bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann
Authors: ZAMUDIO Fernando , SAAVEDRA Rafael , MARTIN Brian Michael , GURROLA‐BRIONES Georgina , HÉRION Pascal , POSSANI Lourival Domingos
Published: March 2005

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides‐suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II–9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross‐reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A–D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross‐reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.
Oxidoreduction of butanol in deermice (Peromyscus maniculatus) lacking hepatic cytosolic alcohol dehydrogenase
Authors: CRONHOLM Tomas , NORSTEN‐HÖÖG Carina , EKSTRÖM Gunilla , HANDLER Jeffrey A. , THURMAN Ronald G. , INGELMAN‐SUNDBERG Magnus
Published: March 2005

In view of conflicting information in the literature regarding enzyme systems responsible for alcohol oxidation in deermice previously reported to lack hepatic alcohol dehydrogenase (ADH) activity, the reversibility of butanol oxidation was studied in vivo and in liver‐perfusion systems. Mixtures of [1,1‐2H2]ethanol and butanol were given intraperitoneally to deermice lacking (ADH−) or possessing (ADH+) ADH activity, followed by analysis of alcohols in blood by GC/MS. 2H exchange between the two alcohols was seen in all experiments. In ADH− deermice, the 2H excess of butanol increased steadily and reached 18 ± 5% after 2.5 h. In ADH+ deermice, butanol was rapidly eliminated and the 2H excess was about 7% after 0.5 h. In similar experiments with rats, the 2H excess was about 40% for 2 h.

Perfusions of livers from ADH− deermice with mixtures of unlabelled and 1‐[2H]butanol showed significant but slow intermolecular hydrogen transfer at C1, indicating oxidoreduction catalyzed by a dehydrogenase. Slow reduction of butanal was observed in mitochondria from ADH− deermice. ADH activity with a pH optimum of 10 and Km for ethanol of 6 mM was detected in the inner mitochondrial membranes from rats and deermice. However, low rates of oxidation observed in experiments carried out with perfused livers and in vitro suggest that this enzyme system does not contribute significantly to alcohol oxidation in vivo. Thus, perfused liver from ADH− deermice appears to be a useful system for studies of ADH‐independent oxidation of alcohols. The 2H exchange between the alcohols seen in vivo indicates that both ethanol and butanol are substrates for a common extrahepatic dehydrogenase in ADH− deermice.
Periportal zonation of the cytosolic acetyl‐CoA synthetase of male rat liver
Authors: KNUDSEN Charlotte T. , IMMERDAL Lissi , GRUNNET Niels , QUISTORFF Bjørn
Published: March 2005

Several important metabolic functions of the mammalian liver have been shown to be located in zones with respect to the complex microcirculation of the organ. The zonal distribution of the cytosolic component of the acetyl‐CoA synthetase activity has been investigated using the dual‐digitonin‐pulse‐perfusion technique, which allows highly zone‐selective sampling of cytosol from the periportal and perivenous zone of rat liver. Approximately 80% of the cytosolic enzymes are eluted from the hepatocytes in the periportal and perivenous sub‐zones affected by digitonin, while less than 1% of the glutamate dehydrogenase activity (a marker enzyme of the mitochondrial compartment) is eluted. A twofold higher activity of the cytosolic form of acetyl‐CoA synthetase is found in the periportal zone compared to the perivenous zone in fed male rats. Following a fasting/refeeding transition, this activity gradient is abolished in a manner similar to that observed for the enzyme acetyl‐CoA carboxylase. Since the latter enzyme is utilizing the product of acetyl‐CoA synthetase, acetyl‐CoA, the similarity in the observed regulation suggests a functional coupling between cytosolic acetate activation and fatty‐acid synthesis.
Mechanism of the stimulatory effect of a potassium‐rich medium on the phosphorylation of glucose in isolated rat hepatocytes
Authors: VAN SCHAFTINGEN Emile , VANDERCAMMEN Annick
Published: March 2005

We have investigated the mechanism by which the replacement of a Na+‐rich medium by a K+‐rich medium causes an increase in the apparent affinity of glucokinase (hexokinase IV or D) for glucose in isolated hepatocytes [Bontemps, F., Hue, L. & Hers, H. G. (1978) Biochem. J. 174, 603–611].

The stimulatory effect of a K+‐rich medium on the rate of glucose phosphorylation, as assessed by the release of tritium from [2‐3H]glucose, was only partially additive with the effect of fructose, suggesting that it was also due to a decrease in the inhibition exerted on glucokinase by its regulatory protein. Measurements of metabolites indicated that the effect of the K+‐rich medium was neither due to the formation of fructose 1‐phosphate, nor to changes in the concentrations of fructose 6‐phosphate or Pi, two other effectors of the regulatory protein.

Replacement of Na+ by K+ in the medium resulted in a time‐dependent and dose‐dependent increase in cell volume that paralleled the changes in the rate of detritiation observed at 5 mM glucose. The water and chloride contents, estimated using radiolabelled compounds, were threefold and tenfold higher, respectively, in K+ cells than in Na+ cells, and the intracellular Cl− concentration about threefold higher (94 versus 29 meq/l). The effects of the K+‐rich medium on cell volume, Cl− concentration and rate of detritiation were greatly reduced by including 80 mM trehalose or sucrose in the medium at the start of the incubation. Addition of trehalose to cells incubated for 45–50 min in the K+‐rich medium caused an immediate decrease in cell volume whereas the rate of detritiation and the Cl− concentration underwent a transient increase followed by a decrease. Replacement of KCl by KBr, potassium acetate or potassium trichloroacetate in the K+‐rich medium resulted in different relationships between cell volume and the rate of detritiation, in agreement with the differential effect of these salts on the activity of purified glucokinase assayed in the presence of regulatory protein.

From these results we conclude that the increase in the activity of glucokinase induced by a KCl‐rich medium is at least partly due to an increase in the concentration of Cl−, which relieves the inhibition exerted by the regulatory protein on purified glucokinase.
Zonation of cytochrome P450 isozyme expression and induction in rat liver
Authors: BÜHLER Rolf , LINDROS Kai O. , NORDLING Åsa , JOHANSSON Inger , INGELMAN‐SUNDBERG Magnus
Published: March 2005

The regional expression of six different cytochrome P450 (CYP) forms in rat liver under constitutive and induced conditions was compared using immunological techniques. Immunostaining of consecutive thin sections from control liver revealed that the same hepatocytes, forming a 6–8 cells thick layer surrounding the terminal hepatic venules, were stained for CYP2B1/2, CYP2E1 and CYP3A1. Staining of CYP2A1 extended further into the midzonal region, whereas all cells of the acinus stained for CYPEtOH2. These results were supported by Western blot analysis of cell lysates from the periportal or perivenous region obtained by zone‐restricted digitonin treatment during in situ perfusion. The data suggest three distinct patterns of constitutive P450 expression: perivenous‐restricted (CYP2B1/2, CYP2E1 and CYP3A1); perivenous‐dominated (CYP2A1) and panacinar (CYPEtOH2).

Chronic exposure to ethanol caused induction of CYP2E1 in the same cells already being constitutively expressed, whereas CYPEtOH2 was more induced in the periportal area. The relative induction of CYP2B1/2, CYP3A1 and CYPEtOH2 after treatment with phenobarbital was stronger in periportal hepatocytes, resulting in levelling out of the initial perivenous dominance of CYP2B1/2 and CYP3A1, whereas CYPEtOH2 became periportal‐dominated. Acetone induced CYP2E1, CYP2C11 and CYP3A1 selectively in the perivenous area. These studies indicate that a particular P450 isozyme is generally induced in the same cells where it is constitutively expressed, and that this regional selectivity is independent of the kind of inducer. The data suggest that, during maturation, the hepatocytes acquire various phenotypes in the periportal and perivenous region, to respond differently to endogenous and exogenous signals in the control of P450 expression.
Immunological characterization of rat kininogens with monoclonal antibodies to T‐kininogen.
Authors: LESAGE Suzanne , BOUHNIK Jacob , RICHOUX Jean‐Pierre , BAUSSANT Thierry , GAUTHIER Francis , EAGER Kendra , CORVOL Pierre , ALHENC‐GELAS François
Published: March 2005

A panel of 16 monoclonal antibodies (mAb) were produced against rat T‐kininogen to characterize this family of proteins. These mAbs bound 125I‐T‐kininogen by radioimmunoassay as well as reacting strongly with immobilized T‐kininogen in an enzyme‐linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T‐kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T‐kininogen encoded by the two different T‐kininogen genes, TI and TII kininogen, except antibody TK 16–3.1 which uniquely reacted with TII kininogen. Two antibodies recognizing domain 2 cross‐reacted with the high‐molecular‐mass kininogen (H‐kininogen), whereas all the other monoclonal antibodies were specific to T‐kininogen and did not recognize the heavy chain of H‐kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T‐kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H‐kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T‐kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T‐kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T‐kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney.
A membrane‐bound metallo‐endopeptidase from rat kidney
Authors: YAMAGUCHI Toru , KIDO Hiroshi , KATUNUMA Nobuhiko
Published: March 2005

A membrane‐bound metallo‐endopeptidase that hydrolyzes human parathyroid hormone (1 – 84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563–571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo‐endopeptidase were examined using an automated gas‐phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat α‐atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5–Leu6 bond in oxidized insulin B chain, the Ile28–Val29 bond in big endothelin‐1 and the Ile5–His6 and Phe8–His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km= 18.1 μM and kcat= 0.473 s−1, giving a ratio of kcat/Km= 2.62 × 104 s−1· M−1. The Km value was about 20‐fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane‐bound metallo‐endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.
Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes
Authors: DALET‐BELUCHE Isabelle , BOULENC Xavier , FABRE Gérard , MAUREL Patrick , BONFILS Claude
Published: March 2005

Two cytochrome P450 isozymes, FA and FI, were isolated and characterized from liver microsomes of phenobarbital‐induced baboons (Papio papio). The cytochrome FA possesses the same N‐terminal amino acid sequence as P450 MK2 from crab‐eating monkeys (Macaca irus) and closely resembles the human P450 3A isozymes. This cytochrome was able to oxidize nifedipine and hydroxylate testosterone at the 6β position. The second baboon cytochrome (FI) is closely related to the P450 2A subfamily and has the same N‐terminal sequence as human P450 2A7. Like human P450 2A forms, it is highly active as a coumarin 7‐hydroxylase. Antibodies against P450 FA and FI crossreact with two human liver proteins of 51 kDa and 49 kDa, respectively. The concentration of the first protein in the human samples, was five‐times greater than the second. However, the latter showed marked interindividual variation. In primary cultures of human hepatocytes, rifampicin is a strong inducer of the 51‐kDa protein and a moderate inducer of the 49‐kDa protein, while phenobarbital has the opposite effect on the two proteins.
The functional role of protein in the organization of bacteriochlorophyll c in chlorosomes of Chloroflexus aurantiacus
Authors: NIEDERMEIER Georg , SCHEER Hugo , FEICK Reiner G.
Published: March 2005

The preparation of five different fractions containing bacteriochlorophyll (Bchl) c and their absorption and circular dichroic properties have been described. The fractions investigated were purified chlorosomes, proteolytically modified chlorosomes, chlorosomes treated with lithium dodecyl sulfate (LDS) which were subsequently subjected to size‐exclusion chromatography, in vitro Bchl c aggregates and, additionally, the so‐called GEF chlorosomes [prepared according to Griebenow and Holzwarth (1989) Biochim. Biophys. Acta 973, 235–240]. Proteolysis of chlorosomes caused a 35–40% decrease in absorption intensity, a 6–8 nm blue shift of the 740‐nm peak and, in particular, a drastic increase of rotational strength as revealed by CD spectroscopy. Although oligomeric Bchl c aggregates and LDS‐treated chlorosomes had absorption characteristics similar to Bchl c in vivo, the data clearly indicated that protein, perhaps the chlorosome‐specific Mr‐3700 polypeptide, was involved in the organization of Bchl c in chlorosomes from C. aurantiacus. Furthermore, the results showed that the LDS‐treated chlorosome fraction was most likely comprised of a micellar complex of Bchl c with LDS which represented an entity entirely different from chlorosomes.
Priming of human polymorphonuclear leukocytes with granulocyte‐macrophage colony‐stimulating factor involves protein kinase C rather than enhanced calcium mobilisation
Authors: SCHATZ‐MUNDING Monika , ULLRICH Volker
Published: March 2005

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N‐formyl‐methionyl‐leucyl‐phenylalanine, fMLP) or a Ca2+‐ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5‐lipoxygenase.

Preincubation of polymorphonuclear leukocytes with GM‐CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca2+‐threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350–400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells.

Protein biosynthesis inhibitors like actinomycin D (10 γg/ml) and cycloheximide (50 γg/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM‐CSF after stimulation with ionomycin.

The priming effect of GM‐CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultanous pretreat‐ment with PMA and GM‐CSF and stimulation with either fMLP or ionomycin.

These results provide evidence that the enhanced arachidonic acid release in GM‐CSF‐primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.
Dissociation of guanosine 5′‐[γ‐thio]triphosphate from guanine‐nucleotide‐binding regulatory proteins in native cardiac membranes
Authors: HILF Gerhard , KUPPRION Christine , WIELAND Thomas , JAKOBS Karl H.
Published: March 2005

Binding of the poorly hydrolyzable GTP analog, guanosine 5′‐[γ‐thio]triphosphate (GTP[S]), to purified guanine‐nucleotide‐binding regulatory proteins (G proteins) has been shown to be non‐reversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of [35S]GTP[S] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] > GTP ≥ guanosine 5′‐[β,γ‐imino]triphosphate > GDP ≥ guanosine 5′‐[β‐thio]diphosphate > GMP, markedly enhanced the observed dissociation, with 20–30% of bound [35S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25°C. Most interestingly, dissociation of [35S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetycholine receptors. Carbachol‐stimulated release of [35S]GTP[S] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [35S]GTP[S] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol‐induced dissociation to the same extent, while GMP and ATP were ineffective. GTP[S]‐binding‐saturation experiments indicated that one agonist‐activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist‐activated receptors can even, either directly or indirectly, interact with GTP[S]‐bound G proteins, resulting in release of bound guanine nucleoside triphosphate.
Production and characterization of recombinant mouse neurotrophin‐3
Authors: GÖTZ Rudolf , KOLBECK Roland , LOTTSPEICH Friedrich , BARDE Yves‐Alain
Published: March 2005

Neurotrophin‐3 (NT‐3) is a neurotrophic factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT‐3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT‐3 that was purified by chromatography on controlled‐pore glass and reversed‐phase and gel‐filtration high‐pressure liquid chromatography. Approximately 200 μg purified NT‐3 was obtained from a single cell‐factory flask (1.6 l conditioned medium). N‐terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT‐3 allowing half‐maximal survival was determined to be 25 μg/ml.
Purification and characterization of a novel FMN‐dependent enzyme
Authors: KATAOKA Michihiko , SHIMIZU Sakayu , YAMADA Hideaki
Published: March 2005

Membrane‐bound L‐(+)‐pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L‐(+)‐pantoyl lactone, was solubilized with Brij 35 and purified 78‐fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE‐Sephacel, Sepharose CL‐6B and Cellulofine GC‐700‐m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high‐performance gel‐permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L‐(+)‐pantoyl lactone as a substrate (Km= 26.8 mM; Vmax= 4.22 μmol μ min−1· mg protein−1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L‐(+)‐pantoyl lactone dehydrogenase was identified as noncovalently bound FMN.
Response coefficients of interconvertible enzyme cascades towards effectors that act on one or both modifier enzymes
Authors: SZEDLACSEK Stefan E. , CÁRDENAS María Luz , CORNISH‐BOWDEN Athel
Published: March 2005

Explicit expressions have been derived for the response coefficients for the effect of activator and inhibitor concentrations on the fraction in the active state of the target enzyme of a monocyclic interconvertible enzyme cascade. These allow one to assess the adequacy of such a cascade for producing a highly sensitive response to an effector. Numerical studies indicate that this type of system can readily generate response coefficients of about seven, even without requiring both modification reactions to be modulated simultaneously, and without requiring all of the parameters that characterize the system to have their optimum values. Thus, a monocyclic cascade can constitute a highly effective on/off switching device in a linear pathway.
1H‐NMR studies on partially and fully reduced 2(4Fe‐4S) ferredoxin from Clostridium pasteurianum
Authors: BERTINI Ivano , BRIGANTI Fabrizio , LUCHINAT Claudio , MESSORI Luigi , MONNANNI Roberto , SCOZZAFAVA Andrea , VALLINI Giovanni
Published: March 2005

The ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated through 1H‐NMR spectroscopy in the reduced and partially oxidized states. The 1H‐NMR spectrum of fully reduced ferredoxin, obtained by addition of stoichiometric amounts of dithionite, has been characterized. One‐ and two‐dimensional NMR saturation transfer experiments on partially reduced samples have allowed the isotropically shifted signals of the reduced form to be correlated to those of the oxidized form, for which the complete assignment of the β‐CH2 cysteinyl residues is available. In addition, observation of the 1H‐NMR signals of the intermediate species with characteristic chemical shift values for each cluster allowed us to assign all the Cys β‐CH2 signals to cluster I or cluster II and to calculate the difference in redox potential between them. Starting from these results, reanalysis of the 1H‐NMR features of the two clusters in the oxidized form showed that they are strikingly similar, supporting the idea of a high degree of internal symmetry between them, in agreement with crystallographic results on an homologous ferredoxin. On the other hand, the 1H‐NMR properties of the two clusters in the reduced form deviate considerably from each other, suggesting that reduction of the clusters brings about different structural changes and loss of internal symmetry. A theoretical approach is reported to account for the isotropic shifts and the temperature dependence of the NMR signals of the reduced protein.
Molecular cloning and characterization of a constitutively expressed heat‐shock‐cognate hsc71 gene from rainbow trout
Authors: ZAFARULLAH Muhammad , WISNIEWSKI Jan , SHWORAK Nicholas W. , SCHIEMAN Steven , MISRA Santosh , GEDAMU Lashitew
Published: March 2005

A rainbow trout major heat‐shock‐protein‐like gene (hsp 70) and corresponding cDNA clones were isolated by hybridization to heterologous hsp70 probes. DNA sequencing revealed that this gene is structurally similar to a mammalian heat‐shock‐cognate hsc70 gene and consists of eight introns. Northern blot and primer extension analyses showed that the corresponding mRNA is constitutively abundant in different trout tissues and salmonid cell lines. Fragments of the isolated gene containing the −900 –+30 and −217 –+58 sequence were linked to a bacterial chloramphenicol acetyltransferase reporter gene and transiently transfected into salmonid cells. The expression pattern of these constructs supports our conclusion that the isolated genomic and cDNA clones correspond to a trout heat‐shock‐cognate hsc70 gene.
Construction and molecular dynamics simulation of calmodulin in the extended and in a bent conformation
Authors: VORHERR Thomas , KESSLER Oliver , MARK Alan , CARAFOLI Ernesto
Published: March 2005

Analysis of sequence similarity and comparison of the three‐dimensional (3D) structures of troponin C and calmodulin have revealed a sequence in the central helix of calmodulin with a high probability for bending. The three amino acids known to form a bend in the N‐terminal portion of troponin C are also found in the central helix of calmodulin. The modelling of a bent calmodulin structure, using the dihedral angles of the three residues in the bend of troponin C as a 3D template, results in a conformation of calmodulin where the N‐ and C‐terminal domains are able to form contacts. Dynamics simulations starting from the X‐ray structure of calmodulin and from the modelled bent calmodulin were carried out to compare flexibility and correlated movements of Ca2+ in the binding loops. Both conformations of calmodulin remained stable during the period of simulation. In the simulation of calmodulin in the extended form, the motions of the Ca2+ atoms in the two domains (Ca2+1 and Ca2+2 in one domain, and Ca2+3 and Ca2+4 in the other) are correlated. In the simulation of the bent form, an additional correlation between the Ca atoms in the two different domains is observed. The results are compatible with the occurrence of a bent conformation of calmodulin in the presence of targets, and with increased Ca2+ affinity and cooperativity of the Ca2+‐binding loops in the calmodulin‐peptide complexes.
Identification of two domains which mediate the binding of activating phospholipids to the plasma‐membrane Ca2+ pump
Authors: BRODIN Peter , FALCHETTO Rocco , VORHERR Thomas , CARAFOLI Ernesto
Published: March 2005

The stimulation of the purified human erythrocyte calcium pump by acidic phospholipids was investigated using synthetic peptides corresponding to a putative phospholipid‐responsive domain [Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N. & Carafoli, E. (1990) Biochemistry 29, 8070–8076] and to the calmodulin‐binding domain of the pump. The peptides interfered with the activation of the enzyme by phosphatidylserine and phosphatidic acid in competition assays. The peptide corresponding to the calmodulin‐binding domain was found to be the most efficient antagonist. Direct binding measurements using fluorescent derivatives of the peptides confirmed the interaction between the acidic phospholipids and the peptides, and fluorescence titrations of dansylated calmodulin with the purified ATPase showed a direct effect of acidic phospholipids on calmodulin binding. A proteolyzed preparation of the Ca2+‐ATPase lacking the calmodulin‐binding domain confirmed that the phospholipid‐induced stimulation is mediated by two sites, one located in the C‐terminal portion of the previously identified 44‐amino‐acid phospholipid‐responsive domain, the other in the calmodulin‐binding domain.
Binding of histones to Xenopus laevis ribosomal genes with different levels of expression
Authors: DIMITROV Stephan I. , TATEOSSYAN Hilda N. , STEFANOVSKY Victor Yu. , RUSSANOVA Valya R. , KARAGYOZOV Luchesar , PASHEV Iliya G.
Published: March 2005

The association of ribosomal RNA genes with histones as a function of their expression has been studied in Xenopus laevis erythrocytes, where the genes are silent, and tadpoles at stage 40, where these genes are actively transcribed. Isolated nuclei were either treated with formaldehyde or irradiated with an ultraviolet laser to cross‐link proteins to DNA. The covalently linked protein‐DNA complexes were purified by centrifugation through CsCl and immunoprecipitated with antibodies against H1, H2A and H4. DNA from the precipitated complexes was analysed for the presence of ribosomal DNA sequences by hybridization to specific probes. The actively transcribed ribosomal genes from X. laevis embryos are associated with H1, H2A and H4 as are the non‐transcribed genes in the erythrocytes.
Probing intercalation and conformational effects of the anticancer drug 2‐methyl‐9‐hydroxyellipticinium acetate in DNA fragments with circular dichroism
Authors: MONNOT Monique , MAUFFRET Olivier , LESCOT Elie , FERMANDJIAN Serge
Published: March 2005

Circular dichroism was applied to the analysis of drug‐DNA associations. With the octanucleotide d(TGACGTCA) (octanucleotide I), which is the cAMP‐responsive element (CRE) in gene promoters and its reverse d(ACTGCAGT) (octanucleotide II), it was demonstrated that the anticancer polyaromatic agent celiptium intercalates into DNA base pairs with its long direction perpendicular to both the DNA‐helix axis and the base‐pair long axis and induces larger conformational changes in the CpG‐containing octanucleotide I CRE than in its reverse‐sequence octanucleotide II.

It was concluded that CD is a powerful and sensitive technique to discriminate between drug‐binding modes of DNA, to define the geometry of the chromophore inserted into base pairs and, finally, to measure sequence‐dependent conformational changes induced by intercalation in DNA.

We anticipate that these studies will contribute to a better understanding of the molecular bases that underlie the mechanism of action of those cytotoxic drugs which interfere with the DNA‐nuclear‐protein recognition.
Characterization of a secreted glycoprotein of the immunoglobulin superfamily inducible by mitogen and oncogene
Authors: WERENSKIOLD Anne Katrin
Published: March 2005

The T1 gene is transiently activated by the Ha‐ras (EJ) or v‐mos oncoproteins and by mitogens in NIH3T3 fibroblasts. Its primary gene product of 337 amino acids (38 kDa) undergoes extensive post‐translational modification. For biochemical analysis, the T1 gene product was over‐expressed in a vaccinia virus system. Cells infected with a recombinant T1‐vaccinia virus produce and secrete multiple proteins of 60–70 kDa which react with polyclonal antisera raised against two T1‐specific peptides. Two lines of evidence suggest that the apparent size heterogeneity of the T1 protein is due to a variable carbohydrate content of 40–50% of the total molecular mass. First, in the presence of an inhibitor of N‐glycosylation (tunicamycin), a single 38‐kDa protein is detected by the antisera in the cells infected with T1‐vaccinia virus. Second, glycosidase digestions show that T1 protein maturation involves glycosylation and sialylation. These post‐translational modification steps appear to be similar in different types of cells.
Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10‐kb DNA fragment
Authors: VAN HUFFEL Christophe , DUBOIS Evelyne , MESSENGUY Francine
Published: March 2005

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl‐phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327‐amino‐acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884‐amino‐acid polypeptide. The acetylglutamate kinase and acetylglutamate‐phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein.

The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11− mutation. In contrast, both ornithine‐carbamoyltransferase‐encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization.

The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.
Interaction of the anthracycline antibiotic nogalamycin with the hexamer duplex d(5′‐GACGTC)2.
Authors: SEARLE Mark S. , BICKNELL Wendy
Published: March 2005

The interaction of the anthracycline antibiotic nogalamycin with the single 5′‐CpG binding site at the centre of the hexamer duplex d(GACGTC)2 has been investigated in solution by NMR spectroscopy. 41 NOE constraints between antibiotic and nucleotide protons provide input parameters for the determination of a family of conformers computed using energy minimisation and restrained molecular dynamics. Nogalamycin adopts a ‘threaded’ binding orientation in which the drug chromophore passes through the DNA helix positioning the nogalose sugar in the minor groove and the positively charged bicycloamino sugar in the major groove. The complex is stabilised by hydrogen bonding, electrostatic and van der Waals interactions. In the major groove hydrogen bonding interactions are identified between the 2′‐OH and 4′‐OH groups, on the rigidly oriented bicyclo sugar, and the guanine N7 and cytosine 4‐NH2 of one G · C base pair and appear to account for the requirement for a G · C base pair at the intercalation site. In the minor groove we identify a hydrogen bond between the carbonyl oxygen of the methyl ester group on ring A and the 2‐NH2 group of guanine at the intercalation site. The nogalose and ring A form a continuous domain that interacts through van der Waals contacts with the floor and walls of the minor groove over a span of four basepairs, d(GACGTC)2. The NOE data together with energy‐minimised structures reveal distortions of the DNA conformation involving a total helix unwinding of about 16° and buckling of the G · C base pairs by about 22° and −9° at the intercalation site. The base pairs effectively wrap around the drug chromophore optimising van der Waals contacts between the two and minimising cavities in the structure. The solution data also reveal that the bound antibiotic substantially slows the rate of exchange of the imino protons of not only the G · C base pairs forming the intercalation site but also that of an A · T base pair which is occluded by the sugar moieties lying in each groove. The data present an opportunity for comparison with two crystal structures of nogalamycin‐DNA complexes in which ligand molecules are bound at terminal 5′‐CpG sites.
Investigating interdomain region mutants Phe194 → Leu and Phe194 → Trp of yeast phosphoglycerate kinase by 1H‐NMR spectroscopy
Authors: JOÃO Heidi C. , TADDEI Niccoló , WILLIAMS Robert J. P.
Published: March 2005

Site‐directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H‐NMR spectroscopy, it was found that the mutations at position 194 induce both local and long‐range conformational changes in the protein. It was also found that 3‐phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild‐type phosphoglycerate kinase. The affinity of mutant Phe194 → Trp for 3‐phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194 → Leu mutant is reduced by about threefold relative to the wild‐type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194 → Leu mutant when compared to wild‐type or Phe194 → Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194 → Leu mutant the Km values for both 3‐phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild‐type enzyme, while for mutant Phe194 → Trp, the Km values for 3‐phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild‐type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194 → Trp and less for mutant Phe194 → Leu, relative to that observed for wild‐type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3‐phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3‐phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.
The hydrogen‐tritium exchange activity of Megasphaera elsdenii hydrogenase
Authors: DOHERTY Gerald M. , MAYHEW Stephen G.
Published: March 2005

The hydrogenase of Megasphaera elsdenii was purified to a specific activity of 350 units/mg. The hydrogen‐tritium exchange assay of Hallahan et al. [Hallahan, D. L., Fernandez, V. M., Hatchikian, E. C. and Hall, D. O. (1986) Biochimie (Paris) 68, 49–54] was adapted to allow its use in the study of the M. elsdenii hydrogenase preparation. Under the assay conditions routinely employed, the enzyme's exchange activity was inhibited by Tris/HCl and MgCl2; it was stimulated by ethylene glycol. Maximal activity in this standard assay occurred at pH 7.1. The effect of the concentration of molecular hydrogen (1H2 plus 3H1H) on the exchange activity was studied. The resulting double‐reciprocal plot was linear; its slope and its intercepts on the ordinate and abscissa were pH‐dependent. The rate equations for a number of models of the exchange activity were derived. Each model gave rise to a linear double‐reciprocal plot at constant pH, but none could explain fully the observed effects of varying pH. The experimental data corresponded most closely to the predictions of models in which protons were treated both as substrates and as regulators of the enzyme's activity.
2′,3′‐Dialdehyde ATP analog labels the Ca2+ ‐ATPase of sarcoplasmic reticulum via the catalytic adenosine‐nucleotide‐binding site
Authors: HOHENEGGER Martin , MAKINOSE Madoka
Published: March 2005

The 2′,3′‐dialdehyde ATP analog (oATP) was synthesized and its ability to activate the Ca2+‐ATPase of skeletal muscle sarcoplasmic reticulum via the adenosine‐nucleotide‐binding site was investigated. After reduction by sodium borohydride, oATP binds covalently to the catalytic adenosine‐nucleotide‐binding site of the enzyme, resulting in 85% loss of acetyl‐phosphate‐driven Ca2+ uptake and ATP‐hydrolysing ability. In the absence of a reducing agent, oATP serves as a substrate for the Ca2+‐ATPase, as indicated by Pi formation (hydrolysis) and Ca2+‐uptake ability. oATP binding to the intact light sarcoplasmic reticulum is observed in the absence and presence of the competitive adenosine nucleotide inhibitor, fluorescine isothiocyanate with apparent affinity constants of 1.2 mM and 2.2 mM, respectively. Autoradiography of tryptic fragments from partially purified Ca2+‐ATPase labeled with [α‐32P]oATP or [γ‐32P]oATP locates the covalent binding site to the A1 fragment, even in the fluorescine‐isothiocyanate‐labeled pump protein. With high probability, a lysine residue in the tryptic A1 fragment is labeled by the ribose‐modified ATP analog close to the phosphorylation site at Asp351.
The effects of ethanol concentration on glycero‐3‐phosphate accumulation in the perfused rat liver
Authors: MASSON Serge , DESMOULIN Franck , SCIAKY Martine , COZZONE Patrick J.
Published: March 2005

The dose‐dependent effect of ethanol on the hepatic metabolism of the perfused rat liver has been investigated by (a) 31P‐NMR spectroscopy for the follow‐up of intracellular phosphorylated metabolites and (b) HPLC for compounds released in the effluents. Perfusion of livers from fed rats with ethanol induced an increase in the level of sn‐glycerol 3‐phosphate and net accumulations of 3.30 ± 0.33 and 0.69 ± 0.15 μmol × g−1 wet liver were reached after 20 min, for 70 mM and 0.5 mM ethanol, respectively. sn‐Glycerol‐3‐phosphate accumulation was fully detected by 31P NMR as indicated by comparing quantitations based on NMR and biochemical assays. Ethanol administration up to a concentration of 10 mM induced a dose‐dependent decrease in the release of lactate + pyruvate by the liver. Lactate release decreased from 1129 ± 39 to 674 ± 84 nmol × min−1× g−1, while pyruvate decreased from 230 ± 9 to 6.2 ± 0.4 nmol × min−1× g−1, after 20 min of perfusion with 10 mM ethanol. Nevertheless, the flux through 6‐phosphofructo‐1‐kinase, as measured by both the accumulation of sn‐glycerol 3‐phosphate and release of lactate + pyruvate, was not affected in the early phase of ethanol oxidation. Finally, data obtained from oxygen consumption, the release of acetate and the accumulation of sn‐glycerol 3‐phosphate do not support the involvement of the microsomal ethanol‐oxidizing system in the catalysis of ethanol oxidation, even at high doses of alcohol.
Structure of the human ferrochelatase gene
Authors: TAKETANI Shigeru , INAZAWA Johji , NAKAHASHI Yoshitsugu , ABE Tatsuo , TOKUNAGA Rikio
Published: March 2005

We have determined the structure of the human ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. This gene was assigned to human chromosome 18 at region q21.3, by fluorescent in situ hybridization. The gene contains a total of 11 exons and has a minimum size of about 45 kb. The exon/intron boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to encode functional protein domains. A major site of the transcription initiation, determined by S1 nuclease mapping, was assigned to an adenine base 89 bases upstream from the adenine base of the translation initiation ATG. The promoter region contains a potential binding site for Sp1, NF‐E2 and erythroid‐specific transcriptional factor GATA‐1, but not a typical TATAA or CCAAT sequence. Analysis of primer extension showed that the transcription starts at the same position between hepatoma HepG2 and erythroleukemia K562 cell mRNA, thereby suggesting that there can be a single transcript in erythroid and non‐erythroid cells.
The nucleotide and deduced amino acid structures of sheep and pig fetuin
Authors: BROWN William M. , DZIEGIELEWSKA Katarzyna M. , SAUNDERS Norman R. , CHRISTIE David L. , NAWRATIL Peter , MÜLLER‐ESTERL Werner
Published: March 2005

This study was initiated to gain further insight into the structural features of the mammalian fetuin family. The cDNA structures of sheep and pig fetuin were determined. The cDNA insert encoding sheep (pig) fetuin comprised 1550 (1470) nucleotides, including 54 (46) nucleotides encoding a signal peptide of 18 (15) residues and 1038 (1041) nucleotides encoding the 346 (347) amino acids of the mature plasma protein. The predicted amino‐terminal sequence of the mature pig fetuin was confirmed by the amino‐terminal sequence of the purified protein. However, two alternative sheep amino‐terminal sequences were found in fetuin purified from the plasma of a single sheep fetus; the minor product was the one predicted by comparison with other fetuin sequences while the major product was two amino acids longer. Comparison of the deduced amino acid sequences of sheep and pig fetuin showed an extensive sequence identity between them (75%) and with other proteins of the mammalian fetuin family, i. e. human α2‐HS glycoprotein, and bovine and rat fetuins. Twelve cysteine residues were found at invariant positions in all fetuin sequences, suggesting strongly that the arrangement of disulphide bridges identified in human α2‐HS glycoprotein is common to the members of the family. Further sequence comparisons revealed that the structures of mammalian fetuins are organised in three domains: two cystatin‐like domains (D1 and D2) and a complex carboxyl‐terminal domain (D3). The proposed three‐domain structure of the protein is reflected in the organisation of the rat fetuin structural gene which has recently been published.