bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacillus Thermoamyloliquefaciens KP1071 α‐Glucosidase II is a Thermostable Mr 540000 Homohexameric α‐Glucosidase with both Exo‐α‐1,4‐Glucosidase and Oligo‐1,6‐Glucosidase Activities
Authors: Suzuki Yuzuru , Nobiki Masayoshi , Matsuda Michio , Sawai Takakazu
Published: July 2004

α‐Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM‐P8477; growth over 30–66°C) was purified to a homogeneous state. Its Mr was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N‐terminal sequence of 20 residues was determined as Ala1‐Ile‐Gln‐Pro‐Glu‐Gln‐Asp‐Asp‐Lys‐Thr‐Gln‐Glu‐Asp‐Gly‐Tyr‐Ile‐Asp‐Ile‐Gly‐Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo‐α‐1,4‐glucosidase and the monomeric oligo‐1,6‐glucosidase from the same microorganism. The α‐glucosidase II had no antigenic group shared with the latter two enzymes.

Analysis of substrate specificity showed that the α‐glucosidase II has dual activity towards oligo‐1,6‐glucosidases and exo‐α‐1,4‐glucosidases, but its preference is for non‐reducing terminal α‐1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 °C and pH 7.0. Its half‐life at pH 6.8 was 10 min at 81 °C, and 5 h at 55 °C in 6.4 M urea, 26% ethanol or 2.5% SDS.

We suggest that the α‐glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo‐α‐1,4‐glucosidase and the oligo‐1,6‐glucosidase present in the same strain.
Selection of RNA Aptamers that Bind Specifically to the NS3 Protease of Hepatitis C Virus
Authors: Urvil Petri T. , Kakiuchi Nobuko , Zhou De‐Min , Shimotohno Kunitada , Kumar Penmetcha K. R. , Nishikawa Satoshi
Published: July 2004

The RNA genome of human hepatitis C virus (HCV) is translated into a large precursor polyprotein. The NS3 protease of HCV has a crucial role in the processing of the polyprotein into functional viral proteins. We have used an in vitro genetic‐selection strategy to isolate high‐affinity RNA aptamers that bind to the NS3 protein, especially to its protease domain. Starting from a RNA pool that had a random sequence core of 12–18 nucleotides, aptamers that bind specifically to the NS3 protein were selected after 10 rounds of selection and amplification. A single aptamer, 10G‐1, was found predominantly (71%) in the selected pool. This aptamer could bind to the NS3 protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro. By phosphate‐modification‐interference analysis we showed that the phosphate residues that are critical for the binding of 10G‐1 to NS3 lie within the selected regions of the aptamer and that binding involves electrostatic contacts with the phosphates of regions G28–U34 and A47–A55. The NS3‐binding region in 10G‐1 can serve as a basis for designing more potential inhibitors of the NS3 protein.
The Mouse Gene for Hypoxia‐Inducible Factor‐1α
Authors: Wenger Roland H. , Rolfs Andreas , Kvietikova Ivica , Spielmann Patrick , Zimmermann Dieter R. , Gassmann Max
Published: July 2004

The ubiquitously expressed hypoxia‐inducible factor‐1 (HIF‐1) is involved in expression of a large number of oxygen‐regulated genes. HIF‐1 is a heterodimer consisting of an α and a β subunit, both belonging to the basic‐helix‐loop‐helix Per‐aryl hydrocarbon receptor nuclear translocator‐Sim (PAS) family of transcription factors. Whereas HIF‐1α is a novel member of this family, HIF‐1β is identical to the aryl hydrocarbon receptor nuclear translocator, previously recognized to be involved in xenobiotic metabolism. cDNA cloning revealed that mouse HIF‐1α can be expressed as two mRNA isoforms containing alternative 5′ untranslated regions and two different predicted translational start sites. We cloned and characterized 20.5 kb of the mouse HIF‐1α gene (Hifla) containing exon II–XV. The two alternative first exons, I.1 and I.2, are separated from exon II by approximately 24 kb and 17 kb, respectively. We also sequenced Hifla exon I.1 and flanking regions, and mapped a single exon I.1 transcription initiation site. Reverse transcription PCR analysis of total RNA derived from normoxic and hypoxic mouse hepatoma and fibroblast cell lines suggested that the two alternative mRNA isoforms are constitutively co‐expressed in these cells, and that two different promoters drive transcription of HIF‐1α. A minimal exon I.1 promoter was identified which moderately activated heterologous gene expression, indicating that additional cis‐elements are required for efficient HIF‐1α transcription in vivo.
Conformational Studies of Human Vitamin‐D Receptor by Antipeptide Antibodies, Partial Proteolytic Digestion and Ligand Binding
Authors: Väisänen Sami , Juntunen Kari , Itkonen Arsi , Vihko Pirkko , Mäenpää Pekka H.
Published: July 2004

We have studied conformational changes of human vitamin‐D receptor by using antipeptide antibodies, partial proteolytic digestion and binding of the natural ligand calcitriol or its synthetic analogs. Before exposing either [35S]methionine‐labelled in vitro translated human vitamin‐D receptor or a recombinant human vitamin‐D receptor produced either in Escherichia coli or in Sf 9 insect cells to limited proteolysis by trypsin or chymotrypsin, the proteins were treated with calcitriol or its synthetic analogs. The digestion products were analyzed by SDS'PAGE, immunoblotting with polyclonal antipeptide antibodies targeted against different domains of the receptor, and Edman N‐terminal sequencing. After limited proteolysis with trypsin, two fragments of M, 21000 and M, 34000 could be localized into N‐terminus and C‐terminus of the receptor, respectively, by antipeptide antibodies. We found that treatment with calcitriol or its synthetic analogs leads to differential resistance of the ligand‐binding domain of the recombinant receptor to partial proteolysis in vitro. We suggest that this is due to distinct conformational changes in the domain induced by the different ligands. The short N‐terminal region and the Zn‐finger domain form, however, a protease‐resistant structure which is independent on the presence or absence of the ligand. When the C‐terminal fragment of M, 34000 was further analyzed by Edman N‐terminal sequencing, the major cleavage site in the receptor between amino acids Argl73 and Hisl74 was revealed.
Up‐Regulation of P‐Glycoprotein Expression in Rat Liver Cells by Acute Doxorubicin Treatment
Authors: Fardel Olivier , Lecureur Valérie , Daval Stephanie , Corlu Anne , Guillouzo André
Published: July 2004

Expression of P‐glycoprotein, a plasma‐membrane glycoprotein involved in multidrug resistance and encoded by mdr genes, was investigated in cultured rat liver cells acutely exposed to doxorubicin. This anticancer drug was shown to increase mdr mRNA levels in a dose‐dependent manner in both rat liver epithelial (RLE) cells and primary rat hepatocytes. This induction of mdr transcripts was detected as early as a 4‐h exposure to doxorubicin used at 0.5 μg/ml. It occurred through increased expression of the mdr1 gene as assessed by northern blot analysis using rat mdr‐gene‐specific probes. In addition, RLE cells exposed to doxorubicin displayed an overexpression of a 140‐kDa P‐glycoprotein as demonstrated by western blotting. Moreover, doxorubicin‐treated RLE cells displayed enhanced cellular efflux of the P‐glycoprotein substrate rhodamine 123 that was inhibited by the P‐glycoprotein blocker verapamil, thus providing evidence that doxorubicin‐induced P‐glycoprotein was functional in liver cells. Doxorubicin‐mediated mdr mRNA induction was found to be fully inhibited by actinomycin D, thus indicating its dependence on RNA synthesis; it was demonstrated to be not associated with alteration of protein synthesis, suggesting it differed from the known mdr mRNA overexpression occurring in response to cycloheximide. In contrast to P‐glycoprotein, other liver detoxification pathways such as cytochromes P‐450 1A were not induced by doxorubicin treatment. These data indicate that doxorubicin can act as a potent acute inducer of functional P‐glycoprotein in rat liver cells and therefore may modulate both chemosensitivity of hepatic cells and P‐glycoprotein‐mediated biliary secretion of xenobiotics.
Structural and Functional Characterization of the Extracellular Calcium‐Binding Protein BM‐40/Secreted Protein, Acidic, Rich in Cysteine/osteonectin from the Nematode Caenorhabditis Elegans
Authors: Maurer Patrik , Sasaki Takako , Mann Karlheinz , Göhring Walter , Schwarzbauer Jean E. , Timpl Rupert
Published: July 2004

Caenorhabditis elegans BM‐40 (positions 19–264) and its extracellular calcium‐binding domain (positions 139–264) were obtained in recombinant form from human kidney cells using an episomal expression vector. The purified proteins showed single bands of 33 kDa [BM‐40‐(19–264)‐peptide] or 14 kDa [BM‐40‐(139–264)‐peptide] on electrophoresis, contained internal disulfide bonds and α helices and were relatively resistant to matrix metalloproteinases. Hexosamine analysis indicated substitution by one N‐linked and two O‐linked oligosaccharides and recombinant BM‐40 was indistinguishable in its immunological epitopes from nematode tissue‐derived BM‐40, suggesting that it was obtained in native form. Both recombinant C. elegans proteins showed a distinct binding activity for human collagens I and IV in solid‐phase and surface‐plasmon‐resonance assays with an affinity (Kd= 1–2 μM), comparable to that of mammalian BM‐40. However, calcium‐binding studies revealed only a low‐affinity site (Kd= 6.2 mM) and failed to show the characteristic conformational change upon addition of EDTA. These and a few other differences are apparently due to two extra disulfide bonds and two deletionslinsertions in C. elegans BM‐40 and can be partly interpreted from the X‐ray structure of a large part of human BM‐40. The immunological assays available and the predictions of the location of the collagen‐binding epitope should facilitate a molecular and genetic approach to understand the function of BM‐40 in the development of C. elegans.
Characterization of a Drosophila Proximal‐Sequence‐Element‐Binding Protein Involved in Transcription of Small Nuclear RNA Genes
Authors: Su Yan , Song Yuru , Wang Yan , Jessop Lea , Zhan Lichun , Stumph William E.
Published: July 2004

In a wide variety of eukaryotic organisms, transcription of small nuclear RNA (snRNA) genes is dependent upon a proximal sequence element (PSE) located upstream of position –40 relative to the transcription start site. There is little or no existent knowledge concerning the PSE‐binding proteins of organisms other than human. Here, we report the purification of a fraction enriched in the Drosophila melanogaster PSE‐binding protein (Dw PBP). Dm PBP forms a highly specific complex with the PSE. The protein stimulates transcription from the Ul gene promoter by RNA polymerase II and from the U6 gene promoter by RNA polymerase III in Drosophila nuclear extracts, and activation is dependent upon the presence of a PSE. The molecular mass of native Dm PBP as measured by gel‐filtration chromatography is 375 kDa. Two polypeptides (apparent molecular masses 59 kDa and 61 kDa) appear to be in close contact with the DNA in that they can be very efficiently and specifically crosslinked to the PSE sequence by ultraviolet irradiation.
HNK‐1 Carbohydrate‐Mediated Cell Adhesion to Laminin‐1 is Different from Heparin‐Mediated and Sulfatide‐Mediated Cell Adhesion
Authors: Hall Heike , Deutzmann Rainer , Timpl Rupert , Vaughan Lloyd , Schmitz Brigitte , Schachner Melitta
Published: July 2004

The sulfated HNK‐1 carbohydrate present on glycolipids and on several neural recognition molecules has been shown to mediate the adhesion of murine small cerebellar neurons and astrocytes to the extracellular matrix molecule laminin‐1. In this study, we characterized the binding of the HNK‐1 carbohydrate to laminin‐1 extracted from the Engelbreth‐Holm‐Swarm (EHS) sarcoma and distinguished it unequivocally from binding sites for other sulfated carbohydrates. Electron microscopic analysis of rotary shadowed complexes of laminin‐1 and a HNK‐1 neoglycoprotein revealed a major binding site on the G domain that comprises the C‐terminal globule of the laminin α1 chain. The HNK‐1 carbohydrate also interacted with placental laminin isoforms containing an α chain variant. It bound to the proteolytic laminin‐1 fragment E8 comprising the domains G1–G3, but not to fragment E3 that carries the major heparin‐binding site on domains G4–G5. No binding was observed to the short arm containing fragments E1XNd or P1. Binding studies with native or denatured laminin E8 fragments and proteolytic or recombinant fragments of the G domain localized the HNK‐1 carbohydrate binding site to domain G2. The binding could be clearly distinguished from binding sites for other sulfated carbohydrates such as heparin and sulfatides. Further, the binding could not be abolished by reduction and alkylation or by urea treatment of laminin‐1 and was independent of the native conformation of laminin‐1 and of Ca2+. The G2 domain is also involved in the adhesion of HNK‐1 carbohydrate expressing early postnatal cerebellar neurons and is different from heparin‐ and sulfatide‐mediated cell adhesion to laminin‐1. HNK‐1 carbohydrate‐mediated cell adhesion appears, however, to be dependent on the native conformation of laminin‐1 indicating a more complex cellular recognition process.
Fluorescence Spectroscopic Studies on Interactions between Liver Annexin VI and Nucleotides
Authors: Bandorowicz‐Pikuła Joanna , Wrzosek Antoni , Pikuła Sławomir , Awasthi Yogesh C.
Published: July 2004

Annexin VI is a 68‐kDa calcium‐, phospholipid‐, and cytoskeletal‐element‐binding protein, which has been implicated in various processes, including calcium release and sequestration in calcifying cartilage, in a receptor‐mediated endocytosis in human fibroblasts, and in secretion from chromaffin granules. In these processes it was found that, in addition to Ca2+ and annexin, the presence of ATP is also a prerequisite. In the present report we show that annexin VI binds ATP and the binding of nucleotide to protein is accompanied by quenching of an intrinsic fluorescence of annexin VI, which was found to be specific for 2'‐(or 3')‐O‐(2,4,6‐trinitrophenyl)adenosine 5'‐triphosphate, GTP and ATP, and dependent on the annexin conformation. The nucleotide‐binding site within an annexin VI molecule is likely to be close to the tryptophan‐containing domain of annexin VI. We propose that ATP plays the role of a physiological ligand for annexin VI, and its binding to annexin VI may represent an alternative cellular mechanism for the regulation of annexin‐membrane interactions coupled to overall energy transitions in the cell.
A Second Isoform of 3‐Ketoacyl‐CoA Thiolase found in Caenorhabditis Elegans, which is Similar to Sterol Carrier Protein x but Lacks the Sequence of Sterol Carrier Protein 2
Authors: Bun‐Ya Masanori , Maebuchi Motohiro , Hashimoto Takashi , Yokota Sadaki , Kamiryo Tatsuyuki
Published: July 2004

We cloned a full‐length cDNA of the nematode Caenorhabditis elegans that encodes a 44‐kDa protein (P‐44, 412 residues) similar to sterol carrier protein x (SCPx). Mammalian SCPx is a bipartite protein: its 404‐residue N‐terminal and 143‐residue C‐terminal domains are similar to 3‐ketoacyl‐CoA thiolase and identical to the precursor of sterol carrier protein 2 (SCP2; also termed non‐specific lipid‐transfer protein), respectively. P‐44 has 56% sequence identity to the thiolase domain of SCPx but lacks the SCP2 sequence. Northern blot analysis revealed only a single mRNA species of 1.4 kb, which agrees well with the length of the cDNA (1371 bp), making it improbable that alternative splicing produces an SCPx‐like fusion protein. The sequence similarities of P‐44 to conventional thiolases are lesser than that to SCPx. Purified recombinant P‐44 cleaved long‐chain 3‐ketoacyl‐CoAs (C8–6) in a thiolytic manner by the ping‐pong bi‐bi reaction mechanism. The inhibition of P‐44 by acetyl‐CoA was competitive with CoA and non‐competitive with 3‐ketooctanoyl‐CoA. This pattern of inhibition is shared with SCPx but not with conventional 3‐ketoacyl‐CoA thiolase, which is inhibited uncompetitively with respect to 3‐ketoacyl‐CoA. From these results, we concluded that nematode P‐44 and mammalian SCPx constitute a second isoform of thiolase, which we propose to term type‐II 3‐ketoacyl‐CoA thiolase.
cDNA Cloning and Expression of Chicken Aminopeptidase H, Possessing Endopeptidase as well as Aminopeptidase Activity
Authors: Adachi Hideki , Tsujimoto Masafumi , Fukasawa Masayoshi , Sato Yuji , Arai Hiroyuki , Inoue Keizo , Nishimura Toshihide
Published: July 2004

Chicken aminopeptidase H is a cysteine protease possessing endopeptidase as well as aminopeptidase activity [Rhyu, M. R., Nishimura, T., Kato, Y., Okitani, A. & Kato, H. (1992)Eur. J. Biochem. 208, 53–59]. This enzyme exhibits molecular masses of 400 kDa on gel filtration and 52 kDa on SDS/PAGE, indicating that it consists of eight subunits with the same molecular mass.

In the current study, we cloned the cDNA for the catalytic subunit of chicken aminopeptidase H. The open reading frame of the aminopeptidase H gene consists of 1362 base pairs encoding a 52‐kDa protein consistent with the molecular mass determined on SDS/PAGE; the deduced amino acid sequence contains all the partial sequences determined for the purified enzyme. The sequence is similar to that of the bleomycin hydrolase of rabbit lung, which has been partially determined. The recombinant 52‐kDa protein expressed in COS7 cells exhibited both aminopeptidase and endopeptidase activities, which were inhibited by monoiodoacetic acid. Furthermore, the expression of aminopeptidase H in COS7 cells was also recognized on immunoblotting. This gene is the first one for aminopeptidase H in an animal tissue whose sequence has been completely determined.
Mapping and Mutational Analysis of the IgE‐Binding Epitopes on Ara h 1, a Legume Vicilin Protein and a Major Allergen in Peanut Hypersensitivity
Authors: Burks A. Wesley , Shin David , Cockrell Gael , Stanley J. Steven , Helm Ricki M. , Bannon Gary A.
Published: July 2004

Peanut allergy is a significant health problem because of the prevelance and potential severity of the allergic reaction. Serum IgE from patients with documented peanut hypersensitivity reactions and overlapping peptides were used to identify the IgE‐binding epitopes on the major peanut allergen, Ara h 1. At least twenty‐three different linear IgE‐binding epitopes, located throughout the length of the Ara h 1 protein, were identified. All of the epitopes were 6–10 amino acids in length, but there was no obvious sequence motif shared by all peptides. Four of the peptides appeared to be immunodominant IgE‐binding epitopes in that they were recognized by serum from more than 80% of the patients tested and bound more IgE than any of the other Ara h 1 epitopes. Mutational analysis of the immunodominant epitopes revealed that single amino acid changes within these peptides had dramatic effects on IgE‐binding characteristics. The identification and determination of the IgE‐binding capabilities of core amino acids in epitopes on the Ara h 1 protein will make it possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.
Characterization of the Equilibrium Intermediates in Acid Denaturation of Human Stefin B
Authors: Žerovnik Eva , Jerala Roman , Kroon‐Žitko Luise , Turk Vito , Lohner Karl
Published: July 2004

Acid‐induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N – IN– I1– U, where N is the native state, INis a native‐like intermediate, I1is an acid intermediate state with properties of a molten globule and U is the unfolded state. State I1exhibits no near‐ultraviolet CD but has some residual far‐ultraviolet CD. It differs from U in its ability to increase fluorescence of 1‐anilino‐naphthalene 8‐sulfonate (ANS). In 0.42 M salt, the pH denaturation is three‐state between the dimeric native state N2and intermediates IN2and I2, which are also dimeric according to size‐exclusion chromatography. The acid intermediate I2is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near‐ultraviolet CD and its far‐ultraviolet CD is even more intense than that for the native state.1H‐NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalori‐metric measurements were performed under conditions where the acid intermediates are maximally populated (18°C): state INfrom pH 5.0 to 4.6, 0.03 M salt; state I2below pH 3.8, 0.42 M salt; and state I1in equilibrium with INat pH 4.05, 0.03 M salt. Enthalpies of unfolding for states INand I2were comparable to those of the native state. The enthalpy of unfolding for state I1could not be determined.
Functional Expression of Fasciola Hepatica Cathepsin L1 in Saccharomyces Cerevisiae
Authors: Roche Leda , Dowd Andrew J. , Tort Jose , McGonigle Sharon , McSweeney Aengus , Curley G. Paul , Ryan Thecla , Dalton John P.
Published: July 2004

A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature‐sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre‐peptide‐encoding and most of the pro‐peptide‐encoding sequences, did not express funtionally active enzyme. The yeast‐expressed enzyme exhibited physico‐chemical properties in common with the native enzyme including, pH optimum for activity, stability at 37°C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast‐expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P2 position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences.
Mössbauer Study of 4‐Hydroxybutyryl‐CoA Dehydratase Probing the Role of an Iron‐Sulfur Cluster in an Overall Non‐Redox Reaction
Authors: Müh Ute , Buckel Wolfgang , Bill Eckhard
Published: July 2004

4‐Hydroxybutyryl‐CoA dehydratase from Clostridium aminobutyricum catalyzes the dehydration of 4‐hydroxybutyryl‐CoA to crotonyl‐CoA. Although dehydration is an overall non‐redox reaction, the enzyme contains FAD and Fe‐S clusters. Previous work has shown that the Fe‐S clusters are difficult to reduce and therefore unlikely to be redox‐active in catalysis. Here, Mössbauer spectroscopy has been used to characterise the Fe‐S clusters in active as well as in air‐inactivated enzyme. In zero magnetic field at 80 K and 4.2 K, the spectra of active dehydratase consisted mainly of one species (95%) with quadrupole splitting, ΔEQ= 1.00 mm s−1 and isomer shift, δ= 0.43 mm s−1 Magnetically perturbed Mössbauer spectra indicated a spin of zero. In the presence of 6 mM crotonyl‐CoA, the spectra remained unchanged. Taken together, the data show that there are [4Fe‐4S]2+ in the enzyme, most probably two clusters/homotetramer, that the four iron atoms in each cluster are coordinated in an identical fashion, and that there is no direct interaction with substrates. We therefore infer that the Fe‐S clusters serve a structural rather than a catalytic role in 4‐hydroxybutyryl‐CoA dehydratase. In air‐inactivated enzyme (10% residual activity), a new doublet appeared (58%) with ΔEQ= 0.72 mm s−1, δ= 0.32 mm s−1 and S= 0. The assignment of this subspectrum to [3Fe‐4S]+ clusters, based on the typical Mössbauer parameters, is contradicted by the finding of spin zero for the species. One possible explanation could be spin‐coupling of two [3Fe‐4S]+ clusters in close proximity.
Identification of Five New Genes, Closely Related to the Interleukin‐1β Converting Enzyme Gene, that do not Encode Functional Proteases
Authors: Rocher C. , Faucheu C. , Blanchet A.‐M. , Claudon M. , Hervé F. , Durand L. , Harnois M. , Diu‐Hercend A. , Lalanne J.‐L.
Published: July 2004

Interleukine‐1β converting enzyme (ICE) was the first identified member of a growing family of cysteine proteases that now includes ten mammalian homologs. Within this large family, two functional proteins, denoted TX and TY share 60% amino‐acid identity with ICE in the mature protein and, together with ICE, constitute the ICE subfamily. The present study describes the identification of five new gene sequences, denoted S1–S5, closely related to ICE and TX and belonging to this subfamily. Sequences were identified using genomic Southern‐blot analysis of human DNA with probes corresponding to ICE and TX exon 6. Using PCR amplification and cloning, the complete exon‐6 sequence of these new genes was identified; three exhibit around 90% identity with Ice within exon 6, whereas the two others share about 70% identity with Ice. Examination of open reading frames and of amino acids essential for ICE activity indicate that none of these genes encodes for a functional protease. In conclusion, extensive analysis of the genes closely related to Ice shows that the Ice subfamily is constituted of eight members. Three of them encode for functional proteases (ICE, TX and TY) whereas the remaining members probably correspond to pseudogenes.
Evolution of Shorter and More Hydrophilic Transthyretin N‐Termini by Stepwise Conversion of Exon 2 into Intron 1 Sequences (Shifting the 3′ Splice Site of Intron 1)
Authors: Aldred Angela R. , Prapunpoj Porntip , Schreiber Gerhard
Published: July 2004

Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396–406], showed that the N‐terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe‐faced Dunnart, Short‐tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N‐termini of transthyretin evolved by successive shifts of the 3’ splice site of intron 1 in the 3’ direction, resulting in successive shortening of the 5’ end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N‐terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection.
The Predominant Protein in Peroxisomal Cores of Sunflower Cotyledons is a Catalase that Differs in Primary Structure from the Catalase in the Peroxisomal Matrix
Authors: Kleff Stefan , Sander Stephanie , Mielke Gregor , Eising Rainer
Published: July 2004

This paper describes a biochemical study on the protein composition of crystalline inclusions (cores) from plant peroxisomes. By SDS/PAGE and immunoblotting, a catalase of 59 kDa was identified as the predominant protein component in purified cores from sunflower (Helianthus annuus L.) cotyledons. A 55‐kDa catalase was the only additional peptide detected. In contrast to in cores, the 55‐kDa catalase was the major catalase protein in matrix fractions obtained from lysed peroxisomes. These findings suggested two peroxisomal populations of catalase differing in molecular structure and subperoxisomal compartmentation in sunflower cotyledons. Evidence for different amino acid sequences of the two catalases was found by peptide mapping with endoproteinase Glu‐C, by expressing a cDNA encoding matrix catalase in Escherichia coli, and by partial amino acid sequencing of peptide fragments from 59‐kDa core catalase. These results contradict the previous view that the formation of cores occurred via condensation of matrix catalase, and indicate that new concepts on the biogenesis and physiological function of plant peroxisomal cores need to be developed.
1‐(5‐Phospho‐β‐d‐Ribosyl)2′‐Phosphoadenosine 5′‐Phosphate Cyclic Anhydride Induced Ca2+ Release in Human T‐Cell Lines
Authors: Guse Andreas H. , Da Silva Cristina P. , Weber Karin , Armah Charlotte N. , Ashamu Gloria A. , Schulze Christian , Potter Barry V. L. , Mayr Georg W. , Hilz Helmuth
Published: July 2004

1‐(5‐Phospho‐β‐d‐ribosyl)2′‐phosphoadenosine 5′‐phosphate cyclic anhydride [2′‐phospho‐cyclic ADP‐ribose, cAdo(2′)P(5′)PP‐Rib] was prepared enzymatically from NADP+ using ADP‐ribosyl‐cyclase from Aplysia californica. The product was purified by HPLC and characterized by NMR and mass spectroscopy, by conversion to 1‐(5‐phospho‐β‐d‐ribosyl)adenosine 5′‐phosphate cyclic anhydride (cADP‐Rib) by alkaline phosphatase and by resistance to snake venom phosphodiesterase. cAdo‐(2′)P(5′)PP‐Rib dose‐dependently released Ca2+ from an intracellular, non‐endoplasmic reticular Ca2+ pool of permeabilized Jurkat and HPB.ALL T‐lymphocytes. In contrast, the closely related compounds 1‐(5‐phospho‐β‐d‐ribosyl)3′phosphoadenosine 5′‐phosphate cyclic anhydride and 1‐(5‐phospho‐β‐d‐ribosyl)cyclic 2′,3′‐phosphoadenosine 5′‐phosphate cyclic anhydride did not induce Ca2+‐release from permeabilized T cells. The Ca2+ pool sensitive to cAdo(2′)P(5′)PP‐Rib partially overlapped with the Ca2+ pool sensitive to cADP‐Rib recently described in T cells [Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L. & Mayr, G. W. (1995) Characterization of cyclic adenosine diphosphate‐ribose‐induced Ca2+‐release in T‐lymphocyte cell lines, J. Immunol. 155, 3353–3359]. Control experiments suggest that the results were neither due to Ca2+ contaminations in the cADP‐Rib preparation nor to catabolism of cAdo(2′)P(5′)PP‐Rib to cADP‐Rib.
Cloning and Characterization of a Testis‐Specific, Developmentally Regulated A‐Kinase‐Anchoring Protein (Takap‐80) Present on the Fibrous Sheath of Rat Sperm
Authors: Mei Xiaohua , Singh Ishwar S. , Erlichman Jack , Orr George A.
Published: July 2004

cAMP is important for the initiation of mammalian sperm motility. Previously we established that a type II cAMP‐dependent protein kinase is tightly associated with the fibrous sheath of rat sperm. This unique cytoskeletal structure surrounds the 9+2 axonemal network in the principal piece of the flagellum. Association of the kinase to the fibrous sheath is mediated via its regulatory subunit, RII. An RII‐binding overlay procedure was used to document that RII could specifically associate with fibrous sheath polypeptides of 120 and 80 kDa. In this study, we report the cloning of a rat testis‐specific, developmentally regulated, RII‐binding protein (TAKAP‐80). A 1.2‐kb cDNA clone, isolated by screening a rat testis expression library with 32P‐labeled RII, hybridized to a 1.8‐kb mRNA transcript present exclusively in testis. This transcript appeared at detectable levels at 30 days after birth. Over the next 10 days the mRNA levels increased greatly. This time interval corresponds to the initiation of spermiogenesis. The complete nucleotide sequence of TAKAP‐80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids. The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues. Amino acids 1–147 of the protein contained 45% charged residues, with lysine and arginine predominating. Similarly, amino acids 268–502 also contained a high percentage of charged amino acids (35%). In contrast, amino acids 148–267 were mostly hydrophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline. The 1.2‐kb cDNA clone was inserted into the pRSET vector and expressed as a His6 tag fusion protein in Escherichia coli. The recombinant protein was soluble and bound RIIα, RIIβ and type IIα holoenzyme by the RII‐binding overlay procedure. Deletion analysis revealed that the high‐affinity interaction site for RII was contained within amino acids 258–378 of TAKAP‐80. Antibodies prepared against the fusion protein recognized an 80‐kDa protein present in the urea‐insoluble particulate fraction of rat testis and in purified fibrous sheath preparations isolated from rat epididymal sperm. Levels of the 80‐kDa immunoreactive protein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.
Phosphorylation Mutants of p53 Show Differential Complex Formation with Putative Dehydrogenase Tms1 of Fission Yeast
Authors: Wagner Peter
Published: July 2004

The yeast tms1 gene was originally identified as a multi‐copy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast. The tms1 gene product (Tms1) was found to form stable complexes with p53 in yeast and in vitro; using purified recombinant proteins, the interaction was mapped to the C‐terminal region of p53. This part is known to be modified by several protein kinases resulting in a transition of p53 from a latent to an activated state capable of transactivating various cellular genes involved in growth suppression or apoptosis. Since there is evidence for an evolutionary conservation of a Tms1‐related protein in mammals, the effect of the phosphorylation status of the C‐terminus of p53 on Tms1/p53 complex formation in vitro has been investigated. Whereas mutants changing the cdc2 phosphorylation site at position 315 of human p53 had only little effect on Tms1/p53 complex formation, we found that mutants involving the protein kinase CK2 site at position 392 showed a significantly decreased relative affinity for the Tms1 protein. The same result was obtained by using a C‐terminal fragment of p53 which was phosphorylated by purified protein kinase CK2, suggesting that the complex formation of p53 with cellular C‐terminal binding proteins like Tms1 impairs regulation by phosphorylation.
The Role of the 90‐kDa Heat‐Shock Protein and its Associated Cohorts in Stabilizing the Heme‐Regulated Eif‐24Al Kinase in Reticulocyte Lysates during Heat Stress
Authors: Xu Zuoyu , Pal Jayanta K. , Thulasiraman Vanitha , Hahn Hejin P. , Chen Jane‐Jane , Matts Robert L.
Published: July 2004

The heme‐regulated eIF‐2α kinase (HRI) is activated not only in heme‐deficient rabbit reticulocyte lysates (RRL), but also in hemin‐supplemented RRL treated with heat‐shock, N‐ethylmaleimide (MalNEt) or heavy metal ions. We have demonstrated previously that heat‐shock proteins, Hsp90, Hsp70 and FKBP52, are associated with HRI in RRL; the association of HRI with Hsp90 and FKBP52, but not Hsp70, is enhanced by hemin. To study the role of Hsp90 and its associated cohorts in the regulation of HRI, we examined the interaction of these proteins with HRI in hemin‐supplemented RRLs during heat or oxidative stress. The association of HRI with Hsp90, FKBP52 and p23 was maintained in heat‐, MalNEt‐ or Hg2+‐treated hemin‐supplemented RRL. Glycerol gradient centrifugation and gel filtration on Sephacryl S‐300 indicated that neither heat shock nor MalNEt‐treatment affected the apparent molecular mass of HRI in hemin supplemented RRL. In addition, active HRI was coimmunoprecipitated with 8D3 anti‐Hsp90 from both heme‐deficient and MalNEt‐treated hemin‐supplemented RRL. These results demonstrate that activation of HRI in response to heat stress and oxidative stress does not require dissociation of Hsp90 from HRI. Furthermore, HRI activity was inhibited upon addition of hemin to Hsp90‐depleted heme‐deficient RRL, indicating that inhibition of HRI activity by hemin is not mediated by the reassociation of Hsp90 with HRI. We also examined the dynamics of the interaction of Hsp90 with HRI. Reconstitution of the interaction of Hsp90 with HRI was stimulated by elevated temperature and required both Mg2+ and ATP. Addition of purified Hsp90 to hemin‐supplemented RRL which had been treated with MalNEt to inactivate its capacity to chaperone protein renaturation, protected HRI from irreversible denaturation and aggregation upon incubation at 41 °C. Our results suggest that Hsp90 interacts with HRI primarily in its capacity as a molecular chaperone, stabilizing HRI from denaturation under conditions of heat stress and oxidative stress.
Structural and Serological Characterization of the O‐Antigenic Polysaccharide of the Lipopolysaccharide from Acinetobacter Strain 90 Belonging to DNA Group 10
Authors: Haseley Simon R. , Holst Otto , Brade Helmut
Published: July 2004

Water‐soluble lipopolysaccharide (phenol/water extraction) isolated from Acinetobacter strain 90, which belongs to DNA group 10, was hydrolysed with 1 % acetic acid, ultracentrifuged, and water‐soluble products finally eluted from a Sephadex G‐50 column. The major fraction, a polysaccharide, contained D‐Gal, D‐GlcNAc, D‐GalNAc, and 4,6‐dideoxy‐4‐[(R)‐3‐hydroxybutyramido]‐D‐galactose (Fuc4NBuOH). The polysaccharide was characterised by means of monosaccharide analyses, Smith‐degradation, N‐deacetylation/deamination, and NMR studies, and was shown to have a branched pentasaccharide repeating unit.
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This structure was specifically recognized in western blots and enzyme immunoassays by polyclonal rabbit antisera.
Anti‐Insect Toxin 5 (Aalts) from Androctonus Australis
Authors: Nakagawa Yoshiaki , Lee Young Moo , Lehmberg Elisabeth , Herrmann Rafael , Herrmann Revital , Moskowitz Haim , Jones A. Daniel , Hammock Bruce D.
Published: July 2004

An insect‐selective scorpion toxin (AaITS) was purified from the venom of the North African scorpion Androctonus australis, and its amino acid sequence was determined by a combination of automated Edman degradation, electrospray‐ionization mass spectrometry, and sequence alignment. This insect toxin is very potent against the tobacco budworm, Heliothis virescens (100% lethal dose < 1.8 μg/100 mg body mass) and shows a distinct insect specificity and various symptoms. It is not toxic to mice after subcutaneous injection. The molecular mass of this toxin is 6882 Da and the amino acid sequence is similar to those of Androctonus australis anti‐insect toxin 4 (AaIT4), Leiurus quinquestriatus depressant anti‐insect toxins (LqhIT2, LqqIT2), and Buthotus judaicus depressant anti‐insect toxin (BJIT2).
Identification of Cdnas Encoding Sterol Methyl‐Transferases involved in the Second Methylation Step of Plant Sterol Biosynthesis
Authors: Bouvier‐Navé Pierrette , Husselstein Tania , Desprez Thierry , Benveniste Pierre
Published: July 2004

Two methyl transfers are involved in the course of plant sterol biosynthesis and responsible for the formation of 24‐alkyl sterols (mainly 24‐ethyl sterols) which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second one to 24‐methylene lophenol. Five cDNA clones encoding two Arabidopsis thaliana, two Nicotiana tabacum and one Ricinus communisS‐adenosyl‐L‐methionine (AdoMet) sterol methyltransferases (SMT) were isolated. The deduced amino acid sequences of A. thaliana and N. tabacum SMT are about 80% identical in all possible combinations. In contrast they are about 40% identical with the deduced amino acid sequence of R. communis SMT and the published Glycine max sequence. Both A. thaliana and one N. tabacum SMT cDNAs were expressed in a yeast null mutant erg6, deficient in AdoMet zymosterol C24‐methyltransferase and containing C24‐non‐alkylated sterols. In all cases, several 24‐ethylidene sterols were synthesized. A thorough study of the sterolic composition of erg6 expressing the A. thaliana cDNA 411 (erg6‐4118‐pYeDP60) showed 24‐methylene and 24‐ethylidene derivatives of 4‐desmethyl, 4α‐methyl and 4,4‐dimethyl sterols as well as 24‐methyl and 24‐ethyl derivatives of 4‐desmethyl sterols. The structure of 5α‐stigmasta‐8, Z‐24(241)‐dien‐3β‐ol, the major sterol of transformed yeasts, was demonstrated by 400 MHz 1H NMR. Microsomes from erg6‐4118‐pYeDP60 were shown to possess AdoMet‐dependent sterol‐C‐methyl‐transferase activity. Delipidated preparations of these microsomes converted cycloartenol into 24‐methylene cycloartanol and 24‐methylene lophenol into 24‐ethylidene lophenol, thus allowing the first identification of a plant sterol‐C‐methyltransferase cDNA. The catalytic efficiency of the expressed SMT was 17‐times higher with 24‐methylene lophenol than with cycloartenol. This result provides evidence that the A. thaliana cDNA 411 (and most probably the 3 plant SMT cDNAs presenting 80% identity with it) encodes a 24‐methylene lophenol‐C‐241 methyltransferase catalyzing the second methylation step of plant sterol biosynthesis.
Specific Interaction of the Streptomyces Chitin‐Binding Protein Chb1 with α‐Chitin
Authors: Zeltins Andris , Schrempp Hildgund
Published: July 2004

Streptomyces olivaceoviridis secretes a so far unique protein of 18.7 kDa (CHB1) which lacks catalytic activity. It interacts highly specifically with α‐chitin, but not with β‐chitin, chitosan, or cellulose. Each of the five codons for tryptophan (Tip) in the chbl gene was replaced by those for leucine (Leu) or tyrosine (Tyr). Eight corresponding mutant proteins and the wild‐type protein were purified to homogeneity and their binding capacity to α‐chitin was determined. The relative affinities to anti‐CHB1 antibodies, the kinetics of binding, the dissociation constants, circular dichroism, and fluorescence emission spectra for three mutant types were compared to the characteristics of CHB1. The presented data lead to the following conclusions, (a) CHB1 presents a highly flexible protein lacking a‐helices. (b) Replacement of each of the buried Trp residues (Trpl34 and Trpl84) leads to conformational alterations and, in due course, to a considerably reduced binding affinity of the protein, (c) The exchange of the exposed Trp 57 by either Leu or Tyr results in relatively slight topological changes, but entails a loss of binding capacity of about 90%. (d) The dissociation constant was highest for the mutant protein [L57]CHB1 (2.17 μM), followed by [L134]CHB1 (0.91 μM) and [L184]CHB1 (0.26 μM), and lowest for the progenitor CHB1 (0.11 μM), indicating its strong affinity to the unsoluble substrate, (e) The data suggest that the exposed Trp57 contributes directly and significantly to the interaction of CHBl with α‐chitin.
69‐kDa and 100‐kDa Isoforms of Interferon‐Induced (2′‐5′)Oligoadenylate Synthetase Exhibit Differential Catalytic Parameters
Authors: Marié Isabelle , Blanco Julià , Rebouillat Dominique , Hovanessian Ara G.
Published: July 2004

The (2′‐5′)oligoadenylate synthetase represents a family of interferon‐induced proteins which when activated by double‐stranded (ds)RNA polymerizes ATP into 2′‐5′‐linked oligomers with the general formula pppA(2′p5′A)n, wheren> 1, which for convenience are referred to as 2–5A. We studied here the influence of pH, dsRNA concentration and time on oligomeric composition of 2–5A synthesized by purified 69‐kDa and 100‐kDa isoforms of (2′‐5′)oligo(adenylate) synthetase. In optimal conditions for activity, the 69‐kDa form synthesized higher oligomers of 2–5A molecules whereas the 100 kDa form synthesized preferentially dimeric molecules, which are known not to be functional for the activation of RNase L. This difference does not reflect a differential affinity of the enzymes for the preformed 2–5A dimer, which is found to be a very poor substrate for both enzymes. This latter strongly suggests that the mechanism of elongation is more likely processive. Moreover, we show that both isoforms have efficient nucleotidyl‐transferase activity and provide evidence that, in optimized conditions, GTP can be used alone as substrate by these enzymes to generate pppG2′p5′G. Our results clearly demonstrate that the 69‐kDa and 100‐kDa forms of (2′–5′)oligoadenylate synthetase manifest various differential catalytic activities, and favor the hypothesis that these enzymes might have other functions in the cell besides those in the 2‐5A system.
The Campylobacter jejuni Porin Trimers Pack into Different Lattice Types when Reconstituted in the Presence of Lipid
Authors: Zhuang Jianping , Engel Andreas , Pagès Jean‐Marie , Bolla Jean‐Michel
Published: July 2004

Purified major outer membrane protein of Campylobacter jejuni exhibited different classes of molecules by SDS/PAGE and immunoblotting. A high‐molecular‐mass product (120–140 kDa) was observed under mild conditions of solubilization, a folded monomeric form of 35 kDa was seen when treated at high SDS concentrations and finally, a single band around 45 kDa occurred when the sample was heated to 96°C [Bolla, J. M., Loret, E., Zalewski, M. & Pagès, J. M. (1995)J. Bacterial. 177, 4266–4271]. The high‐molecular‐mass product was reconstituted into two‐dimensional crystals in the presence of phospholipids and Mg2+. The C jejuni porin required different conditions for successful reconstitution into two‐dimensional crystals than the Escherichia coli porin OmpF. Electron microscopy and digital image processing of negatively stained specimens revealed a rectangular lattice with a unit cells size of a= 8.9 nm, b= 14.9 nm, an oblique lattice with a= 8.9 nm, b= 30.1 nm, γ= 98°, and a trigonal lattice with a=b= 9.6 nm. Projection maps were calculated to a resolution of 2 nm, and exhibited a trimeric protein with three stain‐filled indentations.
Overexpression of Cruzipain, the Major Cysteine Proteinase of Trypanosoma cruzi, is Associated with Enhanced Metacyclogenesis
Authors: Tomas Ana M. , Miles Michael A , Kelly John M.
Published: July 2004

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected T. cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandemly repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life‐cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non‐transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteine proteinase inhibitor Cbz‐Phe‐Phe‐CHN2 (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.