bluuebunny/crossref_metadata_2025 huggingface dataset:
The initial characterization of the iron environment in lipoxygenase by Mössbauer spectroscopy
Authors: DUNHAM William R. , CARROLL Richard T. , THOMPSON John F. , SANDS Richard H. , FUNK Max O.
Published: March 2005

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using Mössbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in Mössbauer measurements. It was unequivocally demonstrated that the native enzyme contains high‐spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect Mössbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high‐spin Fe(II) Mössbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The Mössbauer spectra (4.2–250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these Mössbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III) · diethylenetriaminepentaacetic acid.
Oxalate as synergistic anion for Cd(II) binding to ovotransferrin
Authors: SOLA Marco
Published: March 2005

The Cd(II) derivative of ovotransferrin containing oxalate as synergistic anion was investigated through 113Cd‐ and 13C‐NMR spectroscopy and compared with the analogous derivative obtained in the presence of bicarbonate. Cadmium loaded in the C site gives rise to a 113Cd‐NMR signal at 54 ppm, while that bound to the N site is broadened beyond detection. The two resonances at 168.5 and 169.9 ppm observed in the 13C‐NMR spectrum of the Cd2 derivative obtained with [13C]oxalate each correspond to slowly exchanging oxalate specifically bound to a single site. No splitting of these resonances due to 113Cd–13C magnetic coupling is observed upon insertion of 113Cd‐enriched Cd(II) ion, unlike previous observations for the corresponding derivative obtained with bicarbonate as synergistic anion. It was found that the metal sites in the present derivative are inequivalent, as observed for other metal‐transferrin‐oxalate adducts. The C site is found to be sensitive to a residue ionization with a pKa of 9.5.
The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor
Authors: KARRASCH Marion , BÖRNER Gerhard , ENSSLE Marion , THAUER Rudolf K.
Published: March 2005

Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo‐iron‐sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6‐carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes.

A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5–0.8 mol molybdenum, 0.6–0.9 mol pterin, 28 ± 2 mol non‐heme iron and 28 ± 2 mol acid‐labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 μmol · min−1· mg−1 (kcat= 640 s−1) assayed with methylviologen (app. Km= 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.
Analysis of the membrane potential of rat‐ and mouse‐liver mitochondria by flow cytometry and possible applications
Authors: PETIT Patrice X. , O'CONNOR José E. , GRUNWALD Didier , BROWN Spencer C.
Published: March 2005

Washed and purified rat‐ or mouse‐liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine O and a cyanine derivative, 3,3′‐dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non‐energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non‐energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non‐specific dye binding) and a second population which exhibited high fluorescence.

Flow cytometry of the unpurified, simply washed, rat‐liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light‐scattering properties and membrane‐potential‐related fluorescence. One third of the washed mitochondria were found to be non‐functional by such assays.

The fluorescence of purified rat‐liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogeneous substrates.

The low‐angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization.

The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non‐active mitochondrial population dispersion) point of view could thus be demonstrated by flow‐cytometry analysis.

Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat‐liver mitochondria), and the effects of ammonium toxicity (mouse‐liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies.
Physicochemical and biological characterization of asialoerythropoietin
Authors: IMAI Nobuo , HIGUCHI Masato , KAWAMURA Akinori , TOMONOH Kikuo , OH‐EDA Masayoshi , FUJIWARA Masaaki , SHIMONAKA Yasushi , OCHI Norimichi
Published: March 2005

Various partially or fully desialylated human erythropoietins were obtained by neuraminidase digestion of the hormone, without non‐specific proteolysis and degradation of carbohydrates. Asialoerythropoietin showed a specific activity of 220‐IU/mg protein in vivo, although that of the intact erythropoietin was 2.2 × 105 IU/mg. A linear relationship was found between the logarithm of the specific activity in vivo and the number of sialic acids. The asialoerythropoietin showed a four‐times‐higher specific activity in vitro compared with intact erythropoietin using mouse bone marrow cells. It also showed an approximately six‐times‐higher specific activity in a colony‐forming assay for the erythroid colony‐forming unit and the erythroid burst‐forming unit. Partially or fully de‐N‐glycosylated erythropoietin derivatives also showed lower in vivo activity but higher in vitro activity than the intact erythropoietin, dependent on the number of sialic acids. To clarify the reason for the enhanced biological activity of asialoerythropoietin in vitro, the binding of intact 125I‐erythropoietin or 125I‐asialoerythropoietin to cells containing specific receptors for the hormone was analyzed. 125I‐asialoerythropoietin bound to spleen cells from anemic mice approximately five times faster than did intact 125I‐erythropoietin. The amount of 125I‐asialoerythropoietin internalized by target cells, measured in the absence of NaN3, was four times higher than that of intact erythropoietin. These results demonstrate that asialoerythropoietin binds to its receptor faster than the intact form. This may be the main reason for the increased activity of asialoerythropoietin in vitro.
Modulation of DNA methylation and gene expression in cultured sycamore cells treated by hypomethylating base analog
Authors: NGERNPRASIRTSIRI Jarunya , AKAZAWA Takashi
Published: March 2005

The selective suppression of photosynthetic genes in both the nuclear and plastid genomes of the nonphotosynthetic white wild‐type cell line of sycamore (Acer pseudoplatanus) has been found to be inversely related to the presence of a variety of methylated bases, especially 5‐methylcytosine (5‐MeCyt) and N6‐methyladenine (N6‐MeAde), localized in regions of the plastid genome containing silent genes. We used hypomethylating base analogs to manipulate the level of cytosine and adenine methylation in the white cells of sycamore, and examined the effects of changes in methylation on gene expression. Treatment with 5‐azacytidine (5‐AzaCyd) and N6‐benzyladenine (N6‐BzlAde) decreased cytosine and adenine methylation. This was accompanied by restoration of transcriptional activity in photosynthetic genes which are usually suppressed. Both 5‐MeCyt and N6‐MeAde suppressed nuclear gene expression, but only 5‐MeCyt suppressed plastid gene expression.
Primary structure and inhibition of protein synthesis in eukaryotic cell‐free system of a novel thionin, γ‐hordothionin, from barley endosperm
Authors: MENDEZ Enrique , MORENO Aurora , COLILLA Francisco , PELAEZ Fernando , LIMAS Gabriel G. , MENDEZ Raul , SORIANO Fernando , SALINAS Matilde , de HARO Cesar
Published: March 2005

A new sulfur‐rich and basic polypeptide, designated as γ‐hordothionin, has been isolated from barley endosperm by a semi‐preparative purification consisting of extraction with a volatile salt solution followed by high‐performance liquid chromatography using a reversed‐phase C4 column. The isolated polypeptide was found to be homogeneous by micro‐two‐dimensional gel electrophoresis in the presence of sodium dodecyl sulfate. The complete primary structure of γ‐hordothionin was determined by automatic degradation of the intact, S‐carboxymethylated and S‐pyridylethylated γ‐hordothionin and fragments obtained by proteolytic cleavage. γ‐Hordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. γ‐Hordothionin inhibits translation in cell‐free systems derived from mammalian (rabbit reticulocyte, mouse liver) as well as non‐mammalian (Artemia embryo) cells, at several levels. At low concentrations (1–10 μM) the protein seems to affect mainly the polypeptide‐chain‐initiation process, although it might also act at the elongation level. At higher concentrations (20–80 μM) this inhibitor induces activation of an eukaryotic polypeptide‐chain initiation factor 2 α‐subunit (eIF‐2α) kinase in hemin‐supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in γ‐hordothionin appears to be essential for the eIF‐2α kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (α and β‐thionins, from wheat and barley), γ‐hordothionin is a putative member of the thionin family.
Assignment of the 1H‐NMR spectrum of a lac repressor headpiece‐operator complex in H2O and identification of NOEs
Authors: LAMERICHS Rudolf M. J. N. , BOELENS Rolf , VAN DER MAREL Gijs A. , VAN BOOM Jacques H. , KAPTEIN Robert
Published: March 2005

A complex between the headpiece amino‐terminal residues 1–56 of lac repressor (HP56) and an 11‐bp lac operator fragment was studied by 1H NMR. The sequence specific assignment of the exchangeable and nonexchangeable protons has been accomplished. Several protons have favourable chemical shifts in the complex, therefore new intraprotein NOEs could be found that had not been unambigously identified in the free protein. By comparison, most of these intraprotein NOEs are also present in the spectra of the free headpiece but some are different. Furthermore, several new protein DNA NOEs could be identified. The NOE between the side‐chain amide protons of Gln18 and C5H of C7 confirms the specific contact between these residues which was proposed from genetic experiments [Ebright, R. M. (1985) J. Biomol. Struct. & Dyn. 3, 281–2971. The implications of the new data for the interaction between the lac repressor headpiece and its operator are discussed.
The presence of two classes of high‐affinity cyclosporin A binding sites in mitochondria
Authors: McGUINNESS Orla , YAFEI Nasser , COSTI Andreas , CROMPTON Martin
Published: March 2005

The inner membrane of rat liver mitochondria contains a reversible Ca2+‐dependent pore, opening of which is largely blocked by cyclosporin A. Analyses of [3H]cyclosporin binding to rat liver mitochondria demonstrate two classes of high‐affinity binding site with capacities of < 5 pmol and approximately 60 pmol cyclosporin · mg mitochondrial protein−1 in addition to partitioning into membrane phospholipids (0.03 pmol · mg mitochondrial protein · nM−1). Direct measurement [14C]sucrose entry into the matrix space indicates that cyclosporin A inhibits pore opening by interacting with the low‐capacity sites. The same low‐capacity sites (Ka cyclosporin, 8 nM) are possibly attributable to peptidylprolyl cis‐trans‐isomerase, although investigation of pore state interconversion from the rapid kinetics of [14C]sucrose entrapment in the matrix space does not indicate that cyclosporin‐sensitive prolyl isomerization occurs at the actual step of pore opening/closure. It is suggested that the low‐capacity cyclosporin‐binding component may stabilize the open pore state; this is supported by the observations that Ca2+ decreases cyclosporin binding to this component and that cyclosporin brings about closure of the pre‐opened pore. The implications for the possible number of functional pores in mitochondria are discussed.
Changes in free and bound forms and total amount of hexokinase isozyme II of rat muscle in response to contractile activity
Authors: WEBER Franz E. , PETTE Dirk
Published: March 2005

Increased contractile activity as induced by chronic low‐frequency stimulation evoked in rat fast‐twitch muscle an almost immediate increase in the ratio between structure‐bound and free hexokinase. In addition, an up to 14‐fold rise in total hexokinase activity occurred after two weeks of stimulation indicating that glucose phosphorylation became a limiting step of glucose utilization under these conditions. The increase in hexokinase activity was transitory as prolonged stimulation led to a leveling off and steep decline with an apparent half‐life of 2.5 days after three weeks of stimulation. The transient increase in glucose phosphorylating capacity can be explained by previous observations indicating that prolonged stimulation leads to a shift from a carbohydrate‐based to a fatty‐acid‐based energy metabolism. Using an isozyme‐specific sandwich ELISA, it was shown that both increases and decreases in total hexokinase activity were matched by corresponding changes in the amount of hexokinase isozyme II protein. Increases in both total hexokinase activity (3–4‐fold) and hexokinase II protein content were also observed after denervation in rat fast‐twitch muscle. In view of reports in the literature, it is suggested that the elevations in hexokinase II observed with increased contractile activity and denervation relate to enhanced glucose uptake and utilization.
Structural analysis of the human inter‐ş‐trypsin inhibitor light‐chain gene
Authors: DIARRA‐MEHRPOUR Maryam , BOURGUIGNON Jeannette , SESBOÜÈ Richard , SALIER Jean‐Philippe , LÈVEILLARD Thierry , MARTIN Jean‐Pierre
Published: March 2005

The human inter‐α‐trypsin inhibitor (ITI) light‐chain gene, which codes for the two proteins α1‐microglobulin (protein HC) and ITI‐derived human inhibitor of 30 kDa (HI‐30), was isolated from a human genomic library. This gene, present as a single copy in the human genome, is composed of 10 exons and 9 introns distributed over 20 kbp. A single transcriptional initiation site was identified in the 5′‐flanking region which contained promoter elements, but no typical TATA box. However a sequence equivalent to the TATA box is present on both sense and anti‐sense strands in the 5′‐flanking region of the first exon coding for HI‐3C. The exon‐intron organization suggets that the region coding for protein HC and other members of the lipocalin superfamily evolved from a common ancestral gene that is probabaly differnt from that coding for HI‐30 These data suggest that two distinct ancestral genes could have existed and fused during evolution. Several direct and one inverted repeats are also found within this gene, as well as potential glucorticoid‐receptor binding sites.
Solution NMR studies of colicin E1 C‐terminal thermolytic peptide
Authors: WORMALD Mark R. , MERRILL Allan R. , CRAMER William A. , WILLIAMS Robert J. P.
Published: March 2005

The aqueous solution structure of the C‐terminal thermolytic peptide of colicin E1 has been investigated using both one‐ and two‐dimensional NMR techniques. The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C‐terminal peptide in the crystalline state, although some differences have been noted. The one‐dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH. The in vintro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form. However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions. This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur.
Biochemical and genetic analysis of a maltopentaose‐producing amylase from an alkaliphilic Gram‐positive bacterium
Authors: CANDUSSIO Anton , SCHMID Gerhard , BÖCK August
Published: March 2005

Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A‐60 consists of a single type of polypeptide chain of 60 kDa and exhibits an α‐amylase‐type of starch cleavage. Amylase A‐180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A‐60 and A‐180 are immunologically unrelated enzymes. The structural gene for amylase A‐180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino‐terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino‐terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram‐positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70‐kDa N‐terminal part is similar to classical α‐amylases. The C‐terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial β‐amylases. It appears, therefore, that A‐180 has arisen by gene fusion events.
Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver
Authors: WETTSTEIN Matthias , GEROK Wolfgang , HÄUSSINGER Dieter
Published: March 2005

In single‐pass perfused rat liver, the sinusoidal uptake of infused 3H‐labelled leukotriene (LT) C4 (10nmol · 1−1) was inhibited by sulfobromophthalein. Inhibition was half‐maximal at sulfobromophthalein concentrations of approximately 1.2 μmol · 1−1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34% Sulfobromophthalein (20 μmol · 1−1) also decreased the uptake of infused [3H]LTE4 (10 nmol · 1−1) by 31%. Indocyanine green (10 μmol · 1−1) inhibited the sinusoidal [3H]LTC4 uptake by 19%.

Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol · 1−1) by 56%, but was without effect on the uptake of sulfobromophthalein.

The canalicular excretion of LTC4, LTD4 and N‐acetyl‐LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar ω‐oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4.

Following the infusion of [3H]LTD4 (10 nmol · 1−1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 μmol · 1−1), however, this reconversion was completely inhibited.

The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium‐dependent and a sodium‐independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N‐acetyl‐LTE4, but not with the excretion of ω‐oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.
Translation and regulation of translation in the yeast Saccharomyces cerevisiae
Authors: MÜLLER Peter P. , TRACHSEL Hans
Published: March 2005

In recent years the yeast Saccharomyces cerevisiae has become a model system for studies of eukaryotic translation and translation regulation. Analysis of mRNA structure, translation initiation factor sequences and the translation initiation pathway indicate, that translation in S. cerevisiae is very similar to translation in higher eukaryotes. The availability of powerful genetic techniques lead to the dissection in yeast of individual steps in the translation pathway, the detection of biochemical interactions between components involved in translation and the unravelling of complex regulation phenomena.
Cloning and sequence analysis of the genes encoding the α and β subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus
Authors: HAWKINS Christopher F. , BORGES Adolfo , PERMHAM Richard N.
Published: March 2005

A 4175‐bp EcoRI fragment of DNA that encodes the α and β chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1α subunit (368 amino acids, Mr 41312, without the initiating methionine residue) and E1β subunit (324 amino acids, Mr 35306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1β gene begins just 3 bp downstream from the E1a stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome‐binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1α and E1β subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1α and E1β subunits of pyruvate and branched‐chain 2‐oxo‐acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1α chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.
Peptidoglycan cross‐linking in Staphylococcus aureus
Authors: Snowden Michael A. , Perkins Harold R.
Published: March 2005

The peptidoglycan of Staphylococcus aureus contains relatively short glycan chains and is highly cross‐linked via its peptide chains. The material from wild‐type (strain H) and mutants H28, H4B and MR‐1 was freed from the teichoic‐acid‐linked component and then hydrolysed by Chalaropsis muramidase to yield disaccharide‐repeating units of the glycan with attached peptides either non‐cross‐linked (monomer) or joined to similar units by one (dimer), two (trimer) or more (oligomer) peptide cross links. The resulting fragments were separated by high‐resolution HPLC so that distinguishable components as large as nonamer could be identified. Extrapolation showed that, in S. aureus H, H28 and MR‐1, oligomers at least as large as eicosamer formed part of the smooth distribution of oligomer fragments, whereas in strain H4B (PBP4−) the maximum size was around dodecamer. The oligomer distribution profile was related to the polymerization theories of Flory, which allow a distinction to be made between a monomer addition model, whereby each oligomer can only be synthesized by the addition of a single monomer unit to its next lower homologue, and a random addition model, in which an oligomer can be formed by linkage of any combination of its constituent smaller units. In S. aureus close approximation to the random addition model for oligomer synthesis and hence for peptidoglycan cross‐linking was observed, both in PBP4+ and PBP4− mutants. The implications for secondary cross‐linking in S. aureus cell wall formation are inescapable, although the possibility of an endopeptidase/transpeptidase providing later modification of the peptidoglycan is not completely ruled out.
Covalent modification and metabolic control analysis
Authors: SMALL J. Rankin , FELL David A.
Published: March 2005

A study of the sensitivity properties of metabolic systems containing covalently modifiable enzymes and cascades has been carried out with the aid of metabolic control analysis. We have considered how the theorems of metabolic control analysis must be modified to take into account covalently modifiable enzymes, and have used these results to investigate the effects of increasing the total amount of modifiable enzyme. The sensitivity of system variables to an effector acting through a covalent‐modification cycle has also been investigated.
Stereochemical studies on algal pheromone biosynthesis
Authors: NEUMANN Christoph , BOLAND Wilhelm
Published: March 2005

Unsaturated C8 and C11 hydrocarbons act as chemical signals (chemotaxis) during sexual reproduction of many marine brown algae. One of these compounds, namely (+)‐(6S)‐6‐(1Z‐butenyl)cyclohepta‐1,4‐diene (=ectocarpene) is also formed as a major hydrocarbon by the flowering plant Senecio isatideus (Asteraceae). On administration of enantiospecifically labelled (8R)‐ or (8S)‐[7,8‐2H2]trideca‐3,6,9‐trienoic acid instead of the natural precursor dodeca‐3,6,9‐trienoic acid to this plant, the artificial C12 analogue of ectocarpene is formed. Mass spectroscopic analysis of the metabolites revealed this process to be enantiospecific for the C(8)‐HRe atom of the precursor acid. The stereochemical course of the overall reaction is in agreement with a precise, U‐shaped embedding of the trienoic acid into the active center of the enzyme(s) and Re‐attack onto a hydrogen at the C(8) methylene group of this precursor. The reactive intermediate cyclizes by π‐orbital interaction between C(4) and C(6) of the acid and is accompanied by decarboxylation. The first product is a thermally unstable (S,2R)‐cis‐1‐(1E3Z‐hexadienyl)‐2‐vinylcyclopropane, which immediately rearranges to (+)‐(6S)‐ectocarpene (homo‐Cope rearrangement). The present work provides first experimental evidence for a homo‐Cope rearrangement as a naturally occurring electrocyclic reaction.
Rapid turnover of low‐density lipoprotein receptor by a non‐lysosomal pathway in mouse macrophage J774 cells and inhibitory effect of brefeldin A
Authors: SHITE Satoshi , SEGUCHI Tadashi , SHIMADA Tatsuo , ONO Mayumi , KUWANO Michihiko
Published: March 2005

The low‐density lipoprotein (LDL) receptor of molecular mass 155 kDa was expressed on the cell surface of cultured mouse macrophage J774 cells. The conversion rate of precursor to mature form of LDL receptor in J774 cells was comparable to that in mouse fibroblast L cells. The half‐life of the LDL receptor of J774 cells was about 2 h, that of L cells was about 11 h. The rapid degradation of LDL receptor was not significantly inhibited by the lysosomotropic agents, chloroquine and NH4Cl, nor by the thiol‐protease inhibitors leupeptin and E‐64. By contrast, incubation at 18°C retarded the degradation of LDL receptor. Treatment of J774 cells with brefeldin A, an inhibitor of membrane transport between the endoplasmic reticulum and the Golgi apparatus, inhibited the rapid turnover of the LDL receptor. Even after a 9‐h chase in the presence of brefeldin A, LDL receptor 5–10 kDa smaller than the normal mature form was found to be stable. Rapid turnover of the LDL receptor in the macrophages appeared to occur after exit from the Golgi apparatus, possibly during transport of the LDL receptor to the plasma membrane.
Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain
Authors: DEUTZMANN Rainer , AUMAILLEY Monique , WIEDEMANN Hanna , PYSNY Werner , TIMPL Rupert , EDGAR David
Published: March 2005

The cell adhesion, spreading and neurite‐promoting properties of mouse tumor laminin fragment E8. which contains major site(s) responsible for laminin ‐ cell interactions, were probed by proteolytic degradation, denaturation, synthetic peptides and antibody inhibition. Removal of more than half of the N‐terminal portion contributing to the rod‐like domain did not effect cell attachment or spreading although neurite‐promoting activity was reduced. More extensive degradation of the rod or of the globular domains of E8, or separation of the globule from the rod, also resulted in loss of cell spreading activity although weak attachment was found to an A chain subfragment comprising the globular domain and a short piece of the rod. Exposure of E8 to increasing concentrations of dissociating agents produced an apparently reversible denaturation but an irreversible loss of both attachment and neurite‐promoting activities, as did reduction and alkylation of disulfide bonds in the globular domain. Although cell adhesion and spreading were blocked by antibodies to an α6 integrin subunit, neurite outgrowth was unaffected, indicating two distinct receptors for these two activities. Furthermore, a synthetic peptide, the sequence of which is found in the vicinity of adhesion and neurite‐promoting sites and previously implicated in neurite growth and cell attachment activities, was found to be inactive. These results indicate that the major cell attachment and neurite‐promoting sites of laminin are distinct although both require the native conformation of parts of the rod and the terminal globular domain of the long arm of laminin.
Flavin‐photosensitized oxidation of reduced c‐type cytochromes
Authors: RONCEL Mercedes , HERVÁS Manuel , NAVARRO José A. , de la ROSA Miguel A. , TOLLIN Gordon
Published: March 2005

In order to compare the oxidation and reduction reactions of c‐type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20–100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin–cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.
Primary structure of human placental 5′‐nucleotidase and identification of the glycolipid anchor in the mature form
Authors: MISUMI Yoshio , OGATA Shigenori , OHKUBO Kumiko , HIROSE Shinichi , IKEHARA Yukio
Published: March 2005

A cDNA was cloned coding for human placental 5′‐nucleotidase. The 3547‐bp cDNA contains an open reading frame that encodes a 574‐residue polypeptide with a calculated size of 63375 Da. The NH2‐terminal 26 residues comprise a signal peptide, which is followed by the NH2‐terminal sequence of the purified protein. Four potential N‐linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post‐translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane‐anchoring domain of 5′‐nucleotidase. BrCN‐cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys‐Val‐Ile‐Tyr‐Pro‐Ala‐Val‐Glu‐Gly‐Arg‐Ile‐Lys‐Phe‐Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510–523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5′‐nucleotidase lacks the predicted COOH‐terminal peptide extension (524–548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5′‐nucleotidase.
Suppression of the ‘uncovering’ of mannose‐6‐phosphate residues in lysosomal enzymes in the presence of NH4Cl
Authors: ISIDORO Ciro , RADONS Jürgen , BACCINO Franceso M. , HASILIK Andrej
Published: March 2005

The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is > 95%. It is only slightly decreased in cells incubated in the presence of 1α,25‐dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N‐acetylglucosamine‐1‐phosphodiester α‐N‐acetylglucosaminidase, as determined with UDP–N‐acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic‐pH‐dependent functions of lysosomal compartments and of mannose‐6‐phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose‐6‐phosphate residues.
A novel gene member of the human glycophorin A and B gene family
Authors: VIGNAL Alain , RAHUEL Cécile , LONDON Jacqueline , CHERIF ZAHAR Baya , SCHAFF Sophie , HATTAB Claude , OKUBO Yasuko , CARTRON Jean‐Pierre
Published: March 2005

A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6‐kb message which encodes a 78‐amino‐acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M‐type glycophorin A (GPA), but the C‐terminal domain (residues 27 – 59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5′ region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a‐), S‐s‐U‐ and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.
Structure of the arylsulfatase A gene
Authors: KREYSING Joachim , von FIGURA Kurt , GIESELMANN Volkmap
Published: March 2005

A 14‐kb genomic clone containing the entire gene of human lysosomal arylsulfatase A was isolated. The arylsulfatase A gene is about 3.2 kb long and has eight exons (103–320 nucleotides in size). All intron–exon splice junctions conformed to the GT/AG consensus sequence. S1 nuclease mapping shows multiple transcription initiation sites between nucleotides – 367 and – 387. A fragment encompassing 360 nucleotides of the flanking sequence upstream of the transcription initiation site shows promoter activity when it was transiently expressed in COS cells using the gene for bacterial chloramphenicol acetyltransferase as a reporter gene. This putative promoter region shows four potential Sp1 binding sites but lacks typical TATA and CAAT box sequences. Three different mRNA species of 2.1, 3.7 and 4.8 kb are transcribed from the gene and arise probably from the use of different polyadenylation signals.
Functional properties of phosphorylated elongation factor 2
Authors: CARLBERG Ulf , NILSSON Anders , NYGÅRD Odd
Published: March 2005

The effect of phosphorylation on the functional activity of eukaryotic elongation factor 2 (eEF‐2) was studied using a purified phosphorylated factor. The modified factor was unable to stimulate protein synthesis in an eEF‐2‐dependent rabbit reticulocyte lysate. The functional alteration was further analyzed by measuring the effects of phosphorylation on the ability of the factor to catalyse the ribosome‐dependent hydrolysis of GTP. Kinetic analysis showed that both phosphorylated and unmodified factor was able to hydrolyse GTP with approximately the same maximum rate, indicating that the rate of nucleotide exchange was not impaired by the modification. However, the phosphorylated factor showed a marked reduction in the second‐order rate constant, suggesting that the phosphorylation interfered with ribosome · eEF‐2 complex formation by reducing the affinity of eEF‐2 for the ribosome. This assumption was confirmed by direct measurements of the dissociation constants for the ribosomal complexes containing unmodified and phosphorylated eEF‐2.
Purification and characterisation of TOL plasmid‐encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida
Authors: SHAW Jeffrey P. , HARAYAMA Shigeaki
Published: March 2005

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram‐negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 μM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 μM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH‐blocking agents such as N‐ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro‐R side of the prochiral C4 of the pyridine ring of NAD+.
Purification and characterization of bovine brain type I phosphatidylinositol kinase
Authors: MORGAN Sarah J. , SMITH Anthony D. , PARKER Peter J.
Published: March 2005

Investigation into the phosphatidylinositol kinase activities in bovine brain has revealed the presence of a type I PtdIns kinase activity. This classification is based upon potent inhibition by neutral detergent and the production of a phosphatidylinositol phosphate that can be distinguished from phosphatidyl‐inositol 4‐phosphate [PtdIns(4)P] by thin‐layer chromatography. The enzyme has been substantially purified and the activity is associated with an 85‐kDa polypeptide on SDS/polyacrylamide gel electrophoresis. Analysis of the product confirms the identification of the enzyme as a type I PtdIns kinase. The purified kinase has been characterized with respect to substrate dependence (Mg2+, ATP, PtdIns), substrate presentation (pure lipid versus mixed micelle) and specificity [PtdIns versus PtdIns(4)P and phosphatidylinositol 4,5‐bisphosphate].
Molecular cloning of the gene for the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae
Authors: STEENSMA H. Yde , HOLTERMAN Lennart , DEKKER Iris , van SLUIS Cees A. , WENZEL Thibaut J.
Published: March 2005

The E1α and E1β subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector λgt11.

The gene encoding the E1α subunit was unique and localized on a 1.7‐kb HindIII fragment from chromosome V. The identity of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti‐E1α serum. (b) Gene replacement at the 1.7‐kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti‐E1α serum in haploid cells. In addition, the 1.7‐kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N‐terminal and central regions of the human E1α peptide.

We propose to call the gene encoding the E1α subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the λgt11 library using the anti‐E1β serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.
Protein HMG‐17 is hyper‐expressed in rat glucagonoma
Authors: NIELSEN Egon , WELINDER Benny , MADSEN Ole D.
Published: March 2005

High‐mobility‐group protein 17 (HMG‐17) was identified by reversed‐phase high‐performance liquid chromatography analysis as a major component in acidic extracts of transplantable rat glucagonoma tissue but not in insulinoma tissue of similar origin. The peptide was purified in a single step and the entire sequence of 89 amino acids was determined. Rat HMG‐17 has a molecular mass of 9238 Da and shows strong similarity to human, bovine (94.4%) and chicken (88.8%) HMG‐17. Six of the seven residues which vary among the mammalian sequences are located within a short segment (positions 64–83) present in the acidic, non‐DNA‐binding C‐terminal part of HMG‐17. This region shows least similarity to the otherwise related proteins HMG‐14 and H6 (a trout HMG protein). Interestingly, four of the six variable positions are Asp in rat HMG‐17 which results in an overall net increase in the negative charge of the C‐terminal region. The nature of selective hyper‐expression of HMG‐17 in glucagon but not in insulin‐producing tumor tissue remains to be clarified.