bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomal Localisation, Inducibility, Tissue‐Specific Expression and Strain Differences in Three Murine Peroxisome‐Proliferator‐Activated‐Receptor Genes
Authors: Jones Paul S. , Savory Richard , Barratt Paula , Bell Alex R. , Gray Tim J. B. , Jenkins Nancy A. , Gilbert Debra J. , Copeland Neal G. , Bell David R.
Published: September 2004

Three murine peroxisome‐proliferator‐activated‐receptor (PPAR) genes were localised to chromosome 15 (PPARα), chromosome 17 (PPARβ) and chromosome 6 (PPARγ). The expression of the three PPAR RNAs was determined using a specific RNase protection assay. In liver RNA, PPARα was expressed at the highest level, with 20‐fold lower levels of PPARβ, and very low levels of PPARγ. The three PPAR RNAs showed no sex‐specific differences in expression, and the levels of these transcripts were unaffected by treatment of mice with testosterone or the potent peroxisome proliferator, methylclofenapate. In agreement with this data, the level of PPARα protein in liver was unchanged after treatment of mice with methylclofenapate. Investigation of the tissue‐specific distribution revealed that the PPARα RNA was expressed at highest levels in liver, to moderate levels in kidney and brown adipose tissue, and at low levels elsewhere. PPARβ was expressed at moderate levels in liver, and lower levels in other tissues, including brown adipose tissue. In contrast, PPARγ RNA was expressed at low levels in liver or epididymal white adipose tissue and at very low levels elsewhere, but was expressed at high levels in brown adipose tissue. The tissue distribution of these receptors suggests an important role in lipid metabolism and toxicity for individual members of the PPAR family. The expression of PPARα and PPARβ RNAs was examined in 13 strains of mice, and the levels of expression varied within a fourfold range. Polymorphism in the size of PPARα RNA from Swiss‐Webster mice was detected, and shown to be due to a 2‐bp mutation in the 3′ non‐coding region of PPARα in Swiss Webster mice.
Characterization of a cDNA Encoding a Cytosolic Peptidylprolyl Cis‐Trans ‐Isomerase from Blattella Germanica
Authors: Martinez‐Gonzalez José , Hegardt Fausto G.
Published: September 2004

Cyclophilins are an abundant and ubiquitous class of proteins first identified by their high affinity for the immunosuppressive drug cyclosporin A. Cyclophilins have peptidylprolyl cis/trans ‐isomerase activity in vitro, and thus may be involved in protein folding and trafficking in vivo.

In this study, we report the cloning and characterization of a Blattella germanica cyclophilin cDNA. Analysis of this 846‐bp cDNA reveals an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular mass of 17934 Da. This B. germanica cyclophilin shares a central peptidylprolyl cis/trans ‐isomerase and a cyclosporin‐A‐binding domain with other cyclophilin sequences. The B. germanica cyclophilin amino acid sequence shares 83% identity with the cytosolic cyclophilin isoform from Drosophila melanogaster (Cyp‐1). This identity suggests that B. germanica cyclophilin is a member of the cytosolic cyclophilin A (CyPA) family.

From the alignment of cyclophilin sequences, we have found that 62 residues (positional identity of 40%) have remained invariant in eukaryotes for more than 1 billion years of divergence. We calculated a unit evolutionary period of 30.9 million years for the cytosolic isoform.

Northern‐blot analyses show that B. germanica CyPA mRNA is abundant, and present in all insect organs tested. The highest values for B. germanica cyclophilin mRNA tissue content were found in 6‐day‐old ovary, followed by brain, testis, and gut (15–30% the content of ovary). The muscle, fat body, and colleterial gland contained the lowest cyclophilin mRNA level (1–5% the content of ovary). There is a developmental pattern of gene expression affecting the embryo stages. These results suggest that this ubiquitously expressed B. germanica cyclophilin is subject to a differential regulation in tissues and during development. Southern‐blot analysis of B. germanica DNA shows that only one copy of the CyPA gene is present per genome, whereas at least 20 genes or pseudogenes were detected in the mammalian genome.
Involvement of Phosphorylase Kinase Inhibition in the Effect of Resorcinol and Proglycosyn on Glycogen Metabolism in the Liver
Authors: Van Schaftingen Emile
Published: September 2004

The purpose of this study was to identify the mechanism by which proglycosyn and resorcinol decrease the phosphorylase a content and the fructose 2,6‐bisphosphate concentration in isolated hepatocytes. The intracellular concentrations of the glucuronide derivatives of proglycosyn and resorcinol have been measured by HPLC in hepatocytes incubated for 5 min or 30 min with different concentrations of these agents. At both times, there was a reciprocal relationship between the phosphorylase a content and the intracellular concentration of the glucuronidated metabolites, half‐maximal inactivation being observed at about 2 μmol/g protein and 0.25 μmol/g protein for resorcinylglucuronide and proglycosyn‐glucuronide, respectively. Glycogen synthase was not significantly activated by these agents after 5 min but was well activated after 30 min.

Preincubation of hepatocytes with 1 mM resorcinol or with 100 μM proglycosyn resulted in a decrease in the rate at which phosphorylase was activated following the addition of glucagon, vasopressin, the protein phosphatase inhibitor calyculin A or the calcium ionophore A 23187, but did not reduce the rate of synthase inactivation. Proglycosynglucuronide and resorcinylglucuronide inhibited phosphorylase kinase in liver Sephadex filtrates, with Ki values of about 0.75 mM and 4 mM, respectively. Preincubation of the filtrates with ATP and cAMP decreased the sensitivity of phosphorylase kinase to resorcinylglucuronide by about fourfold. It is concluded that the effect of resorcinol and proglycosyn on the phosphorylase a content is due, at least partly, to an inhibition of phosphorylase kinase by their glucuronidated metabolites.

Resorcinol and proglycosyn caused a parallel decrease in the concentration of fructose 2,6–bisphosphate and of hexose 6‐phosphates, without significantly changing the activity of 6‐phosphofructo‐2‐kinase. The decrease in the fructose 2,6‐bisphosphate concentration appears therefore to be secondary to the decrease in the hexose 6‐phosphate concentration.
Intrinsic Tryptophan Fluorescence of Equinatoxin II, a Pore‐Forming Polypeptide from the Sea Anemone Actinia Equina L, Monitors its Interaction with Lipid Membranes
Authors: Maček Peter , Zecchini Marialucia , Pederzolli Cecilia , Serra Mauro Dalla , Menestrina Gianfranco
Published: September 2004

Equinatoxin II is a cytolytic polypeptide from the sea anemone Actinia equina L. which forms pores in natural and artificial membranes. The intrinsic fluorescence of its five tryptophanyl residues was used to monitor the conformational changes induced by denaturing agents, pH and lipids. In the presence of denaturants, the emitted fluorescence peak, normally occurring at 335 nm, was reduced in height by about 65% and red‐shifted to 354 nm indicating unfolding. The toxin fluorescence intensity reversibly decreased by increasing the pH, whereas lipid vesicles, at every pH, caused an increase and a blue shift. The amount of toxin binding to the lipid vesicle was increased by the presence of sphingomyelin. With sphingomyelin‐containing vesicles half‐saturation occurred at a lipid/toxin molar ratio of about 40, whereas with phosphatidylcholine no saturation appeared up to a ratio of 300. One hydrophilic neutral quencher (acrylamide) and two lipid‐confined phosphatidyltype quenchers [bis(9,10‐dibromostearoyl)‐sn ‐glycero‐3‐phosphocholine and 1‐palmitoyl‐2‐(l‐pyrenedecanoyl)‐sn ‐glycero‐3‐phosphocholinel were used to assess the exposure of the emitting centres to the solvent and/or to the lipid. Most of the indolyl residues were found to be solvent‐exposed in the water‐soluble form of the toxin, as inferred from acrylamide quenching. Upon association with lipid vesicles, the fraction accessible to acrylamide dropped considerably, meanwhile the toxin became sensitive to lipid‐soluble quenchers. Taken together these results suggest that insertion of equinatoxin II into sphingomyelin‐containing bilayers is facilitated by high pH and results in the transfer of one or more exposed tryptophanyl residues into the lipid phase. Calcein‐loaded vesicles, with or without a lipid quencher, were used to monitor simultaneously the formation of pores and the transfer of the tryptophans to the lipid phase. We found that the rate constants for vesicles permeabilization and for changes of intrinsic tryptophanyl fluorescence had a different dependence on the lipid/toxin ratio suggesting they correspond to separate steps in the toxin lipid interaction.
Two Soluble forms of Glutamate Dehydrogenase Isoproteins from Bovine Brain
Authors: Cho Sung‐Woo , Lee Jongweon , Choi Soo Young
Published: September 2004

Two soluble forms of novel glutamate dehydrogenase isoproteins, designated GDH I and GDH II, have been purified from bovine brain. GDH I and GDH II were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50 mM for GDH I and GDH II, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57 500 Da. Differences between the biochemical properties of GDH I and GDH II, such as N‐terminal amino acid sequences of intact and tryptic‐digested enzymes, kinetic parameters, optimum pH and heat stability, were extensively examined in both reductive amination of α‐oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate that GDH I and GDH II, isolated from bovine brain, are novel and distinct polypeptides.
Glycogen Metabolism in Quail Embryo Muscle
Authors: Lomako Joseph , Lomako Wieslawa M. , Whelan William J.
Published: September 2004

Cultured quail embryo muscle has proven to be an excellent model system for studying the synthesis of macromolecular glycogen from, and its degradation to, glycogenin, the autocatalytic, self‐glucosylating primer for glycogen synthesis. We recently demonstrated that proglycogen, a low‐Mr form of glycogen, is an intermediate in the synthesis. Here we show that proglycogen also functions as an intermediate in macroglycogen degradation and, in one set of circumstances, represents an arrest point in glycogen breakdown, which does not continue to glycogenin. We suggest that in the nutritionally dependent turnover of glycogen in tissues, the molecules cycle between proglycogen and macromolecular glycogen and are not normally degraded to glycogenin. Nevertheless, when this does happen, the released glycogenin is active, capable of re‐initiating glycogen synthesis. Under culture conditions where the conversion of proglycogen into glycogenin does take place, the intermediates lying between form a discrete rather than a continuous series, suggestive of a cluster structure for proglycogen and indicating that breakdown is stepwise. Evidence of post‐translational modification of glycogenin was obtained by the finding that, in glycogen from cultured muscle, glycogenin is phosphorylated.
Reaction Mechanism of Thioredoxin: 3′‐Phospho‐adenylylsulfate Reductase Investigated by Site‐Directed Mutagenesis
Authors: Berendt Uwe , Haverkamp Thomas , Prior Antje , Schwenn Jens D.
Published: September 2004

Properties of purified recombinant adenosine 3′‐phosphate 5′‐phosphosulfate (PAdoPS) reductase fromEscherichia coliwere investigated. The Michaelis constants for reduced thioredoxin andPAdoPSare 23 μM and 10 μM, respectively; the enzyme has aVmaxof 94–99 (μmol min−1mg−1and a molecular activity/catalytically active dimer of 95 s−1. Adenosine 3′,5′‐bisphosphate (PAdoP) inhibits competetively (Ki4 μM) with respect toPAdoPS; adenosine 2′,5′‐bisphosphate and sulfite are not inhibitory. Alkylation by SH‐group inhibitors irreversibly inactivates the enzyme.

The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGI/LH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction ofPAdoPSto sulfite was investigated by site‐specific mutagenesis:cysHwas mutated by single‐strand‐overlay extension PCR; the mutated genes were cloned in pBTac1 and expressed inE. coliRL 22 (ΔcysHIJ). Homogenous Cys239Ser and Tyr209Phe mutantPAdoPSreductases were investigated for altered catalytic properties. Mutation of the single Cys reducedVmaxby a factor of 4.5×103(Vmax= 0.02–0.013 μmol min−1mg−1) with marginal effects onKmforPAdoPS(19 μM) and reduced thioredoxin (14 μM). Mutation of Tyr209 drastically affected saturation with thioredoxin (Km1.5 μM) and decreasedVmax(0.22–0.25 μmol min−1mg−1) in addition to a small increase inKmforPAdoPS(31 μM).

Chromophores as prosthetic groups were absent from recombinantPAdoPSreductase. Difference absorption spectra between reduced and oxidized forms of wild‐type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (Δλmax292 nm) appears to be involved in the reduction.

The data suggest a special ping‐pong mechanism withPAdoPSreacting with the reduced enzyme isomer in a Theorell‐Chance type mechanism.
Transient and Steady‐State Kinetics of the Oxidation of Scopoletin by Horseradish Peroxidase Compounds I, II and III in the Presence of NADH
Authors: Marquez Leah A. , Dunford H. Brian
Published: September 2004

Scopoletin, a naturally occurring fluorescent component of some plants and a proven plant growth inhibitor, is a known reactant with peroxidase. However, the kinetics of the elementary steps of the reaction have never been investigated, nor has the quantitative effect of interfering substances ever been explored in detail, despite the fact that scopoletin is widely used in a peroxidase assay for H2O2. In this work, we employed both transient‐state and steady‐state methods to determine the second‐order rate constants for the oxidation of scopoletin by the horseradish peroxidase (HRP) intermediate compounds I and II: (3.7 ± 0.1)×106 M−1 S−1 and (8.5 ± 0.5)×105 M−1 S−1 at 20°C, pH 6.0 and ionic strength of 0.1 M. We investigated the possible inhibitory effect of NADH on the reaction of scopoletin with HRP and also the effect of scopoletin on the NADH reaction. In the presence of NADH the rate constant for the reaction between HRP‐I and scopoletin decreased slightly to (2.8 ± 0.1)×106 M−1 S−1. Thus, although NADH is also a peroxidase substrate, it cannot compete effectively for the oxidized forms of the enzyme. On the other hand, scopoletin stimulates the oxidation of NADH by the HRP/H2O2 system, apparently by forming a phenoxyl radical which then oxidizes NADH to NAD· radicals. We present spectral evidence showing that in the aerobic reaction between HRP and NADH at pH 7.0 (without exogenously added H2O2) HRP‐II is the dominant enzyme intermediate with HRP‐III also detectable. Addition of scopoletin to the HRP/NADH system leads to a biphasic reaction in which HRP‐II and HRP‐III disappear. The rate constants for both phases are linearly dependent on scopoletin concentration. We attribute the faster phase to the HRP‐II reaction with scopoletin with a rate constant of (6.2 ± 0.1)×105 M−1 S−1 and the slower phase to the HRP‐III reaction with scopoletin with rate constant (5.0 ± 0.4)×104 M−1 s−1. Our present work not only provides rate constants for the oxidation of scopoletin by HRP‐I, II and III but also elucidates the interactions that possibly occur physiologically during NADH oxidation in the presence of scopoletin.
Hydrogen Isotope Effects in the Reactions Catalyzed by H2‐forming N5,N10‐Methylenetetrahydromethanopterin Dehydrogenase from Methanogenic Archaea
Authors: Klein Andreas R. , Hartmann Gudrun C. , Thauer Rudolf K.
Published: September 2004

H2‐forming N5,N10‐methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron‐sulfur clusters, catalyzes the reversible reduction of N5,N10‐methenyltetrahydomethanopterin (CH≡H4MPT4) with H2 to N5,N10‐methylenetetrahydromethanopterin (CH2=H4MPT) and a proton (ΔG°′=− 5.5 kJ/mol). The enzyme also catalyzes a CH≡H4MPT+‐dependent H2/H+ exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH≡H4MPT+ reduction was performed with D2 instead of H2, Vmax and the Km did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H2O or D2O was the solvent. The findings indicate that a step other than the activation of H2 was rate‐determining in CH≡H4MPT+ reduction with H2. This was substantiated by the observation that also the CH≡H4MPT+‐dependent H2/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. VMax for CH2=H4MPT dehydrogenation to CH≡H4MPT+ and H2 was only 2–3 times higher than for CD2=H4MPT dehydrogenation to CD≡H4MPT+ and HD. Such a small primary isotope effect indicates that the breakage of the C‐H bond in the methylene group of CH2=H4MPT was only rate‐limiting when hydrogen was substituted by a deuterium.
Structural Elucidation of Novel Methylglucose‐Containing Polysaccharides from Mycobacterium xenopi
Authors: Tuffal Gilles , Ponthus Christian , Picard Claudine , Rivière Michel , Puzo Germain
Published: September 2004

The structures of methylglucose‐containing polysaccharides (MeGlc PS) from Mycobacterium xenopi were investigated using high‐pH anion‐exchange chromatography and liquid secondary‐ion mass spectrometry. We report the structure of two novel MeGlc PS, referred to as A and B. MeGlc PS A is composed of 16 d‐glucopyranose residues, 11 of which are methylated, and MeGlc PS B contains 15 d‐glucopyranose residues, 10 of which are methylated. The main structural feature of both MeGlc PS A and B, compared to the previously described structures, is the absence of the tetrasaccharide non‐reducing end 3‐O‐Me‐D‐Glcp‐[α(1→4)‐d‐Glcp]3. The MeGlc PS A structure is similar to the synthetic polysaccharide [Saïer, M. H. & Ballou, C. E. (1968) J. Biol. Chem. 243, 992–1005], having a lower affinity for fatty acids than the MeGlc PS of Mycobacterium smegmatis [Kiho, T. & Ballou, C. E. (1988) Biochemistry 27, 5824–5828]. Thus, the occurrence of MeGlc PS A and the consequences on the regulation of fatty acid synthetase I activity involved in the biosynthesis of fatty acids, precursors of mycolic acid biosynthesis, is discussed.
Effects of Ionic Strength and pH on the Binding of Medium‐Chain Fatty Acids to Human Serum Albumin
Authors: Pedersen Anders Overgaard , Mensberg Karen‐Lise Dons , Kragh‐Hansen Ulrich
Published: September 2004

Binding equilibria for the interactions of the medium‐chain fatty acid anions, laurate and myristate, with defatted human serum albumin have been investigated under varying environmental conditions such as ionic strength and pH. Since these ligands bind strongly to albumin (Kass≈ 107 M−1), conventional equilibrium dialysis is not a feasible method for these investigations. Consequently, we employed a dialysis method, allowing determination of very low concentrations of unbound ligand by measuring the rate of exchange of labelled ligand across a dialysis membrane under conditions of chemical equilibrium.

Over a range of ionic strength, 8–68 mM, the binding of the first few molecules of laurate to albumin was weakened with increasing ionic strength, whereas the binding of subsequent molecules seemed to proceed independently of ionic strength. The binding of myristate by albumin, however, appeared to be independent of ionic strength in the observed range of concentrations.

The influence of pH in the range 5.1–9.0 on the binding of the two fatty acid anions by albumin was more complicated. The first molecule of laurate appeared to bind with a slightly weaker affinity to albumin at low pH, compared to pH 7 and high pH, while the trends for the following molecules varied. The binding of myristate (irrespective of concentration) seemed to strengthen monotonously with pH, but this conclusion depends critically on the interpretation of the kinetic behaviour of the myristate anion.

We have previously shown [Pedersen, A. O., Honoré, B. & Brodersen, R. (1990) Eur. J. Biochem. 190, 497–502] that the strength of binding of the first few molecules of the two fatty acid anions to albumin decreases with increasing temperature, whereas binding of subsequent molecules seems to proceed independently of temperature.

We explain these findings as follows. The binding of the first few (3 or 4) molecules of the C12 laurate anion is clearly driven by formation of ionic bonds between the fatty acid anion and positively charged groups, such as lysine residues, in the albumin molecule, whereas the binding of subsequent molecules of laurate seems to depend more on hydrophobic interactions. In the case of the C14 myristate anion, the binding of the first few (only 1 or 2) molecules may depend on ionic forces, but binding of the following molecules of myristate seems to depend on hydrophobic interactions only. Thus, multiple binding of both ligands is considered to be an association between fatty acid anions stacked on the protein molecule and/or it may take place in hydrophobic regions of the albumin molecule. This type of binding will contribute entropy rather than enthalpy to the total binding energy.
Molecular Cloning, Expression, and Characterization of a Human Intestinal 15‐kDa Protein
Authors: Fujita Michiyo , Fujii Hiroshi , Kanda Tatsuo , Sato Eiju , Hatakeyama Katsuyoshi , Ono Teruo
Published: September 2004

We have isolated a cDNA encoding a human intestinal 15‐kDa protein (1–15P) from a human ileal λgt 11 cDNA library, using a full‐length rat I‐15P cDNA. One clone encompassed 571 nucleotides and encoded a 128–amino‐acid protein with a calculated molecular mass of 14355 Da. The deduced amino acid sequence of human I‐15P showed high similarity to the rat counterpart (78%), mouse ileal lipid‐binding protein (80%) and porcine gastrotropin (75%). It also exhibited 36% similarity to human liver fatty‐acid‐binding protein (L‐FABP). Northern blot analysis of human I‐15P revealed a single transcript only in ileum, however, the reverse‐transcription/PCR demonstrated expression in ovary and placenta, but it was much lower than in ileum. Transformation of Escherichia coli with the I‐15P cDNA resulted in the efficient expression of a protein that was identical to the ileal cytosolic 1–15P. In vitro binding studies revealed that the bacterially expressed recombinant I‐15P showed much lower affinities for palmitate and oleate than L‐FABP. However, it showed similar affinity for taurocholate, compared with a control, BSA. Comparison of the structural features of human I‐15P and human L‐FABP suggested that loss of a long α‐helix region and hydrophobic profile of I‐15P may be attributable to a unique ligand‐binding specificity of I‐15P.
Properties and Functions of a Neuromedin‐B‐Preferring Bombesin Receptor in Brain Microvascular Endothelial Cells
Authors: Vigne Paul , Feolde Eric , Van Renterghem Catherine , Breittmayer Jean Philippe , Frelin Christian
Published: September 2004

Endothelial cells were isolated from rat brain microvessels and grown in vitro. They expressed a high density of [125I‐Tyr4]bombesin receptors (Bmax= 0.9 pmol/mg protein) with an apparent Kd value of 10nM. The pharmacological profile of inhibition of the specific [125I‐Tyr4]bombesin binding [bombesin = neuromedin B > gastrin releasing peptide (GRP)] was consistent with the presence of a neuromedin‐B‐preferring receptor. Addition of bombesin, neuromedin B and GRP increased the activity of phospholipase C as measured by the production of total inositol phosphates and from intracellular Ca2+ measurements. They increase 86Rb4 uptake by the Na+, K+, 2C1− cotransporter and by a charybdotoxin‐sensitive, Ca2+‐activated K+ channel and 22Na+ uptake by the Na+/H+ exchanger. The pharmacological profiles of activation of phospholipase C., Na+, K+, 2C1 cotransport and Na+/H+ exchange by bombesin‐like peptide were consistent with an involvement of the neuromedin‐B‐preferring receptor characterized in binding experiments. It is suggested that one of the actions of neuromedin B in brain vessels could be to control K+ secretion by the blood/brain barrier.
The Identification of the Single‐Stranded DNA‐Binding Domain of the Escherichia coli RecA Protein
Authors: Gardner Renee V. , Voloshin Oleg N. , Camerini‐Otero R. Daniel
Published: September 2004

To identify the ssDNA‐binding domain of Escherichia coli RecA protein, we examined the ssDNA‐binding capabilities of synthetic peptides, the sequences of which were derived from the C‐ and N‐termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter‐binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coli RecA in this region did not. The binding of RecA to ssDNA examined using a gel‐shift assay was inhibited by a synthetic peptide derived from this ssDNA‐binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C‐terminus or from N‐terminal residues 6–39. A peptide corresponding to amino acid positions 152– 169 of the RecA molecule and spanning loop LI and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20–amino‐acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose‐binding protein‐RecA fusion proteins were made, one containing amino acid residues 185–224 of RecA and the other the last 51 C‐terminal residues of RecA (amino acid residues 303–353). In contrast to the C‐terminus‐derived fusion protein, the fusion protein containing the putative DNA‐binding site demonstrated significant binding to single‐stranded oligonucleotides in both filter‐binding and gel‐shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA‐binding domain of the RecA protein.
The Purification, Characterization and Analysis of Primary and Secondary‐Structure of Prolyl Oligopeptidase from Human Lymphocytes
Authors: Goossens Filip , De Meester Ingrid , Vanhoof Greet , Hendriks Dirk , Vriend Gert , Scharpé Simon
Published: September 2004

Prolyl oligopeptidase was isolated and purified to homogeneity from human lymphocytes, yielding a specific activity of 7780 mU/mg. The molecular mass using size‐exclusion chromatography matches the 76 kDa obtained by SDS/PAGE. This provides evidence that prolyl oligopeptidase is a monomer. The isoelectric point is 4.8 as judged by isoelectric focusing in free solution. Di‐isopropyl fluorophosphate and phenylmethylsulphonyl fluoride completely abolish the activity, classifying the enzyme as a serine proteinase. The inhibition by p–chloromercuribenzoic acid indicates the importance of a free sulfhydryl group near the active‐site. α1‐Casein and ornithine decarboxylase, two proteins containing a PEST sequence, inhibit prolyl oligopeptidase, but were not hydrolyzed. This demonstrates that prolyl oligopeptidase is not participating in the metabolism of proteins according to a PEST‐dependent pathway. α1‐Antitrypsin partially inhibits the enzyme but in contrast, aprotinin does not. Its inability to cleave corticotropin‐releasing factor, ubiquitin, albumin and aprotinin, together with the hydrolysis of bradykinin between Pro7–Arg8 confirms the affinity of prolyl oligopeptidase for small peptides. Multiple sequence alignment does not reveal any similarity with proteases of known tertiary structure. Secondary‐structure prediction displays striking similarity with dipeptidyl peptidase IV and acylaminoacyl peptidase. Two characteristic features of the members of the prolyl oligopeptidase family of serine proteases are highlighted: the linear arrangement of the catalytic triad is nucleophile‐acid‐base and the proteolytic cleavage releasing the catalytically active C‐terminal region of around 500 amino acids from the N‐terminal sequence. Secondary structure prediction and comparison of the active‐site of serine proteinases with known three‐dimensional coordinates prove that Asp641 is the third member of the catalytic triad. The secondary structural organization of the protease domain of prolyl oligopeptidase is in accordance with the α/β hydrolase fold.
The Myristoylated Alanine‐Rich C‐Kinase Substrate (MARCKS) is Sequentially Phosphorylated by Conventional, Novel and Atypical Isotypes of Protein Kinase C
Authors: Herget Thomas , Oehrlein Silke A. , Pappin Dairyl J. C. , Rozengurt Enrique , Parker Peter J.
Published: September 2004

The myristoylated alanine‐rich C‐kinase substrate (MARCKS) is the major protein kinase C (PKC) substrate in many cell types including fibroblasts and brain cells. Here we describe the phosphorylation of MARCKS and the site specificity for different PKC isotypes. Conventional (c)PKC β1, novel (n)PKC δ and nPKC ɛ efficiently phosphorylated the MARCKS protein in vitro. The Km values were extremely low, reflecting a high affinity between kinases and substrate. The apparent affinity of nPKC δ (Km= 0.06 μM) was higher than that of nPKC ɛ and cPKC β1 (Km= 0.32 μM). The rate of substrate phosphorylation was inversely correlated with affinity and decreased in the order nPKC ɛ > cPKC β1 > nPKC δ. Atypical (a)PKC ζ did not phosphorylate the intact MARCKS protein. However, a 25‐amino‐acid peptide deduced from the MARCKS phosphorylation domain, was efficiently phosphorylated by aPKC ζ as well as by the other three PKC.

Site analysis revealed that only serine residues S152, S156 and S163 were phosphorylated, with S163 phosphorylated highest, followed by S156 and S152; in contrast, S160 and S167 were not phosphorylated. No further PKC phosphorylation sites could be detected in MARCKS. The phosphorylation pattern was independent of the type of PKC isotype used. Kinetic analysis showed, that MARCKS is sequentially phosphorylated in the order S156 > S163 > S152 by cPKC, nPKC and aPKC. There was no dramatic difference in the sequential phosphorylation of MARCKS detectable when comparing the four PKC isotypes. The results are discussed in the context of the functional significance of MARCKS phosphorylation.
UDP‐N‐Acetylglucosamine:Dolichyl‐Phosphate N‐Acetylglucosamine‐1‐Phosphate Transferase is Amplified in Tunicamycin‐Resistant Soybean Cells
Authors: Zeng Yucheng , Elbein Alan D.
Published: September 2004

A tunicamycin‐resistant soybean cell line was developed by gradually increasing the concentration of tunicamycin in the growth medium. At the final stage, the resistant cells could survive in media containing 60 μg/ml of tunicamycin, whereas normal cells show a greatly retarded growth rate at 0.5 μg/ml of antibiotic. The tunicamycin‐resistant cells had a greater than 40‐fold increase in the activity of the enzyme UDP‐GlcNAc:dolichyl‐P GlcNAclP transferase, a 2–3‐fold increase in the activity of dolichyl‐P ‐mannose synthase, but no increase in the activities of other enzymes of the lipid‐linked saccharide pathway such as dolichyl‐P ‐glucose synthase or mannosyl transferases. There was also no change in the activities of the glycoprotein‐processing enzymes, glucosidase I or glucosidase II, as compared to wild‐type cells. The increase in GlcNAclP transferase was due to an increased production of enzyme, as seen by a dramatic increase in the amount of a 39–kDa protein, which is presumed to be this enzyme protein. The GlcNAclP transferase from tunicamycin‐resistant cells was equally sensitive to tunicamycin as was the wild‐type enzyme, but was considerably more labile to temperatures above 30°C. The activity in tunicamycin‐resistant cells was greatly stimulated by exogenous dolichyl‐P. The spectrum of oligosaccharides from labeled lipid‐linked oligosaccharides was similar in wild‐type and tunicamycin‐resistant soybean cells, but the resistant cells had significantly greater amounts of the shorter and much lower amounts of the larger‐sized oligosaccharides.
Changes in the Composition of Bilirubin‐IX Isomers During Human Prenatal Development
Authors: Yamaguchi Tokio , Nakajima Hiroshi
Published: September 2004

We analyzed the isomeric composition of bilirubin‐IX in human fetal bile using HPLC. The approximate ratio of the bilirubin‐IX isomers obtained from the fetal bile at 20 weeks of gestation was IXα, 6%; IXβ, 87%; IXγ, 0.5%; and IXδ, 6%.

From 15 to 22 weeks, bilirubin‐IXβ was predominant and bilirubin‐IXδ and bilirubin‐IXα were also present in the bile as minor components. By 28 weeks, bilirubin‐IXα constituted about 50% of the total bilirubin.

There was a general correlation between fetal age and the proportion of bilirubin‐IXβ to bilirubin‐IXβ in the bile and the small intestinal contents of fetuses. As development proceeded from mid‐gestation to near term, the isomeric composition dramatically changed, with a decrease in the IXβ isomer and a subsequent increment of the IXα isomer. In contrast, the IXβ isomer changes little.

Recently, we identified four forms of biliverdin reductase including two biliverdin‐IXα reductases and two biliverdin‐IXβ reductases in human liver cytosolic fractions [Yamaguchi, T., Komoda, Y. & Nakajima, H. (1994) J. Biol. Chem. 269, 24343–24348]. The proportion of the total activity of biliverdin‐IXβ reductases to that of biliverdin‐IXα reductases was considerably higher in the fetal, than in the adult liver.
Reconstitution of the F0 Complex of Escherichia coll ATP Synthase from Isolated Subunits
Authors: Dmitriev Oleg Yu. , Altendorf Karlheinz , Fillingame Robert H.
Published: September 2004

Subunit c of the Escherichia coli F1F0‐ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent‐solubilized F0 subunits a and h to form a functionally active FT channel. The rates of H+ uptake by the proteoliposomes containing the reconstituted F0 complex were comparable to those observed with native F0 reconstituted without subunit dissociation. The F0 reconstituted from purified subunits was also shown to form an active ATP‐driven FT pump upon binding of the F1‐ATPase sector of the complex. Reconstitution of D61N and D61G mutant c subunits with wild‐type subunits a and b produced an inactive F0. Hybrid F0 complexes, formed with mixtures of wild‐type and D61N or D61G mutant c subunits, were also prepared. Formation of an active F0 was prevented by addition of relatively small proportions of D61N or D61G mutant c subunits, i.e. active F0 formation was gradually disrupted as the mutant/wild‐type ratio was increased from 0.05 to 0.2. The hybrid reconstitution studies support a model where inactivation of one of the 9–12 c subunits found in F0 is sufficient to abolish activity.
Involvement of the C‐terminal Tail in the Activity of Drosophila Alcohol Dehydrogenase
Authors: Albalat Ricard , Valls Marc , Fibla Joan , Atrian Sílvia , Gonzàlez‐Duarte Roser
Published: September 2004

Drosophila alcohol dehydrogenase belongs to the heterogeneous family of short‐chain dehydrogenases/reductases, which does not include the well characterized mammalian alcohol dehydrogenases. Although it is clear that the main biological role of this enzyme is in alcohol oxidation, in the absence of the three‐dimensional conformation only partial information on the protein regions involved in the active site, and the coenzyme and substrate interacting cavities is available. Two segments have already been identified, a coenzyme‐binding segment at the N‐terminus, and the reactive Tyr152 and Lys156 residues. Limited proteolytic assays had suggested the involvement of the 13 C‐terminal amino acids in the function of the enzyme. By site‐directed mutagenesis, we have constructed eight different truncated mutant enzymes and expressed them in Escherichia coll. The purified mutant enzymes have been recovered and characterized using monoclonal antibodies. Kinetic analysis and stability assays have been performed, and clearly demonstrate the contribution of the last 13 amino acids to the activity. We hypothesize that the C‐terminal tail constitutes an essential region for maintaining the hydrophobicity of the catalytic pocket needed for binding of the substrate.
Genetic and Biochemical Characterization of the UGP1 Gene Encoding the UDP‐Glucose Pyrophosphorylase from Saccharomyces cerevisiae
Authors: Daran Jean Marc , Dallies Nathalie , Thines‐Sempoux Denise , Paquet Véronique , François Jean
Published: September 2004

We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI [Purnelle B., Skala, J., Van Dijck, L. & Goffeau, A. (1992) Yeast 8, 977–986] encodes UDP‐glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP‐Glc from glucose 1‐phosphate and UTP. Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies. In particular, the amino acid sequence motifs involved in the binding of glucose 1–phosphate and UDP‐Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi‐copy expression of YKL248 resulted in a 40‐fold increase in UGPase activity. This gene was, therefore, renamed UGP1. Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability. Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted. This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild‐type gene under the control of the glucose‐repressible galactose‐inducible promoter. The effects of overproducing UGPase on the cell metabolism and morphology were carbon‐source dependent. On glucose medium, the 40–fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP‐Glc, with no significant effect on growth. In contrast, on galactose, the 40–fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3–5–fold increase in the concentrations of UDP‐Glc, UDP‐Gal and galactose 1–phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation. Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium. Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5–10% wild‐type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium. However, the 10–fold reduction of UGPase activity induced a multi‐budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell‐wall β‐glucan content. All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP‐Glc in yeast metabolism.
Structure of Metal Site in Cd‐Substituted His117Gly Mutant of Azurin with and without Addition of Imidazole Derivatives
Authors: Danielsen Eva , Bauer Rogert , Hemmingsen Lars , Bjerrum Morten J. , Butz Tilman , Tröger Wolfgang , Canters Gerard W. , Den Blaauwen Tanncke , Van Pouderoyen Gertie
Published: September 2004

The present work uses 111mCd‐perturbed angular correlations of ‐γ‐rays (PAC) to investigate the structure of the metal site of the His117Gly mutant of Pseudomonas aeruginosa azurin in aqueous solution and the effect on the structure upon addition of the following exogenous ligands: imidazole, 4–methyl imidazole, 1‐methyl imidazole, 2‐methyl imidazole and histidine. The nuclear quadrupole interaction of cadmium bound to the mutant without addition of exogenous ligands shows a strong pH dependence with three different nuclear quadrupole interactions consistent with two pKa values at about 7.2 and 8.6 at 2°C. Addition of the imidazole derivatives resulted in a significant change in the PAC spectrum showing that they coordinate. This is in accordance with observations by EPR for the same mutant with copper at the metal site [den Blaauwen, T. & Canters, G. W. (1993) J. Am. Chem. Soc. 115, 1121–1129]. However, whereas EPR and ultraviolet/visual absorption show that the characteristics of the wild‐type copper protein are regained by addition of the imidazole derivatives with the exception of the possible bidentates (histidine and histamine), the comparison of the PAC results to model calculations shows that the cadmium ion must be fourfold coordinated in most cases, probably binding an additional water or hydroxide ligand. A fourfold coordination is in contrast to cadmium‐substituted wild‐type azurin where PAC data inferred a threefold coordination by a Cys and two His residues [Danielsen, E., Bauer, R., Hemmingsen, L., Andersen, M., Bjerrum, M. J., Butz, T., Tröger, W., Canters, G. W., Hoitink, C. W. G., Karlsson, G., Hansson, Ö. & Messerschmidt, A. (1995) J. Biol. Chem. 270, 573–580).
Mechanisms Underlying Aberrant Glycosylation of MUC1 Mucin in Breast Cancer Cells
Authors: Brockhausen Inka , Yang Ji‐Mao , Burchell Joy , Whitehouse Caroline , Taylor‐Papadimitriou Joyce
Published: September 2004

The product of the MUC1 gene, the polymorphic epithelial mucin (PEM) is aberrantly glycosylated in breast and other carcinomas, resulting in exposure of normally cryptic peptide epitopes. PEM expressed by breast cancer cells contains more sialylated O‐glycans and has a lower GlcNAc content than that expressed by normal cells. The exposure of peptide epitopes is thus thought to be due to the sugar side chains being shorter on the tumour‐associated mucin. To investigate possible mechanisms underlying the different pattern of glycosylation in breast cancer cells, we analysed the pathways involved in the biosynthesis of O‐glycan chains of mucins in normal and cancerous mammary epithelial cells. An immortalized mammary epithelial cell line originating from normal human milk, MTSV1–7, and three human breast cancer cell lines, BT20, MCF‐7 and T47D, were studied. Glycosyltransferase activities assembling, elongating and terminating O‐glycan core‐1 [Galβl‐3GalNAcα‐R] and core‐2 [GlcNAcβ1‐6 (Galβ1‐3) GalNAcα‐R] were present in the normal mammary cell line. Many of the glycosyltransferase activities were also expressed at variable levels in breast cancer cells. However, a sialyltransferase activity (CMP‐sialic acid Galβ1‐3GalNAc α3–sialyltransferase) was increased several fold in all three cancer cell lines. Moreover, mammary cancer cell lines BT20 and T47D have lost the ability to synthesize core‐2, as shown by the lack of UDP‐GlcNAc: Galβ1‐3GalNAc (GlcNAc to GalNAc) β6–GlcNAc‐transferase activity, which corresponded to the absence of the mRNA transcript. However, MCF‐7 breast cancer cells expressed this enzyme. Thus, the mechanism for the exposure of peptide epitopes in BT20 and T47D cells is proposed to be the loss of core‐2 branching leading to shorter, sialylated O‐glycan chains. A different mechanism is proposed for MCF‐7 breast cancer cells.
Solution Structure of the Na+ form of the Dimeric Guanine Quadruplex [d(G3T4G3)]2
Authors: Keniry Max A. , Strahan Gary D. , Owen Elisabeth A. , Shafer Richard H.
Published: September 2004

The solution structure of the DNA quadruplex formed by the association of two strands of the DNA oligonucleotide, d(G3T4G3), in NaCl solution has been determined by 1H two‐dimensional NMR techniques, full relaxation matrix calculations and restrained molecular dynamics. The refined structure incorporates the sequences 5′‐GlsG2AG3AT4AT5AT6AT7AG8sG9AG10A‐3′ and 5′‐G11sG12AG13AT14AT15A. T16AT17AG18sG19sG20A‐3′ (where S and A denote syn and anti, respectively) in a three‐quartet, diagonal‐looped structure that we [Strahan, G. D., Shafer, R. H. & Keniry, M. A. (1994) Nucleic Acids Res. 22, 5447–5455] and others [Smith, F. W., Lau, F. W. & Feigon, J. (1994) Proc. Natl Acad. Sci. USA 91, 10546–10550] have described. The loop structure is compact and incorporates many of the features found in duplex hairpin loops including base stacking, intraloop hydrogen bonding and extensive van der Waals' interactions. The first and third loop thymines stack over the outermost G‐quartet and are also associated by hydrogen bonding. The second and the fourth loop thymines fold inwards in order to enhance van der Waals' interactions. The unexpected sequential syn‐syn deoxyguanosines in the quadruplex stem appear to be a direct consequence of the way DNA oligonucleotides fold and the subsequent search for the most stable loop structure. The implications of loop sequence and length on the structure of quadruplexes are discussed.
A Fourier‐Transform Infrared Spectroscopic Investigation of the Hydrogen‐Deuterium Exchange and Secondary Structure of the 28‐kDa Channel‐Forming Integral Membrane Protein (CHIP28)
Authors: Haris Parvez I. , Chapman Dennis , Benga Gheorghe
Published: September 2004

Fourier‐transform infrared spectroscopy (FTIR) has been employed to investigate the structural properties of the 28–kDa channel‐forming integral membrane protein (CHIP28) present in phospholipid vesicles suspended in aqueous media. This study reports the FTIR spectra of this membrane protein present in H2O and 2H2O. The secondary structure of the protein was determined and found to consist of 36%α‐helical and 42%β‐sheet structures. These results are in close agreement with the results of a previous CD study [Van Hoek, A. N., Wiener, M., Bicknese, S., Miercke, L., Biwersi, J. & Verkman, A. S. (1993) Biochemistry 32, 11847–11856]. However, the results differ from those given in an FTIR analysis by the same workers who recorded FTIR spectra of the CHIP28 protein in a dehydrated state. An unusually high extent of hydrogen‐deuterium exchange of the peptide groups of this protein occurs. The magnitude of the spectral changes observed upon exposure of the protein to 2H2O is greater than has been observed with any other membrane protein previously studied. Thus, over 80% of the peptide groups exchange within 5 min and the amide I band maximum shifts to low frequency by approximately 20cm−1. This high hydrogen‐deuterium exchange observed with the CHIP28 protein is consistent with the presence of an aqueous pore within the protein structure.