bluuebunny/crossref_metadata_2025 huggingface dataset:
The interferon‐stimulated gene 54 K promoter contains two adjacent functional interferon‐stimulated response elements of different strength, which act synergistically for maximal interferon‐α inducibility
Authors: BLUYSSEN Hans A. R. , VLIETSTRA Remko J. , VAN DER MADE Angelique , TRAPMAN Jan
Published: March 2005

The interferon‐α(IFN‐α)‐regulated hamster ISG‐54 K gene, which is activated in hamster CHO‐12 cells at least 40‐fold, was isolated and the promoter region was characterized in detail. Sequence analysis revealed the presence of two elements, closely related to the interferon‐stimulated‐response‐element (ISRE) consensus sequence [AGTTTCNNTTTC(CT)]. The putative ISRE‐I sequence (GGTTTCAATTTCT) is located at position ‐97 to ‐85; ISRE‐II (AGTTTTACTTTCT), which differs at three positions from ISRE‐I, is found directly upstream of ISRE‐I at position ‐110 to ‐98. In a transient transfection assay in CHO‐12 cells the wild‐type hamster ISG‐54K –promoter–chloramphenicol‐acetyl‐transferase (CAT) reporter construct showed a 40–80‐fold induction, offering an excellent model to study the functional properties of the two ISRE. To find out whether both elements were functional in interferon regulation of the promoter, selected point mutations were introduced in the ‐110 to ‐85 region and in flanking sequences. The (mutated) ISG‐54 K promoter was linked to the CAT reporter gene and transiently expressed in CHO cells in the absence and presence of murine (Mu)IFN‐α6. Transfections showed that both the ‐97 to ‐85 (ISRE‐I) and the ‐110 to ‐98 (ISRE‐II) segment were needed for optimal interferon induction of the ISG‐54 K promoter. However, ISRE‐I has an approximately sevenfold stronger activity compared to ISRE‐II. Sequential substitution of the three ISRE‐I bases, which differ in ISRE‐II showed that the T at position ‐105 causes the lower activity of ISRE‐II. Transfection of ISG‐54 K promoter constructs, in which ISRE‐I was replaced by ISRE‐II, which generates a promoter with two ISRE‐II segments, and vice versa (two ISRE‐I), provided further evidence for a role of both elements in IFN‐α induction. Importantly, all data obtained in transfection studies show that the two ISRE cooperate synergistically. The mechanism of synergism is most probably an indirect interaction between transcription factors binding to the ISRE, because an increase in the spacial arrangement of the two ISRE with a complete helical turn or half a turn did not result in a substantial decrease in promoter activity.
A purified ferredoxin from Giardia duodenalis
Authors: TOWNSON Steven M. , HANSON Graeme R. , UPCROFT Jacqueline A. , UPCROFT Peter
Published: March 2005

A ferredoxin has been purified to homogeneity from the ancient protozoan parasite Giardia duodenalis. As far as we know, this is the first electron transport protein to be characterised from the organism. The ferredoxin exhibits absorption maxima at 296 and 406 nm with molar absorption coefficients of ɛ296= 16650 ± 240 M−1 cm−1 and ɛ406= 13100 ± 370 M−1 cm−1 respectively. The A406/A296 ratio ranged over 0.78–0.82. The molecular mass of the apoprotein calculated by mass spectrometry was 5730 ± 100Da and the minimum molecular mass by amino acid analysis was 5926Da. There were four cysteine residues/molecule protein but no methionine, arginine, histidine or tyrosine. The absence of these latter residues is consistent with the amino acid content of most ferredoxins. The N‐terminal amino acid sequence exhibited greatest similarity to Desulfovibrio gigas ferredoxin II and indicated the potential to coordinate an iron‐sulfur cluster. There were 3.21 ± 0.41 mol sulfide and 2.65 ± 0.06 mol iron/mol protein. Electron paramagnetic resonance studies of this protein have indicated the presence of an iron‐sulfur centre consistent with those of known ferredoxins. Ferredoxin serves as a biological electron acceptor from giardial pyruvate dehydrogenase with metronidazole as a terminal electron acceptor. Such a pathway may serve as a possible mechanism for the reductive activation of metronidazole in this parasite. A second ferredoxin has been purified to homogeneity, but at this stage there is insufficient material to fully characterise this protein. No other low‐molecular‐mass electron transport proteins have been identified in Giardia under the growth conditions described.
Isolation and characterization of a small putative zinc finger protein from cuttlefish epididymal sperm cells
Authors: MARTIN‐PONTHIEU Annie , WOUTERS‐TYROU Danièle , PUDLO Barbara , BUISINE Eric , SAUTIÈRE Pierre
Published: March 2005

At the end of spermiogenesis, sperm chromatin stabilization is ensured by protamine dephosphorylation and, in mammals, by the formation during epididymal transit, of intra‐ and inter‐molecular disulfide bridges between protamines. In cuttlefish, the nuclear protein transition histones → spermtid‐specific protein T→protamine Sp is very similar to that occurring in mammals during spermiogenesis. However, in cuttlefish, the protamine Sp is devoid of cysteine residues. The protein complement of cuttlefish epididymal sperm nuclei has been investigated. A minor basic protein, called protein E, has been isolated. Its primary structure was established from sequence analysis and mass spectrometry data of the protein and its fragments. Protein E contains a motif ‐Cys‐Xaa2‐Cys‐Xaa23‐His‐Cys‐Xaa2‐Cys‐ which is likely to adopt a zinc finger conformation. Reduced protein E does fix zinc whereas alkylation of cysteine residues abolishes this ability. The sequence of protein E does not correspond to that of any known protein, but presents some similarities with a part of ZFY protein, a putative human transcription factor specifically expressed in germinal cells and which could be involved in spermatogenesis.
Formylmethanofuran dehydrogenases from methanogenic Archaea Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron‐sulfur proteins
Authors: BERTRAM Peter A. , KARRASCH Marion , SCHMITZ Ruth A. , BÖCHER Reinhard , ALBRACHT Simon P. J. , THAUER Rudolf K.
Published: March 2005

Formylmethanofuran dehydrogenases, which are found in methanogenic Archaea, are molybdenum or tungsten iron‐sulfur proteins containing a pterin cofactor. We report here on differences in substrate specificity, EPR properties and susceptibility towards cyanide inactivation of the enzymes from Methanosarcina barkeri, Methanobacterium thermoautotrophicum and Methanobacterium wolfei.

The molybdenum enzyme from M. barkeri (relative activity with N‐formylmethanofuran = 100%) was found to catalyze, albeit at considerably reduced apparent Vmax, the dehydrogenation of N‐furfurylformamide (11%), N‐methylformamide (0.2%), formamide (0.1%) and formate (1%). The molybdenum enzyme from M. Wolfei could only use N‐furfurylformamide (1%) and formate (3%) as pseudosubstrates. The molybdenum enzyme from M. thermoautotrophicum and the tungsten enzymes from M. thermoautotrophicum and M. wolfei were specific for N‐formylmethanofuran.

The molybdenum formylmethanofuran dehydrogenases exhibited at 77 K two rhombic EPR signals, designated FMDred and FMDox, both derived from Mo as shown by isotopic substitution with 97Mo. The FMDred signal was only displayed by the active enzyme in the reduced form and was lost upon enzyme oxidation; the FMDox signal was displayed by an inactive form and was not quenched by O2. The tungsten isoenzymes were EPR silent.

The molybdenum formylmethanofuran dehydrogenases were found to be inactivated by cyanide whereas the tungsten isoenzymes, under the same conditions, were not inactivated. Inactivation was associated with a characteristic change in the molybdenum‐derived EPR signal. Reactivation was possible in the presence of sulfide.
Cloning and nucleotide sequence of the Pseudomonas aeruginosa glucose‐selective OprB porin gene and distribution of OprB within the family Pseudomonadaceae
Authors: WYLIE John L. , WOROBEC Elizabeth A.
Published: March 2005

OprB is a glucose‐selective porin known to be produced by Pseudomonas aeruginosa and Pseudomonas putida. We have cloned and sequenced the oprB gene of P. aeruginosa and obtained expression of OprB in Escherichia coli. The mature protein consists of 423 amino acid residues with a deduced molecular mass of 47597 Da. Several clusters of amino acid residues, potentially involved in the structure or function of the protein, were identified. An area of regional homology with E. coli LamB was also identified. Carbohydrate‐inducible proteins, potentially homologous to OprB, were identified in several rRNA homology‐group‐I pseudomonads by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, Western immunoblotting and N‐terminal amino acid sequencing. These species also contained DNA that hybridized to a P. aeruginosa oprB gene probe.
Biosynthesis of casein kinase II in lymphoid cell lines
Authors: LÜSCHER Bernhard , LITCHFIELD David W.
Published: March 2005

We have analyzed the biosynthesis of casein kinase II. In exponentially growing tissue culture cells, the β subunit was synthesized in excess of the catalytic subunit (α). A substantial fraction of newly synthesized β was degraded within the first hour. The remaining fraction of β was incorporated into holoenzyme. In contrast, little degradation of newly synthesized α subunit was observed and most was quickly and efficiently incorporated into holoenzyme. The assembly of β with α was paralleled by an increase in apparent molecular mass of β due to phosphorylation. The subcellular distribution of newly synthesized [35S]Met‐labelled casein kinase II and of enzyme labelled and chased in the presence of excess unlabelled methionine was very similar and compatible with a nuclear localization. The degradation of the excess β subunit occurred through a non‐lysosomal proteolytic system with a very low ATP requirement.
Nucleotide binding by multienzyme peptide synthetases
Authors: PAVELA‐VRANCIC Maja , VAN LIEMPT Henk , PFEIFER Eva , FREIST Wolfgang , VON DÖHREN Hans
Published: March 2005

Peptide synthetases consist of linearly arranged catalytic units, which by sequence alignment show equally spaced amino‐acid‐activating segments/modules of 600–700 amino acid residues. The consensus sequence comprises a new class of sequence motifs which are shared by some carboxyl‐activating enzymes, but which do not occur in aminoacyl‐tRNA synthetases. The catalytic properties of peptide synthetases with respect to the nucleotide substrate were investigated by enzyme kinetic studies. In the activation reaction ATP may be substituted by 2′‐deoxy‐ATP (dATP) and 7‐deazaadenosine 5′‐triphosphate, substrate analogues which are not recognised by many aminoacyl‐tRNA synthetases, and may thus prove useful alternative substrates in the detection of peptide synthetases within complex protein mixtures. ATP derivatives substituted at C2 are substrates, while those substituted at C8 are not, indicating a preference for the anti‐conformation in substrate binding. Kinetic studies revealed that coenzyme A is a non‐competitive inhibitor of the activation reaction, suggesting the presence of a second nucleotide binding site which accommodates nucleotides with phosphate in the C2′ or C3′ position. This substrate and inhibition profile is markedly different from that of aminoacyl‐tRNA synthetases and indicative of a separate homogeneous family of carboxyl‐activating enzymes.
Reconstitution of the myometrial oxytocin receptor into proteoliposomes
Authors: KLEIN Uwe , FAHRENHOLZ Falk
Published: March 2005

The requirements for regaining high‐affinity binding of the myometrial oxytocin receptor after detergent solubilization were investigated by reconstitution experiments. Large unilamellar liposomes were prepared by reverse‐phase evaporation from different mixtures of phospholipids, cholesterol and cholesteryl hemisuccinate. In the presence of the oxytocin receptor solubilized from myometrial membranes from pregnant guinea pig uterus, liposomes were treated with 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐2‐hydroxy‐1‐propanesulfonate (Chapso) throughout the range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles. Detergent removal was achieved using bio‐beads SM‐2 as adsorbent. The presence of cholesterol was a prerequisite for regaining high‐affinity binding of [3H]oxytocin and 125I‐oxytocin antagonist to reconstituted proteoliposomes. Binding of [3H]oxytocin but not of the antagonist was dependent on the presence of Mn2+ ions. Reconstitution after lectin chromatography and photoaffinity labeling of reconstituted vesicles resulted in the exclusive labeling of the oxytocin receptor with a molecular mass of 68–80 kDa.
Transcription of the rat dopamine‐D2‐receptor gene from two promoters
Authors: VALDENAIRE Olivier , VERNIER Philippe , MAUS Marion , DUMAS MILNE EDWARDS Jean‐Baptiste , MALLET Jacques
Published: March 2005

Modulation of the expression of the D2‐dopamine receptor gene is involved in several pathological and developmental circumstances. The gene and the corresponding promoter regions of the rat D2 receptor were isolated and partly characterized to study its regulation. The rat D2‐receptor gene spans at least 50 kb, and possesses eight exons; its organization was compared to those of the other dopamine‐receptor genes in a phylogenetic perspective. The gene contains two transcription‐start sites: the major one is located about 320 bp upstream from the 3′ end of the first exon, and a minor site is 70 bp further upstream. Transient‐expression assays with fusion constructs comprising fragments of the D2‐promoter region and the luciferase reporter gene confirmed the existence of two independent, TATA‐lacking promoters. Both promoters separately induced transcription of the luciferase gene in C6 glioma, primary fibroblasts, GH3 and MMQ pituitary cell lines, among which only the MMQ cells normally express the D2 receptor. Transcription is enhanced by the reunion of the two promoters, and modified by the addition of upstream sequences. Thus the 1‐kb promoter region analysed does not contain all the elements necessary to confer tissue‐specific expression of the gene, but does carry some positive and negative regulatory elements, which remain to be characterized.
Characterization of the ATP‐dependent leukotriene C4 export carrier in mastocytoma cells
Authors: LEIER Inka , JEDLITSCHKY Gabriele , BUCHHOLZ Ulrike , KEPPLER Dietrich
Published: March 2005

The biosynthesis of leukotriene C4 (LTC4) must be followed by an export of this mediator into the extracellular space where it interacts with receptors. Using mastocytoma cells we have demonstrated the existence of a primary‐active, ATP‐dependent transport mediating this export of LTC4 [Schaub, T., Ishikawa, T. & Keppler, D. (1991) FEBS Lett. 279, 83–86]. The following inhibitors served to characterize further this transport system in plasma membrane vesicle from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half‐maximal inhibition of the ATP‐dependent LTC4 transport at 71 μ. Cyclosporin A and its non‐immunosuppressive analog PSC 833 inhibited the ATP‐dependent transport with Ki values of 4.5 μM and 30μM, respectively. The LTD4 receptor antagonist 3‐([{3‐(2‐[7‐chloro‐2‐quinolinyl]ethenyl)phenyl}‐{(3‐dimethylamino‐3‐oxopropyl)‐thio}methyl]thio)propanoic acid (MK 571) was the most potent competitive inhibitor of the export carrier with a Ki value of 0.8 μM.

The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC4‐binding membrane proteins using [3]LTC4 as the photolabile ligand. Proteins with molecular masses of about 190 kDa and 35 kDa were predominantly labeled. In addition, a minor [3H]LTC4 labeling was observed in the molecular mass range of 100kDa.

The [3H]LTC4 labeling of the 190‐kDa protein was competed for by MK 571. The labeled proteins resisted extraction from the membrane with 2% sodium taurocholate suggesting that they are integral membrane proteins. Treatment of the membrane proteins with peptide N‐glycosidase F resulted in the appearance of an additional labeled polypeptide of about 140 kDa suggesting that the 190‐kDa protein is a glycoprotein. Photoaffinity labeling with 8‐azio[α‐32P]ATP predominantly labeled the LTC4‐binding 35‐kDa protein. The [3H]LTC4‐labeled 190‐kDa protein showed a mean isoelectric point at pH 6.3 with a range of pH 5.8–6.7, while the 35‐kDa protein had an isoelectric point at pH 6.8.

Specific labeling of a 190‐kDa membrane glycoprotein by the glutathione conjugate LTC4, which is competed for by a potent inhibitor of the ATP‐dependent LTC4 export carrier, pinpoints its involvement in the ATP‐dependent transport of LTC4 and related conjugates.
Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site‐directed random mutagenesis
Authors: HARUKI Mitsuru , NOGUCHI Eriko , NAKAI Chieko , LIU Yu‐Ying , OOBATAKE Motohisa , ITAYA Mitsuhiro , KANAYA Shigenori
Published: March 2005

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the αV helix and lies close to the active site, was analyzed by means of site‐direected random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease‐H‐dependent, temperature‐sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild‐type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active‐site residues, and/or an enhancement of the stability of the αV helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its δ1 position impacts catalysis.
Properties of multiple G · A mismatches in stable oligonucleotide duplexes
Authors: LANE Andrew , EBEL Susanne , BROWN Tom
Published: March 2005

The solution structure of the deoxydecanucleotide [d(GAGTGAACGA)]·[d(GAGTGAACGA)] has been determined by NMR methods. This duplex, which contains six G · A mismatches and four Watson‐Crick base pairs, is thermodynamically more stable than a decamer where T · A base pairs are substituted for the G · A mismatches, and is less stable than the duplex that contains G · C base pairs. Circular‐dichroism spectroscopy indicates an overall B‐like conformation for the decamer, but stronger than usual base stacking. 1H‐NMR spectroscopy revealed that the N1H groups of the mismatched guanine residues are not hydrogen bonded, and 31P‐NMR showed the presence of BII phosphate conformations for the GpA steps. Detailed analysis of the NMR data showed that all nucleotides have anti glycosidic torsion angles and S type sugar puckers. The G · A mismatches pair in the amino form as originally proposed by Li et al. [Li, Y., Zon, G. & Wilson, W. D. (1991) Proc. Natl Acad. Sci. USA 88, 26–30], which results in extensive base‐base stacking between the tandem G · A base pairs and their nearest neighbours. The terminal G · A base pairs are less stable than the central base pairs and show evidence of an equilibrium between two conformations, one involving BII phosphate.
Heterogeneity of the rat NADH‐cytochrome‐b5‐reductase transcripts resulting from multiple alternative first exons
Authors: MOTA VIEIRA Luisa , KAPLAN Jean‐Claude , KAHN Axel , LEROUX Alena
Published: March 2005

In order to understand the mechanisms responsible for the generation of different isoforms (membrane‐bound and soluble) of NADH‐cytochrome b5 reductase, and the different clinical forms of recessive congenital methemoglobinemia due to the deficiency of this enzyme in humans (type I, without mental retardation; type II, with mental retardation), we have looked for mRNA heterogeneity in various rat tissues.

We have found four types of mRNAs, each with a different first exon (1L, 1R, IX and 1Y), all of which were precisely spliced to join the common second exon. Our results are consistent with a 5′→3′‘scanning’ mechanism for splice‐site selection. The previously characterized 1L and 1R transcripts arise from the alternative use of either a ubiquitous promoter (Pr‐L) or an erythroid‐specific promoter (Pr‐R). In addition, the X and Y RNA species are novel transcripts which are expressed ubiquitously and at a relatively low level. The first alternative exons 1X and 1Y are non‐coding, such that the AUG codon present in the common second exon is functional, as it is in the R mRNA. Thus, the X and Y mRNAs are expected to be translated in vivo into a ubiquitous soluble enzyme. Consequently, the rat NADH‐cytochrome‐b5‐reductase gene is expressed through the use of at least four different promoters, which are probably subjected to different forms of regulation. This model of gene expression in rat could be important in understanding the basis for the different types of the NADH‐cytochrome‐b5‐reductase enzyme and their deficiency in man.
The amine‐donor substrate specificity of tissue‐type transglutaminase
Authors: GROENEN Patricia J. T. A. , SMULDERS Ronald H. P. H. , PETERS Roderick F. R. , GROOTJANS Johan J. , VAN DEN IJSSEL Paul R. L. A. , BLOEMENDAL Hans , DE JONG Wilfried W.
Published: March 2005

The amine‐donor substrate specificity of tissue‐type transglutaminase has been studied in a series of recombinant αA‐crystallin mutants. These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C‐terminal extended arm of αA‐crystallin. A biotinylated amine‐acceptor hexapeptide was used as a probe for labelling the amine‐donor sites. Wild‐type bovine αA‐crystallin does not function as an amine‐donor substrate for tissue‐type transglutaminase. Yet, upon introduction of a lysine residue at the C‐terminal or penultimate position, all mutant αA‐crystallins act as amine‐donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine‐donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.
Human β1,4 galactosyltransferase and α2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum
Authors: KREZDORN Christian H. , KLEENE Ralf B. , WATZELE Manfred , IVANOV Svetoslav X. , HOKKE Cornelis H. , KAMERLING Johannis P. , BERGER Eric G.
Published: March 2005

Biosynthesis and intracellular transport of recombinant human full‐length β1,4 galactosyltransferase (GT) and full‐length α2,6 sialyltransferase (ST) were investigated in Saccharomyces cerevisiae. Recently, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watzele, G., Kleene, R. B., Ivanov, S. X. & Berger, E. G. (1993) Eur. J. Biochem. 212, 113–120]. In the present work, we show that, in yeast strains transformed with plasmid pDPSIA containing the cDNA coding for human ST, rST enzymic activity using asialo‐fetuin or N‐acetyllactosamine as acceptor substrates could readily be detected. Analysis by 1H‐NMR spectroscopy of the disaccharide product of rGT, as recently reported, and the trisaccharide product of rST demonstrated that only the expected glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in membranes of the endoplasmic reticulum (ER) and were activated by Triton X‐100 3–5‐fold. rGT and rST could be immunoprecipitated from their [35S]Met‐labelled transformed yeast extracts using polyclonal antibodies raised against fusion proteins consisting of β‐galactosidase‐GT or β‐galactosidase‐ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa was reported, but for rST two main bands corresponding to apparent molecular masses of 48 kDa and 44 kDa, respectively, were detected. Immunoprecipitation from either tunicamycin‐treated [35S]Met‐labelled transformed yeast cells or labelling with radioactive sugars both indicated that the 44‐kDa form of rST was non‐glycosylated and that the 48‐kDa form of rST was core N‐glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44‐kDa form of rST was converted to the 48‐kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglycosylation of rST and rGT in wild type and lack of the Golgi‐specific man‐α1,6‐man epitope suggest that the recombinant enzymes did not enter the yeast Golgi apparatus.

These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein‐independent import of rST into the ER.
Mössbauer and magnetic susceptibility studies on iron(II) metallothionein from rabbit liver
Authors: DING Xiao‐Qi , BUTZLAFF Christian , BILL Eckhard , POUNTNEY Dean L. , HENKEL Gerald , WINKLER Heiner , VAŠÁK Milan , TRAUTWEIN Alfred Xaver
Published: March 2005

The magnetic properties of the Fe(II)‐binding sites in Fe(II)7‐metallothionein (MT) have been studied using Mössbauer spectroscopy and magnetic‐susceptibility measurements. In agreement with our previous results, simulation of the Mössbauer spectra showed the presence of paramagnetic and diamagnetic subspectra in the ratio 3:4. By comparison with Mössbauer spectra of the inorganic adamantane‐like (Et4N)2[Fe4(SEt)10] model compound, the diamagnetic component in Fe(II)7‐MT has been assigned to a four‐metal cluster in which there is antiferromagnetic coupling between the high‐spin Fe(II) ions. It is suggested that the organization of this cluster is similar to that determined in the three‐dimensional structure of the protein, containing diamagnetic Zn(II) and/or Cd(II) ions. From magnetic‐susceptibility studies, an average magnetic moment of approximately 8.5 μB was obtained for the three remaining bound Fe(II) ions, responsible for the paramagnetic component observed in the Mössbauer studies. This value is slightly lower than that for three completely uncoupled Fe(II) ions, suggesting the existence of a three‐metal cluster within which there is weak exchange coupling between adjacent Fe(II) ions. The spin‐Hamiltonian formalism including, besides zero‐field and Zeeman interaction, also exchange interaction among the three Fe(II) ions in the three‐metal cluster, H = ‐J12 (S1·S2)‐J23 (S2·S3‐J13 (S1·S3), was applied to simulate both magnetic‐Mössbauer and magnetic‐susceptibility data. Reasonable fits were achieved only with values |J12|=|J23|=|J13|=|J| < 1 cm−1. Such a situation could not be reconciled with the chair‐like geometry of the [M3(CysS)9]3‐ cluster determined with paramagnetic metal ions, where significantly stronger coupling would be anticipated (|J|= 50–70 cm−1). However, modest exchange‐coupling properties have been reported for a number of crystallographically characterized trinuclear [Fe3(SR)3X6]3‐ clusters (X = Cl, Br; R = Phe, p‐tolyl, 2,6‐Me2C6H3) distinguished by the preferential formation of a planar Fe3(μ2‐SR)3 ring [Whitener, M. A., Bashkin, J. A., Hagen, K. S., Girerd, J.‐J., Gamp, E. Edelstein, N. & Holm, R. H. (1986) J. Amer. Chem. Soc. 108, 5607–5620]. It is therefore more likely that a pseudo‐planar geometry rather than a chair‐like geometry is present in the Fe3 cluster of Fe(II)7‐MT. This would represent the first example of structural differences on binding divalent metal ions to this protein.
Possible kinetic mechanism of human placental alkaline phosphatase in vivo as implemented in reverse micelles
Authors: CHANG Gu‐Gang , SHIAO Shu‐Li
Published: March 2005

Human placental alkaline phosphatase is an integral membrane protein. It catalyzes the hydrolysis of phosphoester linkage of a broad‐range substrate. We have embedded the enzyme in a reverse micellar system prepared by dissolving the surfactant sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) in isooctane. Linear Lineweaver‐Burk and Eadie‐Hofstee plots for the substrate and linear Arrhenius plot for the temperature‐dependent enzyme reaction were obtained in reverse micelles suggesting that the substrate diffusion limitation was not a rate‐limiting step in the system and exchange of materials between reverse micelles was very rapid. The catalytic constant (kcat) of the enzyme was decreased, and the Km for the substrate was increased in reverse micelles, both in an exponential way with the [H2O]/[AOT] ratio (ωo.) The enzyme was more stable in reverse micelles than in aqueous solution at 30°C but was unstable at higher temperature (65°C). The activation energy of the enzyme in reverse micelles was 46.5 ± 2.6 kJ/mol, which was about 20 kJ/mol higher than that in aqueous solution and reflected in the lower kcat value at low ωo. The binding affinity between the substrate 4‐nitrophenyl phosphate and the enzyme in reverse micelles was decreased as implemented by the higher Km and higher Ki for phosphate values. In aqueous solution, the log kcat/pH plot suggested that amino acid residues with pKa values of 9.03 ± 0.03 and 11.37 ± 0.10 are involved in catalysis. The former should be deprotonated and the latter should be protonated for the reaction to proceed. In reverse micelles, both the above‐mentioned pKa values were detected. However, both groups have to deprotonated to give the optimum catalytic function. In aqueous solution, the enzyme's specificity was highly dependent on pH and buffer. The pKa value of the amino acid residues involved in substrate binding was found to be 8.69 ± 0.07 in carbonate buffer, but changed to 9.80 ± 0.06 in Tris buffer. Our results suggested that the rate‐limiting step of the enzyme‐catalyzed reaction may be changed from phosphate releasing in aqueous solution to another critical step in the reverse micelles.
Direct analysis and significance of cardiolipin transverse distribution in mitochondrial inner membranes
Authors: PETIT Jean‐Michel , HUET Olivier , GALLET Paul François , MAFTAH Abderrahman , RATINAUD Marie‐Hélène , JULIEN Raymond
Published: March 2005

The distribution of cardiolipin across the inner mitochondrial membrane was directly determined by using the ability of the fluorescent dye 10‐N‐nonyl‐3,6‐bis(dimethylamino)acridine (10‐N‐nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed: (a) a spectrophotometric measurement of 10‐N‐nonyl acridine orange binding to isolated rat liver mitochondria, mitoplasts and inside‐out submitochondrial particles, and (b) a flow‐cytometric analysis of specific red fluorescence, emitted when two dye molecules are bound to one membrane cardiolipin; the stoichiometry of 10‐N‐nonyl acridine orange binding to phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisation and subsequent red‐fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organelles, a distribution confirmed by saturation measurements for mitoplasts and inside‐out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1210 cells, and with mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparently tightly correlated with mitochondrial membrane assembly and activity.
Molecular cloning and sequence analysis of the gene of the molybdenum‐containing aldehyde oxido‐reductase of Desulfovibrio gigas
Authors: THOENES Ulrich , FLORES Orfeu L. , NEVES Ana , DEVREESE Bart , VAN BEEUMEN Jozef J. , HUBER Robert , ROMÃO Maria J. , LeGALL Jean , MOURA José J. G. , RODRIGUES‐POUSADA Claudina
Published: March 2005

In this report, we describe the isolation of a 4020‐bp genomic PstI fragment of Desulfovibrio gigas harboring the aldehyde oxido‐reductase gene. The aldehyde oxido‐reductase gene spans 2718 bp of genomic DNA and codes for a protein with 906 residues. The protein sequence shows an average 52% (± 1.5%) similarity to xanthine dehydrogenase from different organisms. The codon usage of the aldehyde oxidoreductase is almost identical to a calculated codon usage of the Desulfovibrio bacteria.
Characterisation and mode of in vitro replication of pea chloroplast OriA sequences
Authors: REDDY Malireddy K. , CHOUDHURY Nirupam Roy , KUMAR Dhirendra , MUKHERJEE Sunil K. , TEWARI K. K.
Published: March 2005

A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5′‐triphosphate, etc. Both strands of template DNA are synthesized and single‐stranded DNA templates undergo more than one round of replication.

When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement‐loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast‐DNA‐specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.
Photolabeling of soybean microsomal membrane proteins with photoreactive acyl‐CoA analogs
Authors: RAJASEKHARAN Ram , NACHIAPPAN Vasanthi , ROYCHOWDHURY Hiranya S.
Published: March 2005

Synthesis of 32P‐labeled 12‐azidooleoyl‐CoA and 125I‐labeled 12‐[(azidosalicyl)amino]dodecanoyl‐CoA (ASD‐CoA) was achieved. The synthesized radioactive, photoreactive reagents were tested as photoaffinity labels for acyl‐CoA:lysophosphatidylcholine acyltransferase from the microsomal membranes of developing soybean cotyledons. When a mixture of microsomal membranes and the azidooleoyl‐CoA or ASD‐CoA were incubated in the dark, the analogs were recognized as substrate and competitive inhibitor, respectively. The enzyme preferentially utilizes unsaturated acyl‐CoAs rather than saturated acyl‐CoAs. Incubation of microsomal membranes with acyl‐CoA analogs and immediately followed by photolysis resulted in an irreversible inhibition of lysophosphatidylcholine acyltransferase activity. Analysis of photolyzed microsomal membranes by SDS/PAGE and autoradiography revealed that azidooleoyl‐CoA preferentially labeled eight acyl‐CoA binding proteins, but ASD‐CoA labeled only three polypeptides with molecular masses of 110, 90 and 32 kDa that are commonly labeled by both the analogs. Oleoyl‐CoA and dodecanoyl‐CoA protect the enzyme against photoinactivation by azidooleoyl‐CoA and ASD‐CoA, respectively. The protection was profound in 110‐kDa polypeptide indicating that this protein could be lysophosphatidylcholine acyltransferase. These results demonstrate that the photoaffinity of acyl‐CoA analogs makes them potential probes to identify and characterize lipid biosynthetic enzymes.
Phospholipid binding and transfer by the nonspecific lipid‐transfer protein (sterol carrier protein 2)
Authors: GADELLA Theodorus W. J. , WIRTZ Karel W. A.
Published: March 2005

The nonspecific lipid‐transfer protein (nsL‐TP) from bovine liver was studied by measuring the binding and transfer of the fluorescent phospholipid 1‐palmitoyl‐2‐[6‐(1‐pyrenyl)‐hexanoyl]‐sn‐glycero‐3‐phosphocholine (PamPyrGroPCho). A kinetic model is presented involving three steps: (a) interaction of nsL‐TP with a membrane surface; (b) equilibration of PamPyrGroPCho monomers between the membrane and nsL‐TP; and (c) dissociation of the nsL‐TP/PamPyrGroPCho complex from the membrane surface. Steady‐state analysis of the model yielded theoretical equations describing both binding and transfer kinetics. Computer analysis, using these equations, showed good fits with the experimental results and several kinetic constants could be calculated. From these constants it was inferred that incorporation of acidic phospholipids into vesicles enhanced the interaction of nsL‐TP with the membrane interface (step a), without affecting the equilibrium binding of phospholipid monomers to nsL‐TP (step b). As a result, the rate of nsL‐TP‐mediated PamPyrGroPCho transfer from donor to acceptor vesicles was greatly affected. Under the conditions of incubation, incorporation of the acidic lipids in the donor membrane vesicles stimulated transfer, whereas incorporation of these lipids in the acceptor membranes could lead to a virtually complete inhibition of transfer. From the results it is concluded that the formation of a soluble lipid–nsL‐TP complex is the key step in nsL‐TP‐mediated phospholipid transfer.
Regulation of the erythrocyte Ca2+‐ATPase at high pH
Authors: DE CARVALHO‐ALVES Paulo Cesar , FREIRE Monica M. , BARRABIN Hector , SCOFANO Helena M.
Published: March 2005

The activation of the Ca2+‐ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the concentration of effector required to elicit half‐maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin‐Sepharose resin and in dependence on calmodulin concentrations when the pH is increased.

In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin‐binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.
Investigation of the effects of phosphorylation of rabbit striated muscle αα‐tropomyosin and rabbit skeletal muscle troponin‐T
Authors: HEELEY David H.
Published: March 2005

FPLC has been employed to prepare the phosphorylated and unphosphorylated forms of rabbit striated muscle αα‐tropomyosin (TM), and the major isoform of rabbit fast‐skeletal‐muscle troponin‐T (Tn‐T2f) and corresponding chymotryptic fragment T1 (residues 1–158), in order to investigate the effects which these in vivo modifications have on thin filament function. In all instances, no significance could be attributed to the presence of a phosphate moiety on acetyl serine 1 of Tn‐T (or fragment T1). As expected, fragment T1 increased the relative viscosities of solutions of unphosphorylated αα‐TM, but this induction was noticably lower for phosphorylated αα‐TM. In affinity chromatography experiments, fragment T1 bound equally well to either form of αα‐TM, but the interaction between fragment T2 (residues 159–259) and phosphorylated αα‐TM was strengthened relative to the control. In the presence of αα‐TM (unphosphorylated), fragment T1 was found to down regulate the actin‐activated myosin‐S1 MgATPase activity, indicating that this portion of Tn‐T possesses modulatory properties. Under the same conditions, less inhibition was observed with phosphorylated αα‐TM. When the two different forms of αα‐TM were reconstituted into a complete regulatory system, the activation of myosin‐S1 was double for those thin filaments containing the phosphorylated molecule. Dephosphorylation of the phospho αα‐TM reduced the rates to control values. In ATPase Ca2+ titrations, these systems exhibited no difference in the co‐operativity of activation and little or no difference in the pCa1/22+ value. Developmentally linked changes in the steady‐state phosphorylation of αα‐TM could be a mechanism to increase the activating propensity of thin filaments, by modifying the functional properties of the T1 section of Tn‐T.
An inducible arylsulfatase of Volvox carteri with properties suitable for a reporter‐gene system
Authors: HALLMANN Armin , SUMPER Manfred
Published: March 2005

The multicellular green flagellate Volvox carteri synthesizes a periplasmic arylsulfatase in response to sulfur deprivation. The inducible enzyme has been purified to homogeneity and characterized. The corresponding gene and cDNA have been cloned. Determination of the sequence of genomic clones and comparisons to the cDNA sequence, revealed sixteen introns and seventeen exons that encode a 649‐amino‐acid polypeptide chain.

Since the arylsulfatase enzyme is readily assayed using chromogenic substrates, but is not detectable in cells grown in sulfate‐containing medium, the gene encoding arylsulfatase may be useful as a reporter gene in V. carteri. In addition, the highly regulated promoter of the arylsufatase gene suggests its suitability as a tool for producing inducible expression vectors for cloned genes.
Isolation and characterization of an olive allergen‐like protein from lilac pollen
Authors: BATANERO Eva , VILLALBA Mayte , LÓPEZ‐OTÍN Carlos , RODRÍGUEZ Rosalía
Published: March 2005

An olive allergen‐like protein has been isolated from lilac (Syringa vulgaris) pollen extract. The protein can be considered as an allergen since is recognized by IgE from olive hypersensitive human sera, and has been called Syr v I (IUIS nomenclature). This protein consists of a glycosylated polypeptide of 20 kDa, which has an amino acid composition, spectroscopic properties, and an N‐terminal sequence similar to the major allergen from olive pollen, Ole e I. The lilac allergen is recognized by rabbit polyclonal antisera raised against olive allergen as well as by an Ole e I‐specific monoclonal antibody. Using a polymerase chain reaction strategy, based on the similarities observed between these olive and lilac proteins, three cDNA clones encoding Syr v I have been isolated and sequenced. These clones code for a polymorphic protein of 145 residues with a derived molecular mass of about 16400Da, which contains a potential N‐glycosylation site. Comparison of the deduced amino acid sequences of these Syr v I isoforms to each other revealed identities of 90–97%. Moreover, these sequences showed a high degree of similarity (85.5–89.6% identity) with Ole e I. The structural and immunological characterization of Syr v I justify the cross‐reactions observed between olive and lilac pollen extracts. The molecular cloning of Syr v I is relevant for the epitope mapping in Oleacease allergens, and may contribute to an improvement in the design of reagents for diagnosis and therapy of IgE‐dependent allergic reactions.
FMRFamide‐related peptides in the sex segmental ganglia of the Pharyngobdellid leech Erpobdella octoculata
Authors: SALZET Michel , BULET Philippe , WATTEZ Christian , MALECHA Jean
Published: March 2005

Using enzyme‐linked immunosorbent assays, a dot‐immunobinding assay and a three‐step reverse‐phase HPLC separation, four Arg‐Phe‐amide (RFamide) peptides were purified from sex segmental ganglia extracts of the leech Erpobdella octoculata; FMRFamide, FM(O)RFamide, FLRFamide and GDPFLRFamide. Their amino acid sequences were elucidated by means of a combined approach using antiserum specificity, synthetic‐peptide coelution, automated Edman degradation and electrospray mass spectrometry. One of these peptides, GDPFLRFamide, is a novel leech RFamide neuropeptide.

Two of the above RFamide peptides are involved in the control of leech hydric balance; one (GDPFLRFamide) is diuretic, the other (FMRFamide) is anti‐diuretic.

Titration of each purified RFamide peptide indicated a similar amount of each tetrapeptide and of tetrapeptides and heptapeptides. A comparison between RFamide peptides of E. octoculata and molluscs reveals structural similarities supporting the hypothesis for the existence of an ancestral RFamide peptide gene common to leeches and molluscs.
Domain B protruding at the third β strand of the α/β barrel in barley α‐amylase confers distinct isozyme‐specific properties
Authors: RODENBURG Kees W. , JUGE Nathalie , GUO Xiao‐Jun , SøGAARD Morten , CHAIX Jean‐Claude , SVENSSON Birte
Published: March 2005

α‐Amylases belong to the α/β‐barrel protein family in which the active site is created by residues located at the C‐terminus of the β strands and in the helix‐connecting loops extending from these ends. In the α‐amylase family, a small separate domain B protrudes at the C‐terminus of the third β strand of the (β/α)8‐barrel framework. The 80% identical barley α‐amylase isozymes 1 and 2 (AMY1 and AMY2, respectively) differ in substrate affinity and turnover rate, CaCl2 stimulation of activity sensitivity to the endogenous 21‐kDa α‐amylase/subtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme‐specific variations, AMY1–AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMY1‐(1‐90)–AMY2‐(90‐403) and AMY1‐(1‐161)–AMY2‐(161‐403) characterized in this study contain the 90‐residue and 161‐residue N‐terminal sequences, respectively, of AMY1 and complementary C‐terminal regions of AMY2. AMY1‐(1‐90)–AMY2‐(90‐403) comprises the 60‐amino‐acid domain B of AMY2 and resembles this isozyme in sensitivity to α‐amylase/subtilisin inhibitor and its low affinity for the substrates p‐nitrophenyl α‐d‐maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1‐(1‐161)–AMY2‐(161‐403) and AMY1, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaCl2. Domain B thus determines several functional and stability properties that distinguish the barley α‐amylase isozymes.