bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence de la polymérisation de la d‐aminoacide oxidase sur le comportement de l'enzyme immobilisée sur chitosane par fixation covalente
Authors: LEMAINQUE Arnaud , BRAUN Jacques , LE GOFFIC François
Published: March 2005

Influence of polymerization of the d‐amino‐acid oxidase on the behaviour of the enzyme covalently immobilized on chitosan.

d‐Amino‐acid oxidase is a flavoprotein using FAD as cofactor. The enzyme has been immobilized in the presence of FAD on a non‐porous matrix: chitosan. This support is covalently bound to the enzyme with glutaraldehyde as cross‐linking reagent. It is characterized by a good mechanical resistance to mechanical stirring. The enzymatic assays have been performed in batch reactor with d‐phenylglycine as substrate by a spectrophotometric method which is based on the variation of the absorbance at 252 or 280 nm.

The behaviour of the biocatalysts has been studied during repeated assays of 1 h at 25 °C in the absence of exogeneous FAD. The experimental results have been compared with those obtained with the soluble enzyme tested in the presence or in the absence of FAD. The dependence of d‐amino‐acid oxidase on FAD concentration has been studied. Immobilized enzyme on chitosan appears to be less sensitive to the association‐dissociation equilibrium of FAD. This property and the capacity of the enzyme to polymerize spontaneously in solution according to the experimental conditions have been established. The fact that the enzyme can exist in various oligomeric forms is of major importance because its catalytic expression is dependent of this phenomenon. The polymerization is known to be responsible for a decrease of the maximal rate V of the enzyme. It has also been shown that in the same way this decrease was accompanied by an improvement of the affinity of enzyme for substrates. Furthermore, the value of the dissociation constant of the apoenzyme‐FAD complex is significantly smaller as the degree of polymerization is high. The conclusion is that the dissociation of the cofactor can be avoided if the immobilization step is carried out at high concentration of enzyme which is favourable to its polymerization.
The NADPH‐linked acetoacetyl‐CoA reductase from Zoogloea ramigera
Authors: PLOUX Olivier , MASAMUNE Satoru , WALSH Christopher T.
Published: March 2005

The NADPH‐linked acetoacetyl‐CoA reductase, (R)‐3‐hydroxyacyl‐CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of d‐3‐hydroxybutyryl‐CoA for poly(d‐3‐hydroxybutyrate) biosynthesis, has been purified from an over‐producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an α4 homotetramer of four 25‐kDa subunits. It has a Km of 2 μM and a kcat/Km of 1.8 × 108 M−1 s−1 for acetoacetyl‐CoA; it is inhibited by acetoacetyl‐CoA above 10 μM. Km is 10−10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride‐equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101–107], we identified a 28‐residue stretch (residues 3–31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3‐oxo‐acyl‐CoA analogs and analogs lacking the adenosine 3′,5′‐bisphosphate moiety of CoA. Only the straight‐chain C5 analog (3‐oxo‐propionyl‐CoA) was found to be an alternative substrate (40%) for the reductase.
Structural analysis of O‐glycosidic type of sialyloligosaccharide‐alditols derived from urinary glycopeptides of a sialidosis patient
Authors: VAN PELT Johannes , VAN BILSEN Daniëlle G. J. L. , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

Sialidosis urine was fractionated by gel filtration on Bio‐Gel P‐6. All pooled fractions containing carbohydrates showed the presence of small amounts of GalNAc in non‐reducing position, besides free N‐acetyllactosamine type of oligosaccharides as major constituents. The fractions were subjected to reductive alkaline borohydride degradation, after which the major part of GalNAc was recovered as N‐acetyl‐d‐galactosaminitol (GalNAc‐ol). The GalNAc‐ol‐containing material was separated from the N‐glycosidic oligosaccharides by a second gel‐filtration step on AcA 202. Subsequently, the O‐glycosidic sialyloligosaccharide‐alditols were subfractionated by anion‐exchange chromatography on Mono Q. Structural analysis by 500‐MHz 1H‐NMR spectroscopy revealed two major components in all fractions, namely: NeuAcα2‐3Galβ1‐3GalNAc‐ol and NeuAcα2‐3Galβ1‐3[NeuAcα2‐6]GalNAc‐ol. Furthermore, NeuAcα2‐3Galβ1‐3[NeuAcα2‐3Galβ1‐4GlcNAcβ1‐6]GalNAc‐ol was found as a minor component in some of the fractions. The presence of these carbohydrate chains in Bio‐Gel fractions differing in molecular mass suggested that they are derived from glycopeptides which are heterogeneous in their peptide part.
Protein‐kinase‐C‐catalyzed phosphorylation of the microtubule‐binding domain of microtubule‐associated protein 2 inhibits its ability to induce tubulin polymerization
Authors: HOSHI Minako , AKIYAMA Tetsu , SHINOHARA Yukiko , MIYATA Yoshihiko , OGAWARA Hiroshi , NISHIDA Eisuke , SAKAI Hikoichi
Published: March 2005

It has previously been demonstrated that microtubule‐associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, C. T., Huang, K.‐P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110–4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648–15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with α‐chymotrypsin; the microtubule‐binding and the non‐binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50–60% of the incorporated phosphates being detected in the microtubule‐binding domain. Polypeptide fragments, containing the microtubule‐binding domain of MAP2 were purified by DEAE‐cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule‐binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule‐binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule‐binding domain catalyzed by protein kinase C.
Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens
Authors: HOOGVLIET Johan C. , LIEVENSE Lou C. , van DIJK Cees , VEEGER Cees
Published: March 2005

The electron transfer kinetics between the hydrogenase from Desulvovibrio vulgaris (strain Hildenborough) and three different viologen mediators has been investigated by cyclic voltammetry. The mediators methyl viologen, di(n‐aminopropyl) viologen and propyl viologen sulfonate differ in redox potential and in net charge. Dependent on the pH both the one‐ and two‐electron‐reduced forms or only the two‐electron‐reduced form of the viologens are effective in electron exchange with hydrogenase. Calculations of the second‐order rate constant k for the reaction between reduced viologen and hydrogenase are based on the theory of the simplest electrocatalytic mechanism. Values for k are in the range of 106–107 M−1 s−1 and increase in the direction propyl viologen sulfonate → methyl viologen → di(n‐aminopropyl) viologen. An explanation is based on electrostatic interactions. It is proposed that the electron transfer reaction is the rate‐determining step in the catalytic mechanism.
Differential acetylation of core histones in rat cerebral cortex neurons during development and aging
Authors: PIÑA Benjamín , MARTÍNEZ Pedro , SUAU Pedro
Published: March 2005

Core histones can be modified by aceylation and this modification has been correlated with the modulation of chromatin condensation and histone deposition. We have now studied the levels of acetylation of the core histones in rat brain cortical neurons from the middle of the period of neuronal proliferation through postnatal development and aging. The results show that the level of acetylation of H4 decreases with age. The kinetics of H4 deacetylation show a perinatal fast phase followed by a much slower phase that spans the rest of the period examined. H4 deacetylation is accounted for by the decrease of the monoacetylated species, the proportions of the more highly acetylated species remaining essentially constant. By contrast to histone H4, the overall levels of acetylation and the proportions of the different acetylated species of H2A, H2B and H3 remain unchanged throughout the period examined. Furthermore, the variants belonging to a given histone class always show the same level of acetylation. The fact that in neurons the level of monoacetylated H4 decreases during development and aging, in sharp contrast with the constancy of the levels of all other acetylated histone species, raises the possibility that in interphase chromatin monoacetylated H4 may have a central role in the modulation of chromatin structure. The results also suggest that the slow decrease of the proportion of monoacetylated H4 may imply a gradual loss of chromatin structural plasticity and thus lead to aging.
Regulation of two interferon‐inducible human genes by interferon, poly(rI) · poly(rC) and viruses
Authors: WATHELET Marc G. , CLAUSS Isabelle M. , CONTENT Jean , HUEZ Georges A.
Published: March 2005

The IFI‐56K and IFI‐54K genes are transcriptionally stimulated when cells are treated by interferon. We have previously shown that the IFI‐56K gene is in addition directly induced by poly(rI) · poly(rC), and inducer of interferon‐β. Since the regulation of the IFI‐56K and IFI‐54K genes by interferon are very much alike, we tested whether the IFI‐54K gene is also directly regulated by poly(rI) · poly(rC). Treatment of various cell lines with poly(rI) · poly(rC) leads to a clear accumulation of the IFI‐54K mRNA to a level which sometimes even exceeds that obtained with high doses of interferon. Several interferon‐resistant cell lines were investigated for the inducibility of both the IFI‐56K and IFI‐54K genes by interferons, poly(rI) · poly(rC) and viruses (which are the natural inducers of interferon‐α and ‐β). Both genes appear to be coordinately regulated by these inducers. It was thus interesting to search for common regulatory element(s) in the control region of these two genes. The IFI‐54K gene promoter region was isolated, from which a 520‐base‐pair segment was sequenced and compared with the promoter region of the IFI‐56K gene that we had previously sequenced. The only homology was found is a well conserved 19‐bp segment located just upstream of the TATA box of these genes; interestingly, this sequence is also homologous to the minimal region needed for the inducibility by poly(rI) · poly(rC) of the interferon‐β gene. This conserved sequence might be responsible for the coordinate induction of the IFI‐56K and IFI‐54K genes by interferon, poly(rI) · poly(rC) and viruses.
Kinetic and spectral studies on the redox forms of methanol dehydrogenase from Hyphomicrobium X
Authors: FRANK Jzn Johannes , DIJKSTRA Maarten , DUINE Johannis A. , BALNY Claude
Published: March 2005

Several reaction rate constants in the catalytic cycle of methanol dehydrogenase (EC 1.1.99.8) in vitro were determined with stopped‐flow spectrophotometry. The studies revealed that the high pH required for adequate activity of the enzyme is related to the strong pH dependency of the oxidation rates of the reduced and semiquinone enzyme forms, MDHred and MDHsem, with the artificial electron acceptor Wurster's blue. The rate‐limiting step in the catalytic cycle is associated with the conversion of oxidized enzyme‐substrate complex (MDHox· s) into reduced enzyme (MDHred) and product. The effect of activator (ammonium salts) was also confined to this step, but even saturating concentrations were unable to remove the limitation completely. Making use of the large deuterium isotope effect associated with substrate oxidation in the absence of activator, the transient MDHox· C2H3OH complex could be isolated and its slow decomposition into MDHred and formaldehyde could be demonstrated. Further evidence is presented to support the view that the different absorption spectra observed originate from genuine redox forms of methanol dehydrogenase with different redox states of the cofactor PQQ and not from enzyme‐electron acceptor complexes. The results confirm and extend our original view on the mechanism of action and contradict the mechanism proposed by others [Parkes, C. & Abeles, R. H. (1984) Biochemistry 23, 6355–6363].
Isolation, structure and expression of cDNA and genomic clones for murine eosinophil differentiation factor
Authors: CAMPBELL Hugh D. , SANDERSON Colin J. , WANG Yao , HORT Yvonne , MARTINSON Mary E. , TUCKER William Q. J. , STELLWAGEN Anne , STRATH Malcolm , YOUNG Ian G.
Published: March 2005

Eosinophil differentiation factor (EDF) is a recently described regulator affecting eosinophil growth and activation. cDNA clones for murine EDF were isolated by direct expression from libraries prepared from the T cell hybrid NIMP‐TH1. The longest cDNA clone obtained was 1534 bp in length encoding a polypeptide of 133 amino acids. Two variant cDNAs suggesting alternative RNA processing events were detected. The EDF gene was cloned from a genomic λ library and a region of 6727 bp encompassing the gene was sequenced. The gene contains three introns 829, 1875 and 79 bases in length and has numerous repetitive sequences. A common, possible regulatory element, including a conserved decamer, lies adjacent to the TATA boxes of the EDF and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) genes and similar sequences are present in some other lymphokine genes. Recombinant EDF produced in monkey COS cells strongly stimulated the eosinophil lineage and also showed B‐cell‐growth factor II (BCGFII) activity whereas recombinant murine interleukin‐3 and GM‐CSF showed much broader activity towards the different myeloid lineages, were less active on eosinophils and had no BCGFII activity. The BCGFII activity of recombinant EDF together with a comparison of the BCGFII (interleukin‐5) cDNA sequence with that of the EDF cDNA establishes that these two activities are the properties of a single polypeptide.
Cross‐linking of α and γ‐thrombin to distinct binding sites on human platelets
Authors: JANDROT‐PERRUS Martine , DIDRY Dominique , GUILLIN Marie‐Claude , NURDEN Alan T.
Published: March 2005

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross‐linking with the membrane‐impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross‐linking of membrane glycoproteins. The proteins covalently linked to 125I‐labelled α and γ‐thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis.

125I‐α‐thrombin was detected in high‐molecular‐mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr∼ 400000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78‐kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active α‐thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine‐protease complex. The 240‐kDa complex was formed with 0.8‐100 nM α‐thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl‐inactivated α‐thrombin. After analysis of Triton‐X‐100‐soluble extracts of cross‐linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I‐α‐thrombin. These were a precipitate containing GPIIb‐IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240‐kDa complex. Significantly, 125I‐γ‐thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240‐kDa and 78‐kDa molecular mass range were not observed.

We conclude that several binding sites for α‐thrombin are present at the platelet surface, and that GPIb is one of them. The studies with γ‐thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.
The stereochemical course of d‐glyceraldehyde‐induced ATPase activity of glycerokinase from Escherichia coli
Authors: BETHELL Richard C. , LOWE Gordon
Published: March 2005

d‐Glyceraldehyde‐induced hydrolysis of adenosine (R)‐5′‐[γ‐17O,18O,thio]triphosphate catalysed by glycerokinase from Escherichia coli gives inorganic [16O,17O,18O]thiophosphate with the (S)‐configuration, showing that the reaction proceeds with inversion of configuration at phosphorus. This result provides powerful support for the chemically most plausible mechanism, namely, that the hydrate of d‐glyceraldehyde is the effective substrate which after phosphorylation or thiophosphorylation eliminates inorganic phosphate or inorganic thiophosphate, respectively, with regeneration of d‐glyceraldehyde.
In vivo and in vitro evidence for a proton leakage through the inner mitochondrial membrane in a mutant of Saccharomyces cerevisiae
Authors: MANON Stephen , RAKOTOMANANA Félicien , GUÉRIN Martine
Published: March 2005

Mutants of Saccharomyces cerevisiae were isolated which supported three mutations: two unlinked chromosomic mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial mutation conferring paromomycin sensitivity. When studied on isolated mitochondria, these mutants exhibited low phosphorylation efficiency and great proton permeability of their inner mitochondrial membrane. Experiments were carried out on whole cells: determination of growth rates, cellular yields and cellular respiration, either in the presence of triethyltin, an ATP synthase inhibitor, or in the presence of uncoupler, demonstrating that the proton leakage is actually a physiological phenomenon linked to the cold‐sensitive phenotype. Experiments performed on isolated mitochondria confirmed the existence of such a proton leakage.
The sheep metallothionein gene family
Authors: PETERSON M. Gregory , HANNAN Frances , MERCER Julian F. B.
Published: March 2005

Southern blot analysis of the sheep genome revealed a metallothionein gene family with at least nine members. Two overlapping cosmid clones spanning approximately 67 kb and containing five metallothionein genes have been isolated. DNA sequence analysis reveals that one of these is a metallothionein I1 variant, three are metallothionein I variants and one is a truncated metallothionein pseudogene containing only the first exon. The predicted amino acid sequence was compared with previously reported amino acid composition data of sheep metallothioneins [Whanger, P. D., Oh, S.‐H. & Deagen, J. T. (1981) J. Nutr. 111, 1207–1215], and this suggests that we have isolated the genes encoding the major protein isoforms found in the sheep liver. The promoter regions of these genes contain many conserved elements, among them metal‐regulatory elements and putative glucocorticoid‐responsive elements. However, there are a number of differences between these genes in the arrangement of these elements. Sequence comparisons indicate that the multiple metallothionein I genes and the pseudogene appear to have resulted from sequential duplication events, and a larger cluster of metallothionein I genes may have been disrupted leading to the formation of the pseudogene.
Differential expression of four linked sheep metallothionein genes
Authors: PETERSON M. Gregory , MERCER Julian F. B.
Published: March 2005

Regulation of the closely linked endogenous sheep MT‐Ia, MT‐Ib, MT‐Ic and MT‐II genes by heavy metals and dexamethasone was studied in cultured sheep fibroblasts. Only MT‐II mRNA was detectable before addition of any inducer. Addition of copper, zinc or cadmium salts to the culture medium increased the level of each mRNA; however, the magnitude of this response varied greatly between the four metallothionein genes. Following induction, levels of MT‐Ia mRNA were the highest, followed by MT‐II and MT‐Ic mRNAs. The MT‐Ib mRNA was only present at low levels. Zinc and cadmium were found to be the most effective inducers of each gene. The maximal response of the sMT‐Ia, Ib and II genes to copper was only 30% of zinc and cadmium. The sMT‐Ic gene responded very weakly to copper, < 5% of the levels achieved with zinc. Only the MT‐II mRNA increased in response to dexamethasone. In the liver of sheep on normal diets, the levels of MT‐Ia, Ic and II mRNAs were found to be unexpectedly high and comparable to induced levels in fibroblasts.
Purification and properties of 4‐hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum
Authors: OBATA Hitoshi , UEBAYASI Masami , KANEDA Toshi
Published: March 2005

4‐Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous state by ammonium sulfate precipitation and chromatography on DEAE‐650s, agarose‐NAD and hydroxyapatite. The enzyme consisted of two identical subunits and had a native relative molecular mass of 53600. There were two residues each of cysteine and tryptophan in the enzyme molecule. Oxo acid rather than hydroxy acid was routinely used as substrate for assay of the enzyme. The enzyme is highly specific for 4‐oxocyclohexanecarboxylic acid: the carboxyl group is essential and the position of carbonyl group is important; neither the 2‐oxo nor the 3‐oxo homologue was used as substrate. A methyl substitution on the ring of 4‐oxocyclohexanecarboxylate resulted in an almost complete loss of its activity. The reduction product was identified as trans‐4‐hydroxycyclohexanecarboxylic acid by gas‐liquid chromatography and mass spectrometry. It was used as a substrate for the reverse reaction in the presence of NAD but not its cis‐isomer. The enzyme was specific for the B‐side (pro‐S) hydrogen of NADH in the hydrogen transfer from NADH to 4‐oxocyclohexanecarboxylate. The Km, values for 4‐oxocyclohexanecarboxylate and NADH in the reduction reaction at pH 6.8 were 0.50 mM and 0.28 mM, respectively, whereas those for trans‐4 hydroxycyclohexanecarboxylate and NAD in the oxidation reaction at pH 8.8 were 0.51 mM and 0.23 mM, respectively. The equilibrium constant of the reaction was 1.79 × 10‐10M. The enzyme was strongly inhibited by N‐bromosuccinimide.
Purification and characterization of cytosolic protein‐tyrosine kinase from bovine platelets
Authors: NAKAMURA Shun‐ichi , YANAGI Shigeru , YAMAMURA Hirohei
Published: March 2005

A cytosolic protein‐tyrosine kinase has been highly purified from bovine platelets using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatography on phosphocellulose, Sephacryl S‐200, poly(l‐lysine)‐agarose, casein‐Sepharose 4B and 2′,5′‐ADP‐Sepharose 4B. Analysis of the most highly purified preparations by SDS/polyacrylamide gel electrophoresis revealed a major silver‐stained band of molecular mass 71 kDa. This molecular mass was consistent with results obtained from sucrose density gradient centrifugation, indicating that the enzyme exists as a monomer. The purified kinase, called CPTK 71, efficiently phosphorylated tubulin and p36 (calpactin 1 heavy chain). However, it did not phosphorylate H1 histone. Half‐maximal enzyme activity was observed at 2.2 μM ATP, and Mn2+, Co2+ and Mg2+ were effective divalent metal ions for the expression of activity. Insulin, epidermal growth factor, and platelet‐derived growth factor had little or no effect on the kinase activity of CPTK 71. CPTK 71 had no immunological cross‐reactivity with pp60src. These results suggest that CPTK 71 is a novel type of protein‐tyrosine kinases among the enzymes so far reported.
Effects of the Xenopus laevis mitochondrial single‐stranded DNA‐binding protein on the activity of DNA polymerase γ
Authors: MIGNOTTE Bernard , MARSAULT Janine , BARAT‐GUERIDE Monique
Published: March 2005

The single‐stranded DNA‐binding protein from Xenopus laevis oocyte mitochondria, which has been found associated with the D loop, binds to ssDNA in stoichiometric amounts and can under certain conditions stimulate the activity of the DNA polymerase γ. Its properties suggest that it is involved in strand displacement during the replication of the mitochondrial genome.
Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli
Authors: HEILMANN Hans J. , MÄGERT Hans J. , GASSEN Hans G.
Published: March 2005

A gene encoding glucose dehydrogenase of Bacillus megaterium M1286 was isolated from a λ‐EMBL3 phage library. It is transcribed and translated in cells of the heterologous organism Escherichia coli by own control regions. The gene is located on a 1126‐bp HindIII fragment. Its nucleotide sequence contains 220 bp in the 5′ non‐coding region, 783 bp in the coding region and 123 bp in the 3′ non‐coding region. The amino acid sequence, as deduced from the coding region, consists of 261 amino acids and is different from the known protein sequence of glucose dehydrogenase from B. megaterium M1286. [Jany, K. D., Ulmer, W., Fröschle, M. & Pfleiderer, G. (1984) FEBS Lett. 165, 6–10]. By using this gene as a hybridization probe a second glucose dehydrogenase gene was isolated, which was also directly expressed in E. coli. Additionally a DNA region with extended sequence homology to the hybridization probe was identified. This work indicates the existence of at least two independent glucose dehydrogenase genes in B. megaterium M1286. Homologies in the primary structures of the two different glucose dehydrogenases of B. megaterium M1286 and of the corresponding Bacillus subtilis enzyme are discussed.
Cloning and expression of a β tubulin gene of Physarum polycephalum
Authors: WERENSKIOLD Anne K. , POETSCH Bettina , HAUGLI Finn
Published: March 2005

A β tubulin gene of Physarum polycephalum has been isolated from a genomic library in the phage EMBL4. Southern‐blot hybridization to genomic DNA indicates that the cloned DNA is derived from the betB1 locus of the β tubulin gene family. A tubulin‐specific subfragment of the phage DNA was used as a hybridization probe to construct a restriction map of the betB1 locus. The probe consisted of the almost complete coding region of the 5′ half of the tubulin gene, interrupted by one intron. The derived amino acid sequence of this subclone deviates from the protein sequence for Physarum amoebal β tubulin (amino acids 4–207) in two of 207 amino acids. We used both recA and recBC sbcB bacterial host strains, which have been recommended for cloning of instability‐conferring sequences of the Physarum genome, but were unable to subclone the 3′ part of the gene from the phage DNA. Primer‐extension analysis indicates that the betB gene is expressed in the vegetatively proliferating amoebal and plasmodial stages of the life cycle as well as in differentiating (sporulating) plasmodia.
Selective distinction at equilibrium between the two α‐neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration
Authors: MARCHOT Pascale , FRACHON Paule , BOUGIS Pierre E.
Published: March 2005

The binding of the monoiodinated α‐neurotoxin I from Naja mossambica mossambica to the membrane‐bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar‐sensitive microtitration assay. From equilibrium binding studies a non‐linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1= 7 ± 4 pM and Kd2= 51 ± 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k−1.1= 25 × 10−6 s−1 and k−1.2= 623 × 10−6 s−1 respectively), but not in their rate of formation of the toxin‐receptor complex (k+1, = 11.7 × 106 M−1 s−1). From these rate constants the same two values of dissociation constant were deduced (Kd1= 2 pM and Kd2= 53 pM). All the specific binding was prevented by the cholinergic antagonists α‐bungarotoxin and d‐tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d‐tubocurarine sites (Kda= 103 nM and Kdb= 13.7 μM respectively). Evidence is provided indicating that these two sites are shared by d‐tubocurarine and α‐neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high‐affinity binding sites for this short α‐neurotoxin are selectively distinguishable.
The control of glycogen metabolism in yeast
Authors: FRANÇOIS Jean , VILLANUEVA Maria Elena , HERS Henri‐Géry
Published: March 2005

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6‐monophosphate and of fructose 2,6‐bisphosphate. The activity of glycogen synthase increased about 4‐fold and that of glycogen phosphorylase decreased 3–5‐fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6‐phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26°C, but were much more pronounced at the nonpermissive temperature of 35°C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate‐cyclase‐deficient mutant (cyr1), able to incorporate exogenous cyclic AMP.

When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic‐AMP‐dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6‐bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6‐bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35°C without affecting cyclic AMP concentration.
The control of glycogen metabolism in yeast
Authors: FRANÇOIS Jean , HERS Henri‐Géry
Published: March 2005

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate‐cyclase‐deficient mutant that had been preincubated at the restrictive temperature of 35°C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20‐fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6‐phosphate, the two forms of enzyme had a similar affinity for UDP‐Glc but differed essentially by their Vmax. Glucose 6‐phosphate had no effect on synthase a, but increased both Vmax and Km, of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km, of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20‐fold more active than synthase b.

When an extract of cells that had been preincubated in the absence of glucose was gel‐filtered and then incubated at 30°C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6‐phosphate. When a gel‐filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30°C in the presence of ATP‐Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6‐phosphate and only slightly and irregularly stimulated by cyclic AMP.
Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+‐dependent phospholipid‐binding protein
Authors: MAURER‐FOGY Ingrid , REUTELINGSPERGER Chris P. M. , PIETERS Jean , BODO Gerhard , STRATOWA Christian , HAUPTMANN Rudolf
Published: March 2005

Based on sequence information from tryptic peptides an almost full‐size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced. The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels. The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays.
On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron‐transfer reactions
Authors: JACQUOT Jean‐Pierre , SUZUKI Akira , PEYRE Jean‐Baptiste , PEYRONNET Rémi , MIGINIAC‐MASLOW Myroslawa , GADAL Pierre
Published: March 2005

Spinach leaf ferredoxin and ferredoxin: NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin‐NADP reductase and adrenodoxin‐NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin‐NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100‐fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin‐Sepharose or ferredoxin‐NADP‐reductase–Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP‐dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.
Topogenic signals in integral membrane proteins
Authors: von HEIJNE Gunnar , GAVEL Ylva
Published: March 2005

Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane‐assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner‐membrane proteins, but also for eukaryotic proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive‐inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.
Post‐translational translocation of polypeptides across the mammalian endoplasmic reticulum membrane is size and ribosome dependent
Authors: ROITSCH T. , LEHLE L.
Published: March 2005

The translation and translocation of two yeast glycoproteins, invertase and carboxypeptidase Y, were studied in a heterologous cell‐free translation system from reticulocytes supplemented with dog pancreas microsomes. Using in vitro synthesized mRNA transcripts, encoding complete or truncated invertase forms, the influence of polypeptide size and ribosome dependence was studied. It was found that C‐terminal truncated fragments of 25 kDa, i.e. a size larger than the average size of a domain structure, are translocated and processed post‐translationally with a similar efficiency to the cotranslational events. Post‐translational import decreases with increasing peptide chain, mature polypeptide (60 kDa) being no longer translocated. Post‐translational competence is only maintained as long as the peptide remains associated with ribosomes. Translocation of invertase depends on the presence of the leader peptide and requires energy independent of protein synthesis. Size dependence of post‐translational import could also be demonstrated for carboxypeptidase Y.
Potential role for prostaglandin F2α, D2, E2 and thromboxane A2 in mediating the metabolic and hemodynamic actions of sympathetic nerves in perfused rat liver
Authors: IWAI Masaru , GARDEMANN Andreas , PÜSCHEL Gerhard , JUNGERMANN Kurt
Published: March 2005

In isolated rat liver perfused at constant pressure perivascular nerve stimulation caused an increase of glucose and lactate output and a reduction of perfusion flow. The metabolic and hemodynamic nerve effects could be inhibited by inhibitors of prostanoid synthesis, which led to the suggestion that the effects of nerve stimulation were, at least partially, mediated by prostanoids [Iwai, M. & Jungermann, K. (1987) FEBS Lett. 221, 155–1601. This suggestion is corroborated by the present study.

Prostaglandin D2, E2 and F2α as well as the thromboxane A2 analogue U46619 enhanced glucose and lactate release and lowered perfusion flow similar to nerve stimulation.

The extents, the kinetics and the concentration dependencies of the metabolic and hemodynamic actions of the various prostanoids were different. Prostaglandin F2α and D2 caused relatively stronger changes of metabolism, while prostaglandin E2 and U46619 had stronger effects on hemodynamics. Prostaglandin F2α, elicited greater maximal alterations than D2 with similar half‐maximally effective concentrations. Prostaglandin F2α mimicked the nerve actions on both metabolism and hemodynamics best with respect to the relative extents and the kinetics of the alterations.

The hemodynamic effects of prostaglandin F2α could be prevented completely by the calcium antagonist nifedipine without impairing the metabolic actions of the prostanoid. Apparently, prostaglandin F2α influenced metabolism directly rather than indirectly via hemodynamic changes.

The present results, together with the previously described effects of prostanoid synthesis inhibitors, suggest that prostanoids, probably prostaglandin F2α and/or D2, could be involved in the actions of sympathetic hepatic nerves on liver carbohydrate metabolism. Since prostanoids are synthesized only in non‐parenchymal cells, nervous control of metabolism appears to depend on complex intra‐organ cell‐cell interactions between the nerve, non‐parenchymal and parenchymal cells.
Excess antisense RNA from infectious recombinant SV40 fails to inhibit expression of a transfected, interferon‐inducible gene
Authors: KERR Shona M. , STARK George R. , KERR Ian M.
Published: March 2005

SV40‐based infectious virus constructs were used to produce a high copy number of full‐length antisense RNA in essentially every cell in a population. Chloramphenicol acetyltransferase (CAT) cDNA was placed in either the sense or antisense orientation relative to the SV40 early promoter in helper‐free recombinant virus. RNA synthesised at high levels from the antisense virus was without effect on the expression of a stably‐transfected CAT mini‐gene controlled by an interferon‐inducible promoter in monkey CV1 and large T antigen‐expressing tsCOS cells. In double infection experiments the antisense RNA was similarly without effect on expression from CAT cDNA placed in the sense orientation in a second virus vector. No activation of the ppp(A2'p)nA(n≥ 2) system was observed after interferon treatment in either type of experiment. There was no evidence, therefore, for the formation of double‐stranded (ds)RNA. It can be concluded that a large excess of a full‐length antisense RNA is not necessarily sufficient to cause inhibition of gene expression even when interferon treatment is used to enhance any effect of dsRNA.
Physiological stresses inhibit guanine‐nucleotide‐exchange factor in Ehrlich cells
Authors: ROWLANDS Anne G. , MONTINE Kathleen S. , HENSHAW Edgar C. , PANNIERS Richard
Published: March 2005

Previously, we have shown that phosphorylation of the eukaryotic initiation factor eIF‐2α increases under several physiological stresses in which protein synthesis is inhibited in Ehrlich ascites tumor cells. As phosphorylated eIF‐2 [eIF‐2(αP)] is a potent inhibitor of guanine nucleotide exchange factor (GEF), it seemed likely that it was responsible for the inhibition. We have assayed GEF activity levels in extracts prepared from Ehrlich cells exposed to three such stresses, namely heat shock, serum deprivation and glutamine deprivation. Activity was estimated by the ability of GEF to enhance the release of [α‐32P]GDP from purified eIF‐2 [a modification of the reticulocyte lysate assay of Matts, R. L. & London, I. M. (1984) J. Biol. Chem. 259, 6708]. GEF activity was reduced from control values in extracts of heat‐shocked cells and serum‐deprived cells, concomitant with increased eIF‐2α phosphorylation. Inhibition of GEF activity in heat‐shocked and serum‐deprived cells was reversed to control levels by increasing the concentration of purified eIF‐2. GDP added as substrate in the GEF assay. Since we have shown elsewhere that eIF‐2(αP). GDP inhibits GEF by competition with eIF‐2. GDP, the complete reversal of inhibition of GEF activity in heat‐shocked and serum‐deprived cells indicates that inhibition is due solely to phosphorylation of eIF‐2α. In glutamine‐deprived cells phosphorylation of eIF‐2α was increased modestly and GEF activity was reduced but GEF activity could not be fully reversed by addition of eIF‐2 GDP, suggesting that GEF may also be regulated in other ways.

There are greater amounts of GEF relative to eIF‐2 in Ehrlich cells (approximately 50%) compared with rabbit reticulocytes (approximately 20%). This explains the efficient rates of protein synthesis in control Ehrlich cells even though they have 30% of their eIF‐2 phosphorylated which is enough to inhibit GEF and initiation in reticulocytes completely but only enough to trap approximately 60% of the GEF in Ehrlich cells.
1H‐NMR assignment and folding of the isolated ribonuclease 21–42 fragment
Authors: JIMÉNEZ M. Angeles , RICO Manuel , HERRANZ José , SANTORO Jorge , NIETO José L.
Published: March 2005

We show in this paper that the isolated bovine ribonuclease 21–42 fragment is able to adopt in water solution a measurable population (14% at 22°C, pH 5.4) of a native‐like α‐helical structure. Strong support for this conclusion is given by the analysis of CD data and 1H chemical shift variations with the temperature and the addition of stabilizing (trifluoroethanol) and denaturing (urea) agents. This result gives experimental support to the idea that native isolated secondary structure elements (at least α helices) are, as a rule, partially stable in solution and therefore they can act as independent protein‐folding nucleation centers.
Amino acid sequences of hemoglobins I and II from root nodules of the non‐leguminous Parasponia rigida‐rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii
Authors: KORTT Alexander A. , TRINICK Michael J. , APPLEBY Cyril A.
Published: March 2005

The amino acid sequence of hemoglobins I (PI 6.15 as oxyhemoglobin) and II (PI 5.64 as oxyhemoglobin) from the nitrogen‐fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (PI 6.16, as oxyhemoglobin) from Parasponia andersonii was re‐examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr∼ 18700 including the heme) with a single cysteine residue and five methionine residues. The N‐terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microhetereogeneity at position 30 (Asp/Glu substitution) and hemoglobin I fom P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.