bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific binding of progesterone receptor to progesterone‐responsive elements does not require prior dimerization
Authors: COHEN‐SOLAL Karine , BAILLY Alain , RAUCH Claudine , QUESNE Monique , MILGROM Edwin
Published: March 2005

Steroid‐hormone receptors undergo, prior to binding to DNA, a hormone‐dependent dimerization. It is generally accepted that this dimerization is indispensable for the high‐affinity binding of hormone receptor to hormone‐responsive elements.

Using a progesterone‐receptor mutant with the complete steroid‐binding domain deleted (positions 663–930), with or without the epitope required for binding the monoclonal antibody Let 126, we have shown that this receptor species was unable to undergo dimerization in solution. However, this mutant retained a high affinity (60–70% of the affinity of the wild‐type receptor) for the progesterone‐responsive elements of the mouse‐mammary‐tumor‐virus long‐terminal‐repeat promoter and for a consensus palindromic progesterone‐responsive element, as measured by both DNase‐I protection experiments and gel‐shift experiments. This mutant also increased gene transcription. Thus, at least in the case of the progesterone receptor, prior dimerization is dispensable for receptor binding to regulatory DNA elements and for subsequent transcription activation.
Novel hirudin variants from the leech Hirudinaria manillensis
Authors: SCACHERI Emanuela , NITTI Gianpaolo , VALSASINA Barbara , ORSINI Gaetano , VISCO Carlo , FERRERA Monica , SAWYER Roy T. , SARMIENTOS Paolo
Published: March 2005

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion‐exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2), and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C‐terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the C‐terminal portion of the molecule which is replaced by aspartic acid.

Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin‐like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre‐hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported.

The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20‐amino‐acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides.

HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.
Testican, a multidomain testicular proteoglycan resembling modulators of cell social behaviour
Authors: ALLIEL Patrick M. , PERIN Jean‐Pierre , JOLLÈS Pierre , BONNET François J.
Published: March 2005

The molecular characterization of a human testicular proteoglycan, the progenitor of a seminal plasma glycosaminoglycan‐bearing peptide, was achieved by cDNA cloning. Its protein core encompasses several domains encountered in various proteins associated with adhesion, migration and cell proliferation. An osteonectin‐like domain, a Kazal‐like sequence and a 46‐amino‐acid motif around a Cys‐Trp‐Cys‐Val peptide encountered in cell‐surface antigens, cell‐adhesion molecules and growth‐factor‐binding proteins are distributed within the testican protein core. Testican is the progenitor of the unique heparan/chondroitin‐sulfate‐bearing peptide present in human seminal plasma, a feature which might confer additional potentialities to this hybrid proteoglycan.
A tetrameric complex of membrane proteins in the endoplasmic reticulum
Authors: HARTMANN Enno , GÖRLICH Dirk , KOSTKA Susanne , OTTO Albrecht , KRAFT Regine , KNESPEL Signe , BÜRGER Elke , RAPOPORT Tom A. , PREHN Siegfried
Published: March 2005

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called ‘signal sequence receptor’ that is now renamed as ‘translocon‐associated protein’ (TRAP). Two glycosylated subunits of the TRAP complex have been identified before (α and β subunits). We now show that the TRAP complex is actually comprised of four membrane proteins (α, β, γ, δ), present in a stoichiometric relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, non‐glycosylated subunits were deduced from cloning of the corresponding cDNAs. The δ subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The γ subunit is predicted to span the membrane four times.
Primary structures and biological activities of substance‐P‐related peptides from the brain of the dogfish, Scyliorhinus canicula
Authors: WAUGH David , WANG Yunxia , HAZON Neil , BALMENT Richard J. , CONLON J. Michael
Published: March 2005

Two peptides with substance‐P‐like immunoreactivity were isolated in pure form from an extract of the brain of the elasmobranch fish, Scyliorhinus canicula (european common dogfish). One peptide was identical to scyliorhinin I, previously identified in dogfish intestine, and the second was the undecapeptide Lys‐Pro‐Arg‐Pro‐Gly‐Gln‐Phe‐Phe‐Gly‐Leu‐Met‐CONH2 which is structurally similar to mammalian substance P. Scyliorhinin II or a peptide analogous to mammalian neurokinin A were not detected in the extract. Synthetic dogfish substance P ([Lys1, Arg3, Gly5]substance P) was approximately threefold more potent than mammalian substance P (Kd= 0.21±0.11 nM versus Kd= 0.74±0.17 nM; mean ± SD; n= 6) in inhibiting the binding of 125I‐labelled substance P to neurokinin (NK1) receptors in rat submandibular gland membranes. The vasodilator action of tachykinins in mammals is mediated primarily through interaction with NK1 receptors. Bolus intravenous injections of [Lys1, Arg3, Gly5]substance P (100 pmol) and scyliorhinin I (100 pmol) produced appreciable (>4kPa) decreases in arterial blood pressure in the rat whereas intravenous injections of up to 5 nmol of the peptides into conscious, unrestrained dogfish produced no change in arterial blood pressure, pulse amplitude or heart rate. Injections of greater amounts of the peptides (10–50 nmol) produced a slight increase (400–667 Pa) in blood pressure. The data indicate that mammalian‐type NK1 tachykinin receptors are not involved in cardiovascular regulation in elasmobranch fish.
Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action
Authors: FAURE Grazyna , HARVEY Alan L. , THOMSON Elisabeth , SALIOU Bernard , RADVANYI François , BON Cassian
Published: March 2005

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase‐A2 subunit (CB) and a non‐enzymic, non‐toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its nonspecific adsorption.

Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase‐A2 activity.

In this study, we demonstrate that the more‐toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less‐toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more‐toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less‐toxic isoforms are less stable (Kd∼25 nM) and dissociate rapidly (half‐life about 1 min), whereas the more‐toxic isoforms are more stable (Kd∼4.5 nM) and dissociate more slowly (half‐life 10–20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles.

The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency.
Integration of a cyanobacterial protein involved in nitrate reduction (narB) into isolated Synechococcus but not into pea thylakoid membranes
Authors: KUMAR Polumetla A. , KRUSE Elisabeth , ANDRIESSE Xanja , WEISBEEK Peter , KLOPPSTECH Klaus
Published: March 2005

Chimeric genes comprised of Rubisco small subunit transit peptide fused in frame with full‐length and truncated sequences of a nitrate reductase (narB) structural gene of Synechococcus were constructed. Fusion proteins were synthesized in a rabbit reticulocyte system. In thylakoido integration of synthetic proteins resulted in the association of the full‐length narB‐coded protein to the Synechococcus photosynthetic membranes. The membrane‐associated protein was sensitive to trypsin treatment but could not be removed by washing in the presence of NaBr. Trypsin pretreatment of thylakoids abolished the capability for association. The association of the narB‐coded protein with thylakoids might require another membrane protein whose identity is not known. It is proposed that the Synechococcus narB polypeptide is a peripheral, membrane bound protein anchored to the thylakoids via a short hydrophobic domain while the major part of the protein resides on the outer side of the thylakoid membranes. The chimeric narB proteins were processed and imported by intact pea chloroplasts in vitro; however, the mature proteins were found localized in the stroma and not in the thylakoid membrane fraction. Similarly, the attempt to integrate the protein in vitro into isolated pea thylakoid membranes failed although these membranes incorporate early light‐inducible proteins.
Co‐reconstitution of plasma membrane H+‐ATPase from yeast and bacteriorhodopsin into liposomes
Authors: WACH Achim , DENCHER Norbert A. , GRÄBER Peter
Published: March 2005

The H+‐ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The enzyme was reconstituted with bacteriorhodopsin into asolectin liposomes by detergent dialysis at a molar ratio of 1 H+‐ATPase to 50 bacteriorhodopsins. The overall orientation of the proteins is such that proton pumping to the vesicle interior occurs upon illumination and after addition of ATP. All liposomes which contain H+‐ATPase also contain bacteriorhodopsin.

The rate of ATP hydrolysis was measured as function of pH in the dark and during illumination of the proteoliposomes. The pH dependency can be described by the protonation of a monovalent group from the outside with an apparent pK of 7.3 and the deprotonation of a monovalent group at the inside with an apparent pK of 3.7. Inside and outside refer to the orientation of the H+‐ATPase in the liposomes which is opposite to that occurring in vivo. It is concluded that the first step in the reaction cycle is the binding of a proton from the cytosol which is followed by ATP binding, ATP hydrolysis on the enzyme and the release of ADP and phosphate, and finally the proton is released from the enzyme into the external medium.
Different in vitro metabolism of 7 α‐methyl‐19‐nortestosterone by human and equine aromatases
Authors: MOSLEMI Safa , DINTINGER Thierry , DEHENNIN Louis , SILBERZAHN Pierre , GAILLARD Jean‐Luc
Published: March 2005

The ability of human and equine placental microsomes to aromatize 7α‐methyl‐19‐nortestoster‐one (MNT) was studied. Kinetic analysis indicates that MNT shares the androgen‐binding site of human and equine placental microsomal aromastases. Human placental microsomal estrogen synthetase had about a 2.5‐fold higher relative affinity for MNT than the equine placental enzyme (KiMNT/Km androstenedione of 32 versus 87). However, MNT was not metabolized by human placental microsomes, whereas it was very actively metabolized by equine placental microsomes. Further studies using purified equine cytochrome P‐450arom indicated that the presence of a 7α‐methyl group and the absence of a C19 methyl group did not impair its conversion by the purified enzyme. The product of this reaction was separated and identified as 7α‐methylestradiol by gas chromatography coupled to mass spectrometry.
Purification and characterization of the α‐agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B
Authors: POTIN Philippe , RICHARD Christophe , ROCHAS Cyrille , KLOAREG Bernard
Published: March 2005

The phenotypic features of strain GJ1B, an unidentified marine bacterium that degrades agar [Young, K. S., Bhattacharjee, S. S. & Yaphe, W. (1978) Carbohydr. Res. 66, 207–212], were investigated and its agarolytic system was characterized using 13C‐NMR spectroscopy to analyse the agarose degradation products. The bacterium was assigned to the genus Alteromonas and the new combination A. agarlyticus (Cataldi) is proposed. An α‐agarase, i.e specific for the α(1→3) linkages present in agarose, was purified to homogeneity from the culture supernatant by affinity chromatography on cross‐linked agarose (Sepharose CL‐6B) and by anion‐exchange chromatograpy (Mono Q column). The major end product of agarose hydrolysis using the purified enzyme was agarotetraose. Using SDS/PAGE, the purified α‐agarase was detected as a single band with a molecular mass of 180 kDa. After the affinity‐chromatography step, however, the native molecular mass was approximately 360 kDa, suggesting that the native enzyme is a dimer which is dissociated to active subunits by anion‐exchange chromatography. The isolectric point was estimated to be 5.3. Enzyme activity was observed using agar as the substrate over the pH range 6.0–9.0 with a maximum value at pH 7.2 in Mops or Tris buffer. The enzyme was inactivated by prolonged treatment at a pH below 6.5, or by temperatures over 45°C or by removing calcium. In addition, a β‐galactosidase specific for the end products of the α‐agarase was present in the α‐agarase affinity‐chromatography fraction, probably as part of a complex with this enzyme. The degradation of agarose by this agarase complex yielded a mixture of oligosaccharides in the agarotetraose series and the agarotriose series, the latter consisting of oligosaccharides with an odd number of galactose residues.
Evolution of pro‐protamine P2 genes in primates
Authors: RETIEF J. D. , DIXON G. H.
Published: March 2005

Protamines P1 and P2 form a family of small basic peptides that represent the major sperm proteins in placental mammals. In human and mouse protamine P2 is one of the most abundant sperm proteins. The protamine P2 gene codes for a P2 precursor, pro‐P2 which is later processed by proteolytic cleavages in its N‐terminal region to form the mature P2 protamines. We have used polymerase chain amplification to directly sequence the pro‐P2 genes of the five major primate families: red howler (Alouatta seniculus) is a New World monkey (Cebidae); the two macaque species, Macaca mulatta and M. nemistrina are Old World monkeys (Cercopithecidae), the gibbon, Hylobates lar, represents one branch of the apes (Hylobatidae); the orangutan, Pongo pygmaeus, gorilla, Gorilla gorilla and two species of chimpanzee Pan paniscus and Pan troglodytes represent a second ape family (Pongidae). These pro‐P2 genes are compared with that of human [Domenjoud, L., Nussbaum, G., Adham, I. M., Greeske, G. & Engel, W. (1990) Genomics 8, 127–133].

The overall size and organization of the genes are conserved within the group. The mean length of pro‐P2 is 101 residues, with an increase to 102 in M. nemistrina and a decrease to 99 residues in red howler (A. seniculus). In gorilla and red howler one of two 79‐bp tandem repeats that occurs 3′ of the gene is deleted. Of the 101 deduced amino acids examined, an amino acid change occurs in one or more primates at 45 positions. Considering only the most recently diverged group, the human/gorilla/chimpanzee clade, this represents a very high mutation rate of 0.99 changes/100 sites in 106 years. This rapid mutation rate is characteristic of both members of the protamine gene family, P1 and P2. Consideration of the variable nature of the sequences at the multiple sites of proteolysis during the processing of the pro‐P2 indicates either that there are several processing enzymes of differing specificities, or more likely that the folded structure of the pro‐P2 limits accessibility of a non‐specific protease to certain exposed sites.
The interferon‐stimulable response elements of two human genes detect overlapping sets of transcription factors
Authors: PARRINGTON John , ROGERS Neil C. , GEWERT Dirk R. , PINE Richard , VEALS Susan A. , LEVY David E. , STARK George R. , KERR Ian M.
Published: March 2005

We have previously reported three types of DNA‐protein complexes, formed specifically with the interferon‐stimulable response elements (ISRE) in the 5′ flanking DNA of the interferon‐inducible 6–16 and 9–27 genes, a type‐I interferon‐Inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type‐I and type‐II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band‐shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe. With a 9–27 ISRE probe the M, G and C1/C2 complexes all contain the γ subunit of ISGF3, which is present constitutively but is induced in response to IFN‐α (to yield M) or IFN‐γ (to yield G). In contrast, a 6–16 ISRE probe forms band‐shift complexes with IFN‐α‐inducible and IFN‐γ‐inducible IRF1 and IRF2. With a 6–16 ISRE probe, therefore, M and G each correspond to two complexes which co‐migrate in band‐shift assays, one corresponding to IRF1, the other to IRF2. With this probe, the constitutive complex C1/C2 corresponds predominantly to IRF2. Consistent with this, IRF1 and IRF2 have lower affinity for the 9–27 ISRE than the 6–16 ISRE, whereas the reverse is true for E (ISGF3) and its γ subunit. Relatively small differences in affinity appear sufficient to determine whether or not a band‐shift complex is detected. In the case of IRF1 and IRF2, the different affinities for the 6–16 and 9–27 probes are dominated by a dinucleotide sequence in the centre of the 14‐nucleotide ‘core’ ISRE. In contrast, preferential binding of E (ISGF3) by the 39‐nucleotide 9–27 ISRE‐containing sequence, although ISRE dependent, appears to be mediated by sequences 3′ of the ‘core’ ISRE. Accordingly, these complexes can be simultaneously assayed using a hybrid probe consisting of the 5′ flanking region and ‘core’ ISRE sequences from the 6–16 gene and sequences immediately 3′ of the ‘core’ 9–27 ISRE sequence. No evidence was obtained for a modulatory role in factor binding for a pseudo‐ISRE sequence close to ISRE in the 9–27 gene.

The precise roles of IRF1 and IRF2 in the induction of IFN‐β and the control of interferon‐inducible gene expression remain to be established. Results from the analysis of the expression of IRF1 and IRF2 in wild‐type and mutant cells argue against a dominant negative role for unmodified IRF2.
Characterization of the receptor and translocator domains of colicin N
Authors: EL KOUHEN Rachid , FIEROBE Henri‐Pierre , SCIANIMANICO Sandra , STEIERT Mathias , PATTUS Franc , PAGES Jean‐Marie
Published: March 2005

Intact colicin N and various colicin derivatives, including a natural fragment lacking the first 36 amino‐acid residues, a chymotryptic fragment lacking the first 66 amino acids and a thermolytic fragment comprising residues 183–387, were used to locate the regions involved in colicin‐N uptake by sensitive Escherichia coli cells. Two separate domains of the molecule participate in colicin‐N entry. Specific binding to OmpF receptor site requires a region located between residues 67–182. A N‐terminal domain, located between residues 17–66, is involved during the translocation step after binding to receptor. Two sub‐regions, residues 17–36 and residues 37–36, can be defined in this domain. The location and interactions between these domains are discussed in comparison to other colicins which use similar cell components for their uptake.
Chemical structure of the lipid A of Escherichia coli J‐5
Authors: HOLST Otto , MÜLLER‐LOENNIES Sven , LINDNER Buko , BRADE Helmut
Published: March 2005

The lipopolysaccharide, and particularly its lipid A moiety, of the J‐5 mutant of Escherichia coli O111 plays a central role in studies on potential induction of cross‐reactive and cross‐protective antibodies, however, its chemical and antigenic structure was hitherto unknown. Here, the chemical structure of the J‐5 lipid A is reported. It is composed of the bisphosphorylated disaccharide β‐d‐GlcpN‐4‐P‐(1‐6)‐α‐d‐GlcpN‐1‐P which carries four residues of 3‐hydroxytetradecanoic acid, one each at positions 2, 3, 2′, and 3′. The hydroxyl groups of the acyl residues at 2′ and 3′ are esterified with dodecanoic and tetradecanoic acid, respectively. The hydroxyl group at C‐6′ functions in the lipopolysaccharide as the attachment site of the core oligosaccharide.

Furthermore, a new method to isolate the hydrophilic backbone, i.e. the 1,4′‐bisphosphorylated glucosamine disaccharide, and its structural analysis by 1H‐, 13C‐, and 31P‐NMR spectroscopy, are described, leading to a new and easier strategy in structural analysis of lipid A from bacterial lipopolysaccharides.
Expression and mutation of soybean β‐amylase in Escherichia coli
Authors: TOTSUKA Atsushi , FUKAZAWA Chikafusa
Published: March 2005

The cDNA clones corresponding to soybean β‐amylase mRNA were isolated and sequenced. The cDNA contained an open‐reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N‐terminal peptide sequence from mature enzyme proved that β‐amylase had no leader sequence. Employing the cDNA, the β‐amylase was directly synthesized in Escherichia coli by the expression vector pKK233‐2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13°C was 130‐fold and 280‐fold, respectively, the value at 37°C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose‐immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the β‐amylases were replaced by Arg (H93R) and Asn (D348N) by site‐directed mutagenesis, respectively. All β‐amylases, including the non‐mutant and mutant β‐amylases, produced in E. coli exhibited lower Vmax values than that of β‐amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]β‐amylase was reduced drastically compared to that of the non‐mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.
Single amino‐acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells
Authors: MIYAZAKI Youichi , MATSUFUJI Senya , MURAKAMI Yasuko , HAYASHI Shin‐ichi
Published: March 2005

The half‐life of ornithine decarboxylase (ODC) in HMOA cells, a variant cell line derived from hepatoma tissue culture (HTC) cells, is markedly increased compared with that in the parental cell line. In the present study, we examined which of the three relevant factors is responsible for the ODC stabilization in HMOA cells, namely ODC itself, a regulatory protein antizyme and an ODC‐degrading activity.

SDS/PAGE analysis of radiolabeled ODC revealed that ODC from HMOA cells migrated somewhat faster than that from HTC cells, suggesting that HMOA ODC was structurally altered. Direct sequencing of reverse‐transcription/polymerase‐chain‐reaction (RT‐PCR) products of ODC mRNA from HMOA cells revealed a T to G replacement, causing a Cys441→Trp replacement near the C‐terminus. No alteration was found in the whole coding region of antizyme mRNA. An authentic mutant ODC cDNA with the same replacement was transfected and expressed in C55.7 ODC‐deficient Chinese hamster ovary cells. Upon cycloheximide treatment, the mutant ODC activity did not decrease appreciably for at least 3 h, whereas wild‐type ODC activity decreased with a half‐life of 1 h. In‐vitro‐synthesized mutant ODC with the Cys441→Trp (or Ala) replacement was also stable in a reticulocyte‐lysate ODC‐degradation system. Metabolically labeled and purified mouse ODC was degraded in HMOA cell extracts in the presence of ATP and antizyme as rapidly as in HTC cell extracts, indicating that HMOA cells have a normal ODC degrading activity. These results indicated that the single amino acid replacement, Cys441→Trp, is responsible for the stabilization of ODC in HMOA cells and that Cys441 is important for rapid ODC turnover.
Effect of bromoconduritol on glucosidase II from rat liver
Authors: ALONSO Josefa María , SANTA‐CECILIA Angelines , CALVO Pedro
Published: March 2005

Bromoconduritol inhibits the p‐nitrophenyl‐glucosidase and maltase activities of glucosidase II purified from rat liver, an enzyme that removes the two α‐1,3‐linked glucose residues of the protein‐bound oligosaccharide Glc2Man9GlcNAc2 in the processing of N‐glycoproteins. The inactivation process exhibits pseudo‐first‐order kinetics. Previously, we have demonstrated the ocurrence of two binding (active) sites in the glucosidase II for the substrates p‐nitrophenyl α‐d‐glucopyranoside (pNphGlc) and maltose (high‐ and low‐affinity sites). The inhibition kinetic studies with bromo‐conduritol indicate that the high‐ and low‐affinity sites for pNphGlc correspond to high‐ and low‐affinity sites for maltose, respectively. Bromoconduritol has no effect on the binding of the substrates (pNphGlc and maltose) to the high‐affinity site, although it does modify the low‐affinity site and hinders the binding of the two indicated substrates to this site. These results, together with previous reports, have prompted us to propose a new kinetic model of binding and hydrolysis of the physiological substrate of the enzyme, in which the outermost glucose residue would bind and be released at the high‐affinity site, whereas the innermost glucose residue would do so at the low‐affinity site.
Structural change of jack bean urease induced by addition surfactants studied with synchrotron‐radiation small‐angle X‐ray scattering
Authors: HIRAI Mitsuhiro , KAWAI‐HIRAI Rika , HIRAI Toshihiro , UEKI Tatsuo
Published: March 2005

Both the quaternary and subunit structures of jack bean urease in solutions with ionic and nonionic surfactants have been studied by small‐angle X‐ray scattering using a synchrotron‐radiation source. The effects of those surfactants on the enzyme activity of urease have also been investigated in the same kind of solvent systems as those used for the scattering experiments. The present results show that the quaternary structure of urease in solution is fairly elongate and that by the relatively monor binding of SDS (SDS/protein = 0.23/1) the native urease molecule is dissociated into six identical subunits with nearly spherical structures. In addition the enzyme activity of urease was mostly retained under every solvent condition investigated, indicating that the subunit found in the present scattering experiments is the fundamental monomeric unit for both the quaternary‐structure formation and enzyme function of urease.
Characterization of a marsupial sperm protamine gene and its transcripts from the North American opossum (Didelphis marsupialis)
Authors: WINKFEIN Robert J. , NISHIKAWA Sandra , CONNOR Wayne , DIXON Gordon H.
Published: March 2005

A synthetic oligonucleotide primer, designed from marsupial protamine protein‐sequence data [Balhorn, R., Corzett, M., Matrimas, J. A., Cummins, J. & Faden, B. (1989) Analysis of protamines isolated from two marsupials, the ring‐tailed wallaby and gray short‐tailed opossum, J. Cell. Biol. 107] was used to amplify, via the polymerase chain reaction, protamine sequences from a North American opossum (Didelphis marsupialis) cDNA. Using the amplified sequences as probes, several protamine cDNA clones were isolated. The protein sequence, predicted from the cDNA sequences, consisted of 57 amino acids, contained a large number of arginine residues and exhibited the sequence ARYR at its amino terminus, which is conserved in avian and most eutherian mammal protamines. Like the true protamines of trout and chicken, the opossum protamine lacked cysteine residues, distinguishing it from placental mammalian protamine 1 (P1 or stable) protamines. Examination of the protamine gene, isolated by polymerase‐chain‐reaction amplification of genomic DNA, revealed the presence of an intron dividing the protamine‐coding region, a common characteristic of all mammalian P1 genes. In addition, extensive sequence identity in the 5′ and 3′ flanking regions between mouse and opossum sequences classify the marsupial protamine as being closely related to placental mammal P1. Protamine transcripts, in both birds and mammals, are present in two size classes, differing by the length of their poly(A) tails (either short or long). Examination of opossum protamine transcripts by Northern hybridization revealed four distinct mRNA species in the total RNA fraction, two of which were enriched in the poly(A)‐rich fraction. Northern‐blot analysis, using an intron‐specific probe, revealed the presence of intron sequences in two of the four protamine transcripts. If expressed, the corresponding protein from intron‐containing transcripts would differ from spliced transcripts by length (49 versus 57 amino acids) and would contain a cysteine residue.
Cloning and expression of phospholipase A2 from guinea pig gastric mucosa, its induction by carbachol and secretion in vivo
Authors: YING Zhao , TOJO Hiromasa , NONAKA Yasuki , OKAMOTO Mitsuhiro
Published: March 2005

A cDNA encoding phospholipase A2 (PLA2) was cloned from guinea pig gastric mucosa using a rat pancreatic‐PLA2‐cDNA fragment as a probe. The cDNA contains an open reading frame sufficient to encode the entire amino‐acid sequence of a PLA2‐precursor protein consisting of 146 amino acids, including a putative 16‐residue signal peptide and a 6‐residue activation peptide at the NH2‐terminus. Its nucleotide sequence exhibits 70% similarity to that of rat pancreatic PLA2 cDNA. The deduced amino‐acid sequence has all the typical pancreatic PLA2 characteristics, with the exception of the substitution of Phe for Tyr at position 28 in the calcium‐binding loop of the mature enzyme. When an expression vector containing the PLA2 cDNA was transfected into COS‐7 cells, a major portion of the proenzyme was secreted into the culture medium. Northern‐blot analysis showed the mRNA was present in guinea pig lung and pancreas at much lower levels than in the stomach. The effect of carbachol, a muscarinic acetylcholine agonist, on the secretion of gastric PLA2 and on its mRNA level in the gastric mucosa were examined. PLA2 secretion into the gastric juice was maximal 30 min after the subcutaneous administration of carbachol (0.4 mg/kg). It also increased the PLA2‐mRNA level in the tissue, the maximal mRNA level being delayed about 15 min compared with that in PLA2 secretion. These results suggest that vagal stimuli may contribute to PLA2 secretion and its compensatory synthesis, and that the secreted PLA2 may participate in the digestion of dietary and biliary phospholipids in the small intestine of guinea pig.
Inhibition of hepatocytic autophagy by okadaic acid and other protein phosphatase inhibitors
Authors: HOLEN Ingunn , GORDON Paul B. , SEGLEN Per O.
Published: March 2005

Autophagy, measured as the sequestration of electroinjected [3H]raffinose or endogenous lactate dehydrogenase, was inhibited in isolated rat hepatocytes by the protein phosphatase inhibitors okadaic acid, calyculin A and microcystin‐LR. Okadaic acid, the most potent inhibitor, suppressed autophagy almost completely at 15 nM, suggesting inhibition of a protein phosphatase of type 2A. Okadaic acid had no effect on ATP levels, protein synthesis or cellular viability at this concentration, but caused a disruption of the hepatocytic cytoskeleton and a consequent reduction in organelle sedimentability, potentially interfering with the autophagy assay unless the necessary precautions are taken. Lysosomal (propylamine‐sensitive) degradation of endogenous protein was inhibited by okadaic acid, whereas non‐lysosomal (propylamine‐resistant) degradation was unaffected. The autophagy‐inhibitory effect of okadaic acid was not affected by inhibitors of cAMP‐dependent protein kinase or protein kinase C (H‐7, H‐89, calphostin C) but eliminated by the non‐specific inhibitor K‐252a and its analogues (KT‐5720, KT‐5823, KT‐5926) and by KN‐62, a specific inhibitor of Ca2+/calmodulin‐dependent protein kinase II. Protein phosphorylation by this kinase would thus seem to play a role in regulation of the autophagic‐lysosomal degradation pathway.
Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27‐kDa heat‐shock protein
Authors: MEHLEN Patrick , BRIOLAY Jérome , SMITH Leila , DIAZ‐LATOUD Chantal , FABRE Nathalie , PAULI Daniel , ARRIGO Andre‐Patrick
Published: March 2005

The Drosophila melanogaster small heat‐shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to α‐crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved α‐crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian‐Virus‐40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen‐peroxide stresses as judged by Trypan‐blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5′ end of the conserved α‐crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N‐terminal half of the conserved α‐crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the α‐crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.
Studies on the induction and phosphorylation of xanthine dehydrogenase in cultured chick embryo hepatocytes
Authors: SCHIEBER Andrea , EDMONDSON Dale E.
Published: March 2005

Chick embryo hepatocytes, cultured in a chemically defined medium, were used to investigate hormonal requirements for xanthine‐dehydrogenase induction and to determine whether the enzyme is phosphorylated. Triiodothyronine is found to be required to induce the synthesis of active enzyme. Inclusion of sodium tungstate in the medium resulted in the complete loss of enyme activity but no decrease of immunochemically detectable levels of enzyme. Immunoprecipitated xanthine dehydrogenase from cell extracts migrates with enzyme purified from adult chicken liver on SDS/PAGE. Both the native 150‐kDa subunit and the 130‐kDa form of the enzyme is observed. N‐terminal sequence analysis of the 150‐kDa subunit shows the following; Ala‐Pro‐Pro‐Glu‐Thr‐Gly‐Asp‐Glu‐Leu‐Val‐Phe‐Phe‐Val‐Asn‐Gly‐Lys‐Lys ‐Val‐Val which is similar to the published N‐terminal sequences of rat, mouse and insect xanthine dehydrogenases. Autoradiography of denaturing gels of xanthine dehydrogenase isolated from 32Pi‐labeled hepatocytes demonstrates that the 150‐kDa and the 130‐kDa forms of the enzyme are phosphorylated. Chemical phosphate analysis of acid‐precipitated, electrophoretically pure chicken liver xanthine dehydrogenase also shows the presence of covalently bound phosphate. Phosphoamino acid analysis of both 32‐P labeled forms of the enzyme demonstrates the presence of phosphoserine. Thus, chicken liver xanthine dehydrogenase contains a phosphoserine residue as found previously in bovine milk xanthine oxidase [Davis, M. D., Edmondson, D. E. & Müller, F. (1984) Eur. J. Biochem. 145, 237–250].
Calcium binding by chick calretinin and rat calbindin D28k synthesised in bacteria
Authors: CHEUNG Wing‐Tai , RICHARDS Donald E. , ROGERS John H.
Published: March 2005

Calretinin is a member of the EF‐hand calcium‐binding protein family, with a high similarity with calbindin D28k. The chick calretinin cDNA sequence was reconstructed in a M13 vector and transferred into an expression plasmid derived from the pET series. The calretinin gene was expressed in Escherichia coli and produced immunoreactive calretinin of the expected size. Bacterially expressed calretinin was purified with successive ammonium‐sulfate precipitation, DEAE chromatography, hydroxyapatite chromatography, Sephadex G‐75 chromatography and Mono‐Q chromatography. Normally, 1.0–1.5 mg calretinin was obtained from 1 I bacterial culture with a protein recovery of 0.5–1.5%. Calbindin D28k was purified similarly from bacteria using an expression plasmid provided by W. Hunziker. Calcium‐binding activity of purified proteins was measured by equilibrium dialysis in calcium/EGTA mixtures with 45Ca as tracer. Both calretinin and calbindin D28k bound 3–4 Ca2+/molecule (calretinin, 4.0±0.5; calbindin D28k, 3.5±0.4), implying that at least one of the canonical EF‐hand domains does not bind calcium. The Kd was 0.3–0.5 μM with little difference between the values for the two proteins.
The glucagon‐insulin antagonism in the regulation of cytosolic protein binding to the 3′ end of phosphoenolpyruvate carboxykinase mRNA in cultured rat hepatocytes Possible involvement in the stabilization of the mRNA
Authors: CHRIST Bruno , NATH Annegret
Published: March 2005

Since protein binding to the 3′ end of mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in glucagon‐induced phosphoenolpyruvate‐carboxykinase‐mRNA (PCK) levels, in the absence and presence of insulin, was correlated with the time course of changes in the binding of cytosolic protein from 24‐h cultured rat hepatocytes to the 3′ end of PCK mRNA. PCK‐mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility‐shift assay.

In 24‐h cultured rat hepatocytes, binding of cytosolic protein to the PCK‐mRNA 3′ end and PCK‐mRNA levels were increased to a transient maximum at 2 h and 2–4 h, respectively, by a 1‐nM glucagon treatment, added with a change of medium. 100 nM insulin, added simultaneously with glucagon, reduced the glucagon‐induced maximum of protein binding by 80% and the increase of PCK mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by insulin.

100 nM insulin, added 1 h after glucagon, reversed protein binding to the 3′ end of PCK mRNA to nearly initial levels within 1 h and impaired the glucagon‐induced increase in PCK‐mRNA levels by 30%.

The transcriptional inhibitor cordycepin, added 1 h after glucagon, did not prevent the further increase in glucagon‐enhanced protein binding nor its reversal by insulin. It did, however, prevent a further significant increase in PCK mRNA.

Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK‐mRNA 3′ end. The proximal 466 bases of the PCK‐mRNA 3′ end as well as the 1050 bases of the histone‐H1°‐mRNA 3′ end and the 1200 bases of the arylsulfatase‐A‐mRNA 3′ end also bound cytosolic protein(s), but this protein binding was not altered by treatment with glucagon or insulin.

The 3′ end of PCK, arylsulfatase A and H1° mRNA exhibited strong binding of cytosolic protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat hepatoma cells. Cytosolic protein(s) from spleen showed weak binding and proteins from HeLa and U937 tumor cells did not bind. Protein binding was most prominent with the 3′ end of PCK mRNA and cytosolic extracts from liver.

The results support the hypothesis that the induction by glucagon and reversal by insulin of both protein binding to the 3′ end of PCK mRNA and the increase in PCK‐mRNA levels are correlated. This correlation suggests a functional relationship, possibly the regulation of mRNA stability by protein/mRNA interactions. Regulation of protein binding by glucagon and insulin is specific for the 3′ end of PCK mRNA and occurs possibly via covalent modification of the binding protein.
Isoforms of fatty‐acid‐binding protein in bovine heart are coded by distinct mRNA
Authors: BARTETZKO Norbert , LEZIUS Axel G. , SPENER Friedrich
Published: March 2005

In the course of our studies on structure/function relationships of fatty‐acid‐binding proteins, we reported earlier that the two isoforms of the 15‐kDa cardiac fatty‐acid‐binding protein (cFABP) from bovine heart only differ in one position; Asn98 in pl 5.1‐cFABP; Asp98 in pI 4.9‐cFABP [Unterberg, C., Börchers, T., Højrup, P., Knudsen, J. and Spener, F. (1990) J. Biol. Chem. 265, 16 255–16 261]. In the present study, we elucidate the origin for this heterogeneity. Isoelectric focusing analysis of immunoprecipitated in vitro translation products from total mRNA and positivehybrid‐selected cFABP/mRNA revealed two l‐[35S]methionine‐labeled proteins corresponding to pI 5.1‐cFABP and pI 4.9‐cFABP. In a control experiment, recombinant mRNA derived from cDNA encoding pI 5.1‐cFABP was translated and produced only pI 5.1‐cFABP as shown by isoelectric focusing of the translation products. We could observe co‐translational acetylation but not posttranslational deamidation of the cFABP isoforms. Taken together, our results demonstrate that the isoforms of cardiac fatty‐acid‐binding protein found in bovine heart are coded by distinct mRNA species.
Enzymes of a novel autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus, the 3‐hydroxypropionate cycle
Authors: STRAUSS Gerhard , FUCHS Georg
Published: March 2005

The phototrophic bacterium Chloroflexus aurantiacus can grow autotrophically but seems not to assimilate CO2 via any of the known autotrophic pathways. Holo [Holo, H. (1989) Arch. Microbiol. 151, 252–256] proposed a new pathway in which 3‐hydroxypropionate is formed from acetyl‐CoA. Previous studies excluded the operation of known CO2 fixation pathways and provided indirect evidence for the suggested pathway based on 13C‐labelling experiments. Here all enzyme activities of the postulated cyclic CO2 fixation mechanism are demonstrated in vitro. In essence, acetyl‐CoA is carboxylated and reductively converted via 3‐hydroxypropionate to propionyl‐CoA. Propionyl‐CoA is carboxylated and converted via succinyl‐CoA and CoA transfer to malyl‐CoA. Malyl‐CoA is cleaved to acetyl‐CoA and glyoxylate. Thereby, the first CO2 acceptor molecule acetyl‐CoA is regenerated, completing the cycle and the net CO2 fixation product glyoxylate is released. This cycle represents the fourth autotrophic pathway in nature and is designated the 3‐hydroxypropionate cycle.
cDNA nucleotide sequence and primary structure of mouse uterine peptidylarginine deiminase
Authors: TSUCHIDA Mamoru , TAKAHARA Hidenari , MINAMI Naoko , ARAI Tomoji , KOBAYASHI Yoshimi , TSUJIMOTO Hiroaki , FUKAZAWA Chikafusa , SUGAWARA Kiyoshi
Published: March 2005

Peptidylarginine deiminase is a protein‐modulating enzyme which converts the arginine residues in proteins to citrulline residues. This study describes the complete primary structure of mouse peptidylarginine deiminase, which was deduced from nucleotide sequence analysis of cDNA clones plus proteochemical analysis of the purified enzyme. The composite cDNA sequence contained a 5′ untranslated region of 7 bases, an open reading frame of 2019 bases that encoded 673 amino acids, a 3′ untranslated region of 2662 bases, and part of a poly(A) tail. The N‐terminal and C‐terminal sequences of the enzyme matched the sequences deduced from nucleotide analysis. Furthermore, we determined that the N‐terminal sequence was Nα‐acetyl‐Met‐Gln‐, a sequence which has never previously been reported among Nα‐acetyl‐Met proteins. The Arg 352 of the enzyme was converted to a citrulline residue and the potential Asn‐linked glycosylation site (Asn542‐Glu543‐Ser544) had no carbohydrate moiety. Thus, mouse peptidylarginine deiminase consists of 673 amino acids with a molecular mass of 76 260. Mouse peptidylarginine deiminase mRNA has two AU‐rich structures in the 3′ untranslated region which exhibit a high degree of similarity to those in lymphokine, cytokine and proto‐oncogene mRNA species. Since the rat enzyme (previously reported) does not possess these characteristic structures, we compared the levels of enzyme activity and mRNA in the mouse and rat uterus at four defined phases of the estrous cycle. The degradation of peptidylarginine deiminase and its mRNA proceeded significantly faster in the mouse than in the rat. We speculate that the unusual structure of the mouse enzyme and its mRNA be involved in this species‐specific rapid degradation.
Molecular evolution of biotin‐dependent carboxylases
Authors: TOH Hiroyuki , KONDO Hiroki , TANABE Tadashi
Published: March 2005

Amino‐acid sequences of three functional units from various biotin‐dependent carboxylases, biotin carboxylase, biotin‐carboxyl‐carrier protein and carboxyl transferase, were investigated by computer‐assisted sequence comparison to obtain information about the structure, function, and molecular evolution of the enzymes. Biotin‐dependent carboxylases, except transcarboxylase and oxaloacetate decarboxylase which lack biotin carboxylase, exert their catalytic activities through the three functional units. The three functional units correspond with functional domains or subunits of the enzymes, and the genetic information for the units is encoded in different ways from enzyme to enzyme. It is known that biotin carboxylase is homologous to carbamoyl‐phosphate synthetase, and that the biotin‐carboxyl‐carrier protein is homologous to lipoic‐acid‐binding domain. The evolutionary relationships between the functional units and their homologues were described. A model for the evolutionary history of the enzymes was proposed by molecular phylogenetic analysis, which shows how a wide variety of domain and/or subunit structures for the enzymes may have been established. A repeated structure was found in biotin‐carboxyl‐carrier protein, and the secondary structure of the protein was predicted using the observed sequence similarity with a lipoic‐acid‐binding domain.