of the dihydrolipoyl transacetylase componenent (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over‐expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine‐proline‐rich sequence between a putative binding region for the peripheral components and the catalytic domain (apa‐4). Most of the catalytic domain from A. vinelandii without the apa‐4 sequence was degraded intracellularly, probably due to incorrect folding.

Fusion proteins of six amino acids from β‐galactosidase, the apa‐4 region and the catalytic domains of A. vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24‐subunit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild‐type E2p. This can be explained by intracellular degradation of overexpressed wild‐type E2p, whereas the catalytic domains are stable against proteolysis in vivo and in vitro.

The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose‐6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable.

Crystals of the catalytic domain of A. vinelandii E2p could be grown to a maximum size of 0.6 × 0.6 × 0.4 mm. They diffract up to a resolution of 0.28 nm.
Purification and characteriation of 3‐hydroxyphenylacetate 6‐hydroxylase: a novel FAD‐dependent monooxygenase from a Flavobacterium species
Authors: VAN BERKEL Willem J. H. , VAN DEN TWEEL Will J. J.
Published: March 2005

3‐Hydroxyphenylacetate 6‐hyroxylase was purified 70‐fold from a Flavobacterium sp. grown upon phenylactic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme.

SDS/PAGE of purified enzyme reveals a single band with a minimum molecuarl mass of 63 KDa. Analytical gel‐filtration, sedimentation‐equilibrium and sedimentation‐velocity experiments indicate that the purified enzyme exists in solultion manily as a dimer, containing 1 molecule non‐covalently bound FAD/sununit.

3‐Hydroxyphenylacetate 6‐hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3‐hydroxyphenylacetate is hydroxylated efficiently, yielding 2.5‐dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4‐dihydroxyphenylacetate and 4‐hydroxyphenylacetate are partially hydroxylated, exculsively at the 6′ (2′) positition. The physiological product 2.5‐dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3‐hydroxyphenylacetate 6‐hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate.

In the native state of enzyme, two sulfhydryl groups are accessible to 5.5′‐dithiobis(2‐nitrobenzoate). Titration with Stoichiometric amounts of either 5.5′‐dithiobis(2‐nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3‐hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol.

The N‐terminal amino acid sequence of the enzyme lacks the consenusus sequence GXGXXG, found at the termini of all faavin‐dependent external mnooxygenses sequenced so far. The amino acid composition of 3‐hydroxyphenylacetate 6‐hydroxylase is also presented.
Effect of retinoic acid on the synthesis of tissue‐type plasminogen activator and plasminogen activator inhibitor‐1 in human endothelial cells
Authors: THOMPSON E. Anne , NELLES Luc , COLLEN Désiré
Published: March 2005

The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue‐type plasminogen activator (t‐PA) and of plasminogen activator inhibitor‐1 (PAI‐1) by cultured human umbilical vein endothelial cells (HUVEC).

Retinoic acid produced a time‐ and concentration‐dependent increase in the secretion of t‐PA‐related antigen but not of PAI‐1 related antigen into the culture medium. A maximal sevenfold increase of t‐PA antigen after 24 h was observed with 10 μM and a half‐maximal increase with 0.1 μM retinoic acid. Retinoic acid induced a time‐dependent increase of the t‐PA mRNA, with a maximum at 8 h and returning to normal at 24 h.

The protein kinase inhibitor H7 decreased the t‐PA antigen induced by both retinoic acid and phorbol 12‐myristate 13‐acetate. These results suggest that treatment of HUVEC with retinoic acid increases t‐PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t‐PA synthesis in the absence of altered PAI‐1 synthesis, which may enhance the fibrinolytic potential of the endothelium.
Tricholongins BI and BII, 19‐residue peptaibols from Trichoderma longibrachiatum
Authors: REBUFFAT Sylvie , PRIGENT Yann , AUVIN‐GUETTE Catherine , BODO Bernard
Published: March 2005

Two nonadecapeptides, tricholongins BI and BII, which display antifungal and antibacterial activities, have been isolated from in vitro cultures of the fungus Trichoderma longibrachiatum. The peptides were separated by reversed‐phase HPLC; their amino acid compositions were determined by gas chromatography and their sequences by positive‐ion fast‐atom‐bombardment mass spectrometry and high‐field NMR. These linear peptides, containing mainly hydrophobic l‐amino acids, 8–9 2‐aminoisobutyric acid residues and exhibiting an acetylated N‐terminal residue and an amino alcohol C‐terminal leucinol belong to the peptaibol class. The methanol solution structure of tricholongins BI and BII has been investigated using both one‐ and two‐dimensional NMR techniques. The total 1H‐NMR and 13C‐NMR assignments are given. By a combination of the 3JNH,CαH coupling constant values, temperature coefficients of the NH and CO groups, amide hydrogen/deuterium‐exchange rate measurements and NOE data, a secondary structure for tricholongins in solution has been proposed. Both peptides adopt a similar α‐helical conformation with a hinge around Pro13 resulting from two 310 bonds. The results suggest that the N‐terminus contains mixed α/310bonds. The membrane permeability modifications induced by tricholongins have been assayed by the use of liposomes composed of egg phosphatidylcholine with 20–30% cholesterol. The peptide‐induced leakage of an entrapped fluorescent probe has been followed by fluorescence spectroscopy. In a concentration range of 0.13–0.31 μM, tricholongins induce the leakage of 50% of the entrapped material in 20 min.
1,25‐Dihydroxycholecalciferol‐related Na+/D‐glucose transport in brush‐border membrane vesicles from embryonic chick jejunum
Authors: DEBIEC Hanna , CROSS Heide S. , PETERLIK Meinrad
Published: March 2005

1,25‐Dihydroxycholecalciferol, when present at and above 10nM in an organ‐culture system of embryonic chick jejunum, approximately doubled the rate of Na+ ‐gradient‐driven D‐glucose uptake by brush‐border membrane vesicles, but had no effect on Na+ ‐independent D‐glucose transfer. The sterol also had no effet on Na+ influx along an outside/inside Na+ gradient ([Na+]o= 100mM; [Na+]i= 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na+ ‐dependent D‐glucose transport through changes in the electrochemical Na+ gradient. D‐[U‐14C] Glucose tracer exchange, measured under voltage‐clamp condition at Na+/D‐glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D‐glucose transporter in the brush‐border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D‐glucose transport through increasing the number of carrier molecules in the brush‐border membrane.

10 nM triiodothyronine, which by itself has no effect on Na+ ‐dependent D‐glucose transport, potentiated the effect of 1,25‐dihydroxycholecalciferol such that in the presence of both hormones, Na+/D‐glucose‐carrier activity was increased fourfold above control levels.
Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H‐NMR and 13C‐NMR spectroscopy
Authors: TRAN‐DINH Son , HERVE Martine , WIETZERBIN Jaime
Published: March 2005

We propose an experimental approach combining 1H‐NMR and 13C‐NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters: 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyle‐CoA and glutamate via the Krebs cycle in glucose‐grown repressed Saccharomyces cerevisiae cells fed with [1‐13C] glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2‐13C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acety‐CoA as expected, these results indicate that (a) the probability for citrate and 2‐oxoglutarte to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent.

The flux of ethanol formation from pyruvate is about three‐times and nine‐times greater than that of ethanol consumption and acetyl‐CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2+C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1.1. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data from similar experiments using unlabeled ethanol and non‐NMR techniques.
Complete sequence and model for the C1 subunit of the carotenoprotein crustacyanin, and model for the dimer, β‐crustacyanin, formed from the C1 and A2 subunits with astaxanthin
Authors: KEEN Jeffrey N. , CACERES Isabel , ELIOPOULOS Elias E. , ZAGALSKY Peter F. , FINDLAY John B. C.
Published: March 2005

The complete sequence has been determined for the C1 subunit of crustacyanin, an astaxanthin‐binding protein from the carapace of the lobster Homarus gammarus (L.). The polypeptide, 181 residues long, is similar (38% identity) to the other main subunit, A2 and to plasma retinol‐binding protein. The tertiary structure of the C1 subunit has been modelled on that derived for the A2 subunit from the coordinates of retinol‐binding protein. Residues lining the putative binding cavities and at the putative carotenoid binding sites of the two subunits are highly conserved. The carotenoid environments are characterized by a preponderance of aromatic and polar residues and the absence of charged side‐chains. A tentative model for the dimer, β‐crustacyanin, formed between the two subunits with their associated carotenoid ligands, is discussed. The model is based on the crystal structure of the dimer of bilin‐binding protein, a member of the same superfamily. This structure has enabled us to examine mechanisms for the bathochromic spectral shift of the protein‐bound carotenoid and to identify likely contact regions between dimers in octameric α‐crustacyanin.