bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial thioredoxin reductase in bovine adrenal cortex
Authors: Watabe Shoji , Makino Yumiko , Ogawa Kazuo , Hiroi Tomoko , Yamamoto Yoshimi , Takahashi Susumu Y.
Published: March 2003

Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin‐dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH‐dependent reduction of 5,5′dithiobis(2‐nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, dl‐α‐lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C‐terminal region of mitochondrial (mt)‐thioredoxin reductase by means of MS and amino acid sequence analyses of the C‐terminal fragment. The four‐amino acid motif, Gly‐Cys‐Sec‐Gly, which is conserved among all Sec‐containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1‐chloro‐2,4‐dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.
Exocytosis of active cathepsin B
Authors: Linebaugh Bruce E. , Sameni Mansoureh , Day Nancy A. , Sloane Bonnie F. , Keppler Daniel
Published: March 2003

Lysosomal cathepsin B has been implicated in parasitic, inflammatory and neoplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is not well defined. The activity of extracellular cathepsin B is difficult to assess because of the presence of inhibitors and inactivation of the enzyme by oxidizing agents. Therefore, we have developed a continuous assay for measurement of cathepsin B activity produced pericellularly by living cells. The kinetic rate of Z‐Arg‐Arg‐NHMec conversion was monitored and the assay optimized for enzyme stability, cell viability and sensitivity. To validate the assay, we determined that human liver cathepsin B was stable and active under the conditions of the assay and its activity could be inhibited by the selective epoxide derivative CA‐074. Via this assay, we were able to demonstrate that active cathepsin B was secreted pericellularly by viable cells. Both preneoplastic and malignant cells secreted active cathepsin B. Pretreatment of cells with the membrane‐permeant proinhibitor CA‐074Me completely abolished pericellular and total cathepsin B activity whereas pretreatment with the active drug CA‐074 had no effect. Immunoprecipitation and immunoblotting experiments suggested that the active enzyme species was 31‐kDa single‐chain cathepsin B. Exocytosis of cathepsin B was not related to secretion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.
Feedback regulation of DNA methyltransferase gene expression by methylation
Authors: Slack Andrew , Cervoni Nadia , Pinard Marc , Szyf Moshe
Published: March 2003

This paper tests the hypothesis that expression of the DNA methyltransferase, dnmt1, gene is regulated by a methylation‐sensitive DNA element. Methylation of DNA is an attractive system for feedback regulation of DNA methyltransferase as the final product of the reaction, methylated DNA, can regulate gene expression in cis. We show that an AP‐1‐dependent regulatory element of dnmt1 is heavily methylated in most somatic tissues and in the mouse embryonal cell line, P19, and completely unmethylated in a mouse adrenal carcinoma cell line, Y1. dnmt1 is highly over expressed in Y1 relative to P19 cell lines. Global inhibition of DNA methylation in P19 cells by 5‐azadeoxycytidine results in demethylation of the AP‐1 regulatory region and induction of dnmt1 expression in P19cells, but not Y1 cells. We propose that this regulatory region of dnmt1 acts as a sensor of the DNA methylation capacity of the cell. These results provide an explanation for the documented coexistence of global hypomethylation and high levels of DNA methyltransferase activity in many cancer cells and for the carcinogenic effect of hypomethylating diets.
Determining and understanding the control of flux
Authors: Moreno‐Sánchez Rafael , Bravo Concepción , Westerhoff Hans V.
Published: March 2003

Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, ‘direct control analysis’ method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 °C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, ‘elasticity based’ method, the elasticity coefficients of the respiratory chain or the H+‐ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH‐CoQ oxidoreductase, and H+‐ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.
Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase
Authors: Alexandrov Kirill , Simon Iris , Yurchenko Vyacheslav , Iakovenko Andrei , Rostkova Elena , Scheidig Axel J. , Goody Roger S.
Published: March 2003

Geranylgeranylation is a post‐translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7–REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7–REP complex. For this interaction we determined a Kd value of about 120 nm.

 Association of RabGGTase with the Rab7–REP complex occurs with a rate constant of ≈ 108 m−1·s−1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7–REP‐1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C‐terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C‐terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7–REP‐1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C‐terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then ‘scans’ the flexible C‐terminus to position the proximal cysteines into the active site.
Modulation of aldosterone biosynthesis by adrenodoxin mutants with different electron transport efficiencies
Authors: Cao Pei‐rang , Bernhardt Rita
Published: March 2003

Aldosterone biosynthesis is highly regulated on different levels by hormones, potassium, lipid composition of the membrane and the molecular structure of its gene. Here, the influence of the electron transport efficiency from adrenodoxin (Adx) to CYP11B1 on the activities of bovine CYP11B1 has been investigated using a liposomal reconstitution system with truncated mutants of Adx. It could be clearly demonstrated that Adx mutants Adx 4‐114 and Adx 4‐108, possessing enhanced electron transfer abilities, produce increases in corticosterone and aldosterone biosynthesis. Based on the Vmax values of corticosterone and aldosterone formation, Adx 4‐108 and Adx 4‐114 enhance corticosterone synthesis 1.3‐fold and aldosterone formation threefold and twofold, respectively. The production of 18‐hydroxycorticosterone was changed only slightly in these Adx mutants. The effect of Adx 1‐108 on the product patterns of bovine CYP11B1, human CYP11B1 and human CYP11B2 was confirmed in COS‐1 cells by cotransfection of CYP11B‐ and Adx‐containing expression vectors. It could be shown that Adx 1‐108 enhances the formation of aldosterone by bovine CYP11B1 and by human CYP11B2, and stimulates the production of corticosterone by bovine CYP11B1 and human CYP11B1 and CYP11B2 also.
Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues
Authors: Kapurniotu Aphrodite , Kayed Rakez , Taylor John W. , Voelter Wolfgang
Published: March 2003

Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget’s disease. Our studies on the conformational features of human calcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21‐[Asp17,Lys21]hCt (1), which had a 5–10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem.38, 836–847]. We hypothesized that a stabilized, possibly type I β turn/β sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19‐member to 17‐member ring‐size analogues of 1 with the structure cyclo17,21‐[Asp17,XX21]hCt with XX = Orn (2), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19‐member cyclo17,21‐[Glu17,Dab21]hCt (6). Analyses of the far‐UV CD spectra indicated increased type I β turn and antiparallel β sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21‐[Asp17,Orn21]hCt (2) was found to have a 400‐fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30‐fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21‐[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in ‘freezing’ hCt in a bioactive conformation. Our findings provide evidence for the first time that a β turn/β sheet conformation in region 17–21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead structure for a hCt‐based drug for the treatment of osteoporosis and other bone‐disorder‐related diseases.
The phosphotransferase system (PTS) of Streptomyces coelicolor
Authors: Parche Stephan , Schmid Roland , Titgemeyer Fritz
Published: March 2003

HPr, the histidine‐containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low‐GC Gram‐positive bacteria and in Gram‐negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N‐terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr‐like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well‐conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine‐tagged fusion protein. Histidine‐tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate‐dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose‐specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr‐P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP‐dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.
Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris
Authors: Raemaekers Romaan J. M. , de Muro Laura , Gatehouse John A. , Fordham‐Skelton Anthony P.
Published: March 2003

Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E‐ and L‐forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomycesα‐factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the α‐factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA‐E, PHA‐L and GNA with heterogenous N‐termini, with the majority of the protein containing N‐terminal extensions derived from the α‐factor prosequence. Polypeptides in which most of the α‐factor prosequence was present were also glycosylated. Inclusion of Glu‐Ala repeats at the C‐terminal end of the α‐factor preprosequence led to efficient processing N‐terminal to the Glu‐Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N‐termini still containing N‐terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA‐E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.
Genomic analysis of a murine cell‐surface sialomucin, MGC‐24/CD164
Authors: Kurosawa Nobuyuki , Kanemitsu Yukihide , Matsui Takanori , Shimada Kazuyuki , Ishihama Hidenori , Muramatsu Takashi
Published: March 2003

MGC‐24 is a sialomucin originally found in human gastic carcinoma cells, and in human hematopoietic progenitor cells. In the human, soluble and transmembrane forms of MGC‐24 are present, and the transmembrane form has been implicated in adhesion of hematopoietic progenitor cells to marrow stroma cells. In the mouse, we found that only the transmembrane form was expressed in many organs. Northern blotting and in situ hybridization analysis showed that MGC‐24 mRNA was widely expressed in various adult and embryonic tissues. The mouse MGC‐24 gene, which we isolated, spanned about 12 kb and was comprised of six exons. The transmembrane domain and the cytoplasmic domain were encoded by a single exon; the finding agrees with the absence of an alternatively spliced product of mouse MGC‐24. The minimal promoter of mouse MGC‐24 was embedded in GC‐rich sequences, in which two Sp1 binding motifs were found, but it lacked TATA and CAAT boxes. That the promoter resembles that of house‐keeping genes is consistent with the broad expression of mouse MGC‐24 mRNA.
Kinetics of prostanoid synthesis by macrophages is regulated by arachidonic acid sources
Authors: Gonchar Maria , Sergeeva Marina , Mevkh Alevtina , Varfolomeyev Sergey
Published: March 2003

The dependence of prostanoid synthesis on the nature of free arachidonic acid (AA) appearance was investigated in mouse peritoneal macrophages. AA delivery from intracellular sources to the constitutive prostaglandin (PG)H synthase was provided by action of calcium‐ionophore A23187; and from extracellular sources by AA addition to the culture medium. It was found that the kinetics of prostanoid synthesis dramatically depends on the sources of AA. Free AA concentration used for prostanoid synthesis is either a constant or a variable value depending upon the sources. The kinetics of cellular prostanoid synthesis can be regulated by the following processes: (a) the irreversible inactivation of PGH‐synthase in the course of the reaction (kin), (b) prostanoid metabolism (kr), and (c) incorporation of exogenous AA into cellular membranes (ka). From our experiments and mathematical calculation these parameters were found to be kin = 0.20 ± 0.02 min−1, kr = 0.17 ± 0.03 min−1 in the case of stimulation with A23187, and kin = 0.0156 min−1, kr = 0.0134 min−1, ka = 0.0025 min−1 in the case of exogenous AA addition. The studies of prostanoid biosynthesis by macrophage microsomes led to independent determination of kin = 0.20 ± 0.02 min−1. This value perfectly fits the kinetics of the prostanoid cell synthesis under endogenous AA supply but shows a 10‐fold decrease in the case of exogenous AA supply. Our study on the kinetics of prostanoid synthesis by mouse peritoneal macrophages clearly demonstrate that AA is able to regulate cellular prostanoid synthesis in the presence of constitutive PGH‐synthase only. A regulation mechanism based on the co‐operation of the constitutive PGH‐synthase isoform and the availability of free AA is proposed and could be confirmed by mathematical modelling.
How do acetylcholine receptor ligands reach their binding sites?
Authors: Sáez‐Briones Patricio , Krauss Michael , Dreger Mathias , Herrmann Andreas , Tsetlin Victor I. , Hucho Ferdinand
Published: March 2003

The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with α‐neurotoxins. Binding assays with [125I]α‐bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor‐rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically ‘hindered’ in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the α/γ‐ but not to the α/δ‐binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor’s binding sites. Together, our data suggest (a) that α‐neurotoxins approach their binding sites from the membrane‐facing periphery of the receptor’s extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.
Structure and transcriptional regulation of the ovine placental lactogen gene
Authors: Liang Rongti , Limesand Sean W. , Anthony Russell V.
Published: March 2003

Ovine placental lactogen (oPL), a member of the growth hormone/prolactin gene family, is produced by chorionic binucleate cells at the maternal–fetal interface, and is thought to modulate metabolic processes and enhance fetal growth. We have determined that the oPL gene contains five exons and four introns, and the transcriptional start site was mapped 91 bp 5′ of the initiation codon (AUG). An additional 4.5 kb of 5′‐flanking sequence was sequenced and used for transient transfection analysis in human (BeWo) and rat (Rcho‐1) choriocarcinoma cell lines to examine trophoblast cell‐specific activity. Trophoblast cell‐specific transactivation of the reporter gene was conferred by the proximal 1.1 kb of oPL gene 5′‐flanking sequence. Transfection of deletion constructs derived from the 1.1 kb of 5′‐flanking sequence resulted in varying profiles of transactivation between the two choriocarcinoma cell lines, but maximal activation in both cell lines resided within the proximal 383 bp of oPL gene 5′‐flanking sequence. DNase I protection analysis using ovine chorionic binucleate cell nuclear protein, identified 19 footprints within the 1.1‐kb sequence, six of which are located within the 383‐bp region. Electrophoretic mobility‐shift assays and mutational analysis identified two functional GATA (−67, −102) sequences as transactivators of the oPL gene. However, a previously undefined element (GAGGAG) residing at −338 and −283 is required for full transactivation, and mutation of either significantly reduces reporter activity. In addition, an AP‐2 site (−58) and an E‐box (−163) were identified and may coordinate oPL transactivation. Transcriptional regulation of human and rodent PL genes has been previously characterized, and our results indicate that tissue‐specific regulation of oPL expression may result from cis‐acting elements in common with human and rat genes expressed within the placenta. However, our data indicate that regulation of oPL also results from novel cis‐acting elements.
Mutations of glutamate‐84 at the putative potassium‐binding site affect camphor binding and oxidation by cytochrome P450cam
Authors: Westlake Andrew C. G. , Harford‐Cross Charles F. , Donovan Jacqueline , Wong Luet‐Lok
Published: March 2003

Cytochrome P450cam(CYP101) fromPseudomonas putidais unusual among P450 enzymes in that it exhibits co‐operative binding between the substrate camphor and a potassium ion. This behaviour has been investigated by mutagenesis of Glu84, a surface residue which forms part of the cation‐binding site. Substitutions that neutralize or reverse the charge of this side chain are shown to disrupt the co‐operativity of potassium and camphor binding by P450cam, and also to influence the catalytic activity. In particular, replacement of Glu84 by positively charged residues such as lysine results in increased high‐spin haem fractions and camphor turnover activities in the absence of potassium, along with decreased camphor dissociation constants. However, in the presence of potassium the camphor dissociation constants of these mutants are significantly increased compared with the wild‐type, although the camphor turnover activities remain marginally higher. In contrast, substitution by aspartate results in tighter binding of both potassium and camphor, but has little effect on the enzymatic activity. In all cases the reaction remains essentially 100% coupled and gives 5‐exo‐hydroxycamphor as the only product. These results suggest that an anionic side chain at the 84 position is crucial for the co‐operativity of camphor and cation binding, and that the physiological role for potassium binding by cytochrome P450camis to promote camphor binding even at the expense of turnover rate, thus allowing the organism to utilize low environmental concentrations of this substrate for growth.
Postsynaptic short‐chain neurotoxins from Pseudonaja textilis
Authors: Gong Nanling , Armugam Arunmozhiarasi , Jeyaseelan Kandiah
Published: March 2003

Two lethal proteins, which specifically bind to the nAChR from Torpedo californica, were isolated from the venom of Pseudonaja textilis, the common brown snake from Australia. The isolated proteins have masses of 6236 and 6345 Da and are structurally related to short‐chain neurotoxins from other elapids. Six cDNAs encoding isoforms of related neurotoxins were cloned using the RT‐PCR of the venom gland mRNAs. The sequences of the corresponding proteins consist of 57–58 amino acid residues and display several unique features when compared with all known short‐chain neurotoxins. Accordingly, they grouped separately in phylogenetic analysis. The six cDNAs were expressed in Escherichia coli and the recombinant proteins were characterized. They have similar masses and display similar toxicities and binding constants to the nAChR as the native toxins isolated from the venom. Thus, a new group of short‐chain postsynaptic neurotoxins from the venom of an Australian elapid has been characterized.
Essential involvement of 12‐lipoxygenase in regiospecific andstereospecific oxidation of low density lipoprotein by macrophages
Authors: Sakashita Toshiki , Takahashi Yoshitaka , Kinoshita Takahiro , Yoshimoto Tanihiro
Published: March 2003

To establish a role of the 12‐lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12‐lipoxygenases in a macrophage‐like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12‐lipoxygenase‐expressing cells were incubated with 400 µg·mL−1 LDL in Dulbecco’s modified Eagle’s medium at 37 °C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock‐transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S‐hydroxyoctadeca‐9Z,11E‐dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12‐lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12‐lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.
The main fatty acid‐binding protein in the liver of the shark (Halaetunus bivius) belongs to the liver basic type
Authors: Córdoba Osvaldo L. , Sánchez Eduardo I. , Santomé José A.
Published: March 2003

Three fatty acid‐binding proteins (FABPs) from the liver of the shark Halaetunus bivius were isolated and characterized: one of them belongs to the liver‐type FABP family and the other two to the heart‐type FABP family. The complete primary structure of the first FABP, and partial primary structures of the two others, were determined. The liver‐type FABP constitutes 69% of the total FABPs, and its amino acid sequence presents the highest identity with chicken, catfish, iguana and elephant fish liver basic FABPs. The L‐FABP protein has low affinity for palmitic and oleic acids and high affinity for linoleic and arachidonic acids and other hydrophobic ligands, all of them important for the metabolic functions of the liver. In contrast, both heart‐type FABPs have the highest affinity for palmitic acid, the principal fatty acid mobilized from fat deposits for β‐oxidation.
Characterization and molecular cloning of an adenosine kinase from Babesia canis rossi
Authors: Carret Céline , Delbecq Stéphane , Labesse Gilles , Carcy Bernard , Precigout Eric , Moubri Karina , Schetters Theo P. M. , Gorenflot André
Published: March 2003

In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA was cloned and sequenced. Protein sequence database searches suggested that the 41‐kDa protein belongs to the phosphofructokinase B type family (PFK‐B). However, because of the low level sequence identity (< 20%) of the protein both with adenosine and sugar kinases from this family, its structural and functional features were further investigated using molecular modelling and enzymatic assays. The sequence/structure comparison of the protein with the crystal structure of a member of the PFK‐B family, Escherichia coli ribokinase (EcRK), suggested that it might also form a stable and active dimer and revealed conservation of the ATP‐binding site. However, residues specifically involved in the ribose‐binding sites in the EcRK sequence (S and N) were substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds rather than sugar ones. Enzymatic assays using a purified glutathione S‐transferase fusion protein revealed that this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nm, whereas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of the PFK‐B family corresponds to an adenosine kinase from B. canis rossi. It was named BcrAK.
Farnesylation of Ras is important for the interaction with phosphoinositide 3‐kinase γ
Authors: Rubio Ignacio , Wittig Ute , Meyer Claudia , Heinze Regina , Kadereit Dieter , Waldmann Herbert , Downward Julian , Wetzker Reinhard
Published: March 2003

The correct functioning of Ras proteins requires post‐translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras‐effector proteins. We describe a specific, GTP‐independent interaction between post‐translationally modified Ha‐ and Ki‐Ras4B and the G‐protein responsive phosphoinositide 3‐kinase p110γ. Our data demonstrate that post‐translational processing increases markedly the binding of Ras to p110γin vitro and in Sf9 cells, whereas the interaction with p110α is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110γ and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf‐1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110γ.
The responses of rat hepatocytes to glucagon and adrenaline
Authors: Ainscow Edward K. , Brand Martin D.
Published: March 2003

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus.

 Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6‐phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels.

 The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6‐phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.
Cloning and expression of succinic semialdehyde reductase from human brain
Authors: Schaller Micheline , Schaffhauser Matteo , Sans Notker , Wermuth Bendicht
Published: March 2003

The neuromodulator γ‐hydroxybutyrate is synthesized in vivo from γ‐aminobutyrate by transamination to succinic semialdehyde and subsequent reduction of the aldehyde group. In human brain, succinic semialdehyde reductase is thought to be responsible for the conversion of succinic semialdehyde to γ‐hydroxybutyrate. In the present work, we cloned the cDNA coding for succinic semialdehyde reductase and expressed it in Escherichia coli. A data bank search indicated that the enzyme is identical with aflatoxin B1‐aldehyde reductase, an enzyme implicated in the detoxification of xenobiotic carbonyl compounds. Structurally, succinic semialdehyde reductase thus belongs to the aldo–keto reductase superfamily. The recombinant protein was indistinguishable from native human brain succinic semialdehyde reductase by SDS/PAGE. In addition to succinic semialdehyde, it readily catalyzed the reduction 9,10‐phenanthrene quinone, phenylglyoxal and 4‐nitrobenzaldehyde, typical substrates of aflatoxin B1 aldehyde reductase. The results suggest multiple functions of succinic semialdehyde reductase/aflatoxin B1 aldehyde reductase in the biosynthesis of γ‐hydroxybutyrate and the detoxification of xenobiotic carbonyl compounds, respectively.
Is β‐poly(L‐malate) synthesis catalysed by a combination of β‐L‐malyl‐AMP‐ligase and β‐poly(L‐malate) polymerase?
Authors: Willibald Bertram , Bildl Wolfgang , Lee Bong‐Seop , Holler Eggehard
Published: March 2003

β‐Poly(l‐malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [14C]β‐poly(l‐malate) from injected l‐[14C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5′‐(α,β‐methylene)triphosphate, adenosine 5′‐(β,γ‐methylene)triphosphate, guanosine 5′‐(β,γ‐methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on β‐poly(l‐malate) synthesis were studied after their coinjection with l‐[14C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5′‐(α,β‐methylene)triphosphate. The plasmodium lysate catalysed an l‐malate‐dependent ATP‐[32P]pyrophosphate exchange, but was devoid of β‐poly(l‐malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of β‐poly(l‐malate), involving the polymerization of β‐l‐malyl‐AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of β‐poly(l‐malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5′‐(β,γ‐methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.
GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the ω subunit in vivo
Authors: Mukherjee Kakoli , Nagai Hiroki , Shimamoto Nobuo , Chatterji Dipankar
Published: March 2003

Highly purified Escherichia coli RNA polymerase contains a small subunit termed ω that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. E. coli strains lacking ω (ω‐less) are viable, but exhibit a slow‐growth phenotype. Renaturation of RNA polymerase isolated from an ω‐less mutant, in the presence of ω, resulted in maximum recovery of activity. The ω‐less RNA polymerase from ω‐less strains recruits the chaperonin, GroEL (unlike the wild‐type enzyme), suggesting a structural deformity of the mutant enzyme. The GroEL‐containing core RNA polymerase interacts efficiently with σ70 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to σ70. The damaged enzyme regained activity after going through a cycle of denaturation and reconstitution in the presence of ω or GroEL. GroES was found to have an inhibitory effect on the core–σ70 association unlike the ω subunit. The ω subunit may therefore be needed for stabilization of the structure of RNA polymerase.
Structural studies utilizing 13C‐enrichment of the O‐antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross‐reacting with Shigella dysenteriae type 4
Authors: Linnerborg Malin , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigen polysaccharide from Escherichia coli O159 has been determined using primarily NMR spectroscopy of the 13C‐enriched polysaccharide. The sequence of the sugar residues could be determined by heteronuclear multiple bond connectivity NMR experiments. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure:

Matrix assisted laser desorption ionization mass spectrometry was performed on intact lipopolysaccharide and from the resulting molecular mass the O‐antigen part was estimated to contain approximately 23 repeating units. Cross‐reactivity of this O‐antigen to that of Shigella dysenteriae type 4 was confirmed using enzyme‐linked immunoabsorbant assay.
Expression and compartmentation of the glucose repressor CRE1 from the phytopathogenic fungus Sclerotinia sclerotiorum
Authors: Vautard‐Mey Géraldine , Cotton Pascale , Fèvre Michel
Published: March 2003

The glucose repressor from the phytopathogenic fungus Sclerotinia sclerotiorum is encoded by the cre1 gene. Polyclonal antibodies were raised against a fusion protein (gluthathione S‐transferase) GST–CRE1 in order to study cre1 expression. Western blot analyses revealed that CRE1 synthesis is regulated by the nature of the extracellular carbon source. High CRE1 levels are induced by glucose and remain stable after transfer into pectin medium, suggesting the existence of post‐translational mechanisms which inactivate CRE1 to allow transcription of glucose‐repressed genes. Subcellular fractionation demonstrated that CRE1 is localized in the nuclei of glucose grown hyphae and in the cytoplasm when glucose is removed from the culture medium. CRE1 fused to green fluorescent protein (GFP) was introduced into Aspergillus nidulans. Fluorescence microscopy showed the nuclear localization of the GFP–CRE1 fusion protein according to the presence of glucose in the culture medium, suggesting homologous post‐translational regulations of glucose repressors in fungi. We propose that filamentous fungi regulate the activity of the glucose repressor by controlling its nuclear translocation.
α‐Fetoprotein causes apoptosis in tumor cells via a pathway independent of CD95, TNFR1 and TNFR2 through activation of caspase‐3‐like proteases
Authors: Dudich Elena , Semenkova Lidia , Dudich Igor , Gorbatova Elena , Tochtamisheva Natasha , Tatulov Edward , Nikolaeva Marina , Sukhikh Gennady
Published: March 2003

α‐Fetoprotein (AFP) is an oncoembryonal protein with multiple cell growth regulating, differentiating and immunosuppressive activities. Previous studies have shown that treatment of tumor cells in vitro with 1–10 μm AFP produces significant suppression of tumor cell growth by inducing dose‐dependent cytotoxicity, but the molecular mechanisms underlying these AFP functions are obscure. Here, we show that AFP cytotoxicity is closely related to apoptosis, as shown by cell morphology, nuclear DNA fragmentation and caspase‐3‐like activity resulting in cleavage of poly(ADP‐ribose) polymerase. Apoptosis was significantly inhibited by a CPP32 family protease inhibitor whereas a general caspase inhibitor had no inhibitory effect, showing some enhancement of AFP‐mediated cell death. Using fluorogenic caspase substrates, we found that caspase‐3‐like proteases were activated as early as 4 h after treatment of Raji cells with 15 μm AFP, whereas caspase‐1, caspase‐8, and caspase‐9‐like activity was not detected during the time interval 0.5–17 h. AFP treatment of Raji cells increased Bcl‐2 protein, showing that AFP‐induced apoptosis is not explained by downregulation of the Bcl‐2 gene. This also suggests that AFP operates downstream of the Bcl‐2‐sensitive step. AFP notably decreased basal levels of soluble and membrane‐bound Fas ligand. Incubation of AFP‐sensitive tumor cells (HepG2, Raji) with neutralizing anti‐Fas, anti‐tumor necrosis factor receptor (TNFR)1 or anti‐TNFR2 mAb did not prevent AFP‐induced apoptosis, demonstrating its independence of Fas‐dependent and TNFR‐dependent signaling. In addition, it was found that cells resistant to TNF‐induced (Raji) or Fas‐induced (MCF‐7) apoptosis are, nevertheless, sensitive to AFP‐mediated cell death. In contrast, cells sensitive to Fas‐mediated cell death (Jurkat) are completely resistant to AFP. Taken as a whole, our data demonstrate that: (a) AFP induces apoptosis in tumor cells independently of Fas/Fas ligand or TNFR/TNF signaling pathways, and (b) AFP‐mediated cell death involves activation of the effector caspase‐3‐like proteases, but is independent of upstream activation of the initiator caspase‐1, caspase‐8, and caspase‐9‐like proteases.
Effects of ligand binding on the internal dynamics of maltose‐binding protein
Authors: Döring Klaus , Surrey Thomas , Nollert Peter , Jähnig Fritz
Published: March 2003

Ligand binding to proteins often causes large conformational changes. A typical example is maltose‐binding protein (MBP), a member of the family of periplasmic binding proteins of Gram‐negative bacteria. Upon binding of maltose, MBP undergoes a large structural change that closes the binding cleft, i.e. the distance between its two domains decreases. In contrast, binding of the larger, nonphysiological ligand β‐cyclodextrin does not result in closure of the binding cleft. We have investigated the dynamic properties of MBP in its different states using time‐resolved tryptophan fluorescence anisotropy. We found that the ‘empty’ protein exhibits strong internal fluctuations that almost vanish upon ligand binding. The measured relaxation times corresponding to internal fluctuations can be interpreted as originating from two types of motion: wobbling of tryptophan side‐chains relative to the protein backbone, and orientational fluctuations of entire domains. After binding of a ligand, domain motions are no longer detectable and the fluctuations of some of the tryptophan side‐chains become rather restricted. This transformation into a more rigid state is observed upon binding of both ligands, maltose and the larger β‐cyclodextrin. The fluctuations of tryptophan side‐chains in direct contact with the ligand, however, are affected in a slightly different way by the two ligands.
Conformations in solution of the fuscopeptins
Authors: Baré Simona , Coiro Vincenza M. , Scaloni Andrea , Di Nola Alfredo , Paci Maurizio , Segre Anna L. , Ballio Alessandro
Published: March 2003

Fuscopeptins are phytotoxic amphiphilic lipodepsipeptides containing 19 amino acid residues. They are produced by the plant pathogenic bacterium Pseudomonas fuscovaginae in two forms, A and B, which differ only in the number of methylene groups in the fatty acid chain. Their covalent structure and biological properties have been reported previously. CD and NMR spectroscopy investigations in solution revealed the absence of identifiable elements of secondary and tertiary structure for these molecules. Fuscopeptin B appears to be completely unstructured in aqueous solution, and has a large molecular flexibility. A dramatic conformational change was observed upon addition of trifluoroethanol. This study reports the complete interpretation of the two‐dimensional NMR spectra and the NOE results obtained for fuscopeptin B in water/trifluoroethanol solutions; the signals relative to the peptidic moiety are identical to those observed for fuscopeptin A. The results of this investigation were used to determine the solution structure of fuscopeptin B by computer simulations applying distance geometry and simulated annealing procedures. In water/trifluoroethanol solutions the peptidic region appears to have a partly helical structure. The lactonic ring assumes defined conformations very similar to those already reported for other lipodepsipeptides. The structure for fuscopeptin B in solution is also valid for fuscopeptin A because of the negligible structural difference between the two metabolites.
Effect of foreign N‐terminal residues on the conformational stability of human lysozyme
Authors: Takano Kazufumi , Tsuchimori Kimiko , Yamagata Yuriko , Yutani Katsuhide
Published: March 2003

To minutely understand the effect of foreign N‐terminal residues on the conformational stability of human lysozyme, five mutant proteins were constructed: two had Met or Ala in place of the N‐terminal Lys residue (K1M and K1A, respectively), and others had one additional residue, Met, Gly or Pro, to the N‐terminal Lys residue (Met(−1), Gly(−1) and Pro(−1), respectively). The thermodynamic parameters for denaturation of these mutant proteins were examined by differential scanning calorimetry and were compared with that of the wild‐type protein. Three mutants with the extra residue were significantly destabilized: the changes in unfolding Gibbs energy (ΔΔG) were −9.1 to −12.2 kJ·mol−1. However, the stability of two single substitutions at the N‐terminal slightly decreased; the ΔΔG values were only −0.5 to −2.5 kJ·mol−1. The results indicate that human lysozyme is destabilized by an expanded N‐terminal residue. The crystal structural analyses of K1M, K1A and Gly(−1) revealed that the introduction of a residue at the N‐terminal of human lysozyme caused the destruction of hydrogen bond networks with ordered water molecules, resulting in the destabilization of the protein.