bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Characterization of Cathepsin B mRNA 3′‐Untranslated‐Region‐Binding Protein (CBBP), A Protein that Represses Cathepsin B mRNA Translation
Authors: Yano Tamaki , Kobayashi Ayako , Kurata Shoichiro , Natori Shunji
Published: July 2004

We reported that the 3′‐untranslated region (3′‐UTR) of cathepsin B mRNA of Sarcophaga peregrina (flesh fly) is necessary for the repression of its translation, and detected the 3′‐UTR‐binding protein in lysates of larval hemocytes in which cathepsin B mRNA translation was repressed [Yano, T., Kurata, S. & Natori, S. (1995)Eur. J. Biochem. 234, 39–43]. In this study, we purified the 3′‐UTR‐binding protein from an embryonic cell line of Sarcophaga. The purified protein (CBBP) was found to repress cathepsin B mRNA translation in a rabbit reticulocyte lysate. We found that the CBBP contents of the hemocytes did not change during metamorphosis, although the cathepsin B mRNA became translatable only at the pupal stage. Moreover, we found that pupal, but not larval hemocytes, contained a factor that inhibited the binding of CBBP to the 3′‐UTR. A regulatory mechanism of cathepsin B expression in Sarcophaga hemocytes is discussed.
GTP‐Binding–Protein‐Coupled Receptor Kinases Two Mechanistic Models
Authors: Palczewski Krzysztof
Published: July 2004

Six vertebrate protein kinases (G‐protein‐coupled receptor kinases; GRKs) that regulate the function of G‐protein‐coupled receptors (GPCRs) were recently cloned; several distinct properties set them apart from conventional second‐messenger regulated protein kinases. It appears that GRKs bind GPCR* through two separate sites: a high‐affinity site, which involves intracellular loops of the activated receptor, and the lower‐affinity site, encompassing the phosphorylation region. The high‐affinity interaction may involve complementary structural elements of GRKs and GPCRs* rather than precise amino acid alignment, thus allowing broad and overlapping specificities of these kinases, in spite of differences in the sequences of GPCRs. In addition, GRK structures are modified by several posttranslational modifications, including phosphorylation, autophosphorylation, prenylation, carboxymethylation, and palmitoylation, probably affecting properties of these enzymes. While GRKs phosphorylate and inactivate receptor molecules which are engaged in G‐protein activation, controversy surrounds whether GRKs might be activated and phosphorylate unstimulated GPCRs, leading to a desensitization of a larger population of the receptors. In this review, mechanistic aspects of GPCR* phosphorylation related to the distinct properties, regulation and modes of action of GRKs are described.
Purification and Properties of a Cellobiose Phosphorylase (CepA) and a Cellodextrin Phosphorylase (CepB) from the Cellulolytic Thermophile Clostridium Stercorarium
Authors: Reichenbecher Marisa , Lottspeich Friedrich , Bronnenmeier Karin
Published: July 2004

Two phosphorolytic enzymes displaying activity towards the soluble cellulose degradation products cellobiose and cellodextrins were purified from the crude extract of the cellulolytic thermophile Clostridium stercorarium. Both phosphorylases have monomeric structures with molecular masses of 93 and 91 kDa, respectively. Although the N‐terminal amino acid sequences are highly similar, a clear distinction of the two enzymes could be made on the basis of their substrate specificities: the enzyme designated cellobiose phosphorylase cleaved exclusively the disaccharide substrate, whereas the enzyme designated cellodextrin phosphorylase accepted only oligosaccharides as substrates. Kinetic constants were determined for the cleavage of cellobiose and cellodextrins. Maximal activity was observed at 65 °C in the pH range 6.0–7.0 for both enzymes. The sequences of the genes cepA and cepB encoding the cellobiose phosphorylase and the cellodextrin phosphorylase, respectively, have been submitted to the GenBank database.
Tubulin Post‐Translational Modifications
Authors: MacRae Thomas H.
Published: July 2004

This review describes the enzymes responsible for the post‐translational modifications of tubulin, including detyrosination/tyrosination, acetylation/deacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non‐tyrosinatable α‐tubulin. Tubulin tyrosineligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin‐specific carboxypeptidase and α‐tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulinlmicrotubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non‐tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post‐translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post‐translational modifications.
Synthesis, Sorting, and Processing into Distinct Isoforms of Human Macrophage Chitotriosidase
Authors: Renkema G. Herma , Boot Rolf G. , Strijland Anneke , Donker‐Koopman Wilma E. , Van Den Berg Marlene , Muijsers Anton O. , Aerts Johannes M. F. G.
Published: July 2004

Chitotriosidase, the human analogue of chitinases from non‐vertebrate species, has recently been identified. The macrophage‐derived enzyme is remarkably heterogeneous in molecular mass and isoelectric point. The synthesis and odification of the enzyme in cultured macrophages is reported. Chitotriosidase is synthesized as a 50‐kDa protein with a PI of about 6.5 and 7.2. It is predominantly secreted, but in part processed into a 39‐kDa form with a PI of 8.0 that accumulates in lysosomes. In the C‐terminal extension of the 50‐kDa chitotriosidase, sialic‐acid containing O‐linked glycans are present, causing its heterogeneous acidic isoelectric point. Chitotriosidase lacks N‐linked lycans and the mechanism of routing to lysosomes proves to be distinct from that of soluble, N‐glycosylated, lysosomal enzymes. It was observed that, in acrophages, alternative splicing generates a distinct chitotriosidase mRNA species, encoding a 40‐kDa chitotriosidase that is C‐terminally truncated. This enzyme is almost identical to the 39‐kDa chitotriosidase formed from the 50‐kDa precursor by proteolytic processing. It is concluded that the C‐terminus present in the 50‐kDa chitotriosidase, but absent in the 39‐kDa isoform, was found to mediate tight binding to chitin. In the blood stream the secretory 50‐kDa chitotriosidase occurs predominantly, whilst in tissues the 39‐kDa form is also abundant. These findings are consistent with the data on the synthesis and processing of chitotriosidase in the cultured macrophage model.
Mycothiol‐Dependent Formaldehyde Dehydrogenase, A Prokaryotic Medium‐Chain Dehydrogenase/Reductase, Phylogenetically Links Different Eukaroytic Alcohol Dehydrogenases Primary Structure, Conformational Modelling and Functional Correlations
Authors: Norin Annika , Van Ophem Peter W. , Piersma Sander R. , Persson Bengt , Duine Johannis A. , Jörnvall Hans
Published: July 2004

Prokaryotic mycothiol‐dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360‐residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc‐containing alcohol dehydrogenases. The structure is found to be a divergent medium‐chain dehydrogenase/reductase (MDR), at a phylogenetic position intermediate between the cluster of dimeric alcohol dehydrogenases of all classes (including the human forms), and several tetrameric reductases/dehydrogenases. Molecular modelling and functionally important residues suggest a fold of the mycothiol‐dependent formaldehyde dehydrogenase related overall to that of MDR alcohol dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active‐site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure. Residues typical of glutathione binding in class‐III alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione. The molecular architecture is different from that of the ‘constant’ alcohol dehydrogenases (of class‐III type) and the ‘variable’ alcohol dehydrogenases (of class‐I and class‐II types), further supporting the unique structure of mycothiol‐dependent formaldehyde dehydrogenase. Borders of internal chain‐length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins.
Evidence for Channeling of Intermediates in the Oxidative Pentose Phosphate Pathway by Soybean and Pea Nodule Extracts, Yeast Extracts, and Purified Yeast Enzymes
Authors: Debnam Phillip M. , Shearer Georgia , Blackwood Linda , Kohl Daniel H.
Published: July 2004

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1‐14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway‐produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6‐phosphate (Glc6P), 6‐phosphogluconolactone, or 6‐phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C‐labelled and unlabelled intermediates are discussed, together with the following results, (a) 14CO2 production by pea nodules in the presence of 3 mM 6‐phosphogluconate was higher than in its absence, (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract, (c) 6‐Phosphogluconate and 6‐phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6‐phosphogluconate dehydrogenase despite the absence of 6‐phosphogluconolactonase in the purified yeast enzyme mixture, (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6‐phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling, (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.
The Promoter of the Human Type I 5′‐Deiodinase Gene — Mapping of the Transcription Start Site and Identification of a DR+4 Thyroid‐Hormone‐Responsive Element
Authors: Jakobs Tatjana C. , Schmutzler Cornelia , Meissner Jutta , Köhrle Josef
Published: July 2004

The selenoenzyme thyroxine 5′‐deiodinase type I deiodinates the prohormone thyroxine to the active thyroid hormone 3,3′,5‐triiodothyronine. It is thus one of the key enzymes involved in the triiodothyro‐nine‐mediated control of growth, differentiation and basal metabolism in vertebrates. We report here the identification of the transcription start site and the cloning of 1500 bases of the upstream regulatory region of the human 5′‐deiodinase gene. They contain a complex triiodothyronine‐responsive element at nucleotides –696 to –673, consisting of an ideal direct repeat (DR) of two AGGTCA half‐sites with a spacing of four nucleotides (DR+4) and a third putative AGTTCA half‐site with a spacing of another two nucleotides (DR+2). The whole DR+4+2 specifically bound to thyroid hormone receptor and retinoid X receptor in electrophoretic mobility shift assays. The DR+4+2 mediates triiodothyronine‐responsiveness in cotransfection experiments of constructs containing the 5′‐deiodinase upstream promoter and enhancer region fused to luciferase or chloramphenicol acetyltransferase reporter genes with expression plasmids of thyroid hormone receptor subtypes. Also, an about 2.5‐fold induction of the 5′‐deiodinase‐promoter–luciferase‐reporter construct by all‐trans retinoic acid was observed in a cotransfection assay with retinoic acid receptors. Point mutation analysis of the DR+4+2 type hormone‐responsive element, however, revealed that it does not alone mediate the retinoic acid effect. The transcription start point of the 5′‐deiodinase gene was mapped to nucleotides ‐23 and ‐24. No CAAT or TATA box is located within the usual distance to the transcription initiation site. Two GC boxes were found at nucleotides ‐68 to ‐63 and ‐39 to ‐34. Transfection analysis revealed that the proximal 105 nucleotides in the 5′‐flanking region of the 5′‐deiodinase gene act as a functional core promoter. This data indicates that triiodothyronine, the end product of thyroid hormone synthesis, positively regulates one of the key enzymes in its production.
Basic Residues in the 74–83 and 191–198 Segments of Protein Kinase CK2 Catalytic Subunit are Implicated in Negative but not in Positive Regulation by the β‐Subunit
Authors: Sarno Stefania , Vaglio Philippe , Marin Oriano , Meggio Flavio , Issinger Olaf‐Georg , Pinna Lorenzo A.
Published: July 2004

Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (α and/or α') and two non‐catalytic, β‐subunits. The β‐subunit possesses antagonist functions that can be physically dissected by generating synthetic fragments encompassing its N‐terminal and C‐terminal domains. Here we show that by mutating basic residues in the 74‐77 and in the 191‐198 regions of the α‐subunit, the negative regulation by the β‐subunit and by its N‐terminal synthetic fragment CK2β‐(1–77), which is observable using calmodulin as a substrate for phosphorylation, is drastically reduced. In contrast, the positive regulation by a C‐terminal, CK2β‐(155–215)‐peptide is unaffected or even increased. Moreover, the basal activity of α mutants K74–77A, K79R80K83A, and R191R195K198A toward specific peptide substrates is stimulated by the β‐subunit many fold more than that of α wild type, while extrastimulation by β mutant D55L56E57A, observable with α wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N‐terminal moiety of β is mediated by basic residues in the 74–83 and in the 191–198 sequences of the α‐subunit. These are also implicated in substrate recognition consistent with the concept that the N‐terminal acidic region of the β subunit operates as a pseudosubstrate. In contrast, another CK2α mutant, V66A, is more sensitive to inhibition by either β‐subunit or its N‐terminal, CK2β‐(1–77)‐peptide, while its stimulation by the C‐terminal peptide, CK2β‐(155–215), is comparable to that of α wild type. These observations suggest an indirect role of Val66 in conferring to the α‐subunit a conformation less sensitive to down regulation by β‐subunit.
Two Types of Polyadenated mRNAs are Synthesized from Drosophila Replication‐Dependent Histone Genes
Authors: Akhmanova Anna , Miedema Koos , Kremer Hannie , Hennig Wolfgang
Published: July 2004

The polyadenylation of replication‐dependent histone H2B, H3 and H4 mRNAs in Drosophila melanogaster was analysed. Two types of mRNAs, containing a poly(A) tail, can be detected in addition to non‐polyadenylated messengers, which represent the majority of replication‐dependent histone mRNAs. Firstly, conventional polyadenylation signals, localized downstream from the stem‐loop region, are used to produce polyadenylated mRNAs, The messengers of this type, generated from the D. metanogaster H2B gene, are preferentially synthesized in the testis of the fly. Secondly, a distinct type of polyadenylated histone mRNA has been identified. This mRNA, which is present in many different tissues and constitutes a minor part of the total histone mRNA pool, contains a short poly(A) tail, added to the end of the 3′ terminal stem‐loop structure, which is in most cases lacking several nucleotides from its 3′ end. The sites of polyadenylation within the stem‐loop are not preceded by a normal polyadenylation signal. The possible functions of the polyadenylated histone transcripts are discussed.
Molecular Cloning and Functional Characterization of the Paracoccus Denitrificans Porin
Authors: Saxena Krishna , Richter Oliver‐Matthias H. , Ludwig Bernd , Benz Roland
Published: July 2004

Bacterial porins facilitate the passive uptake of small solutes across the outer membrane of the cell. The channel properties and the primary structure of the porin from Paracoccus denitrificans were investigated. As judged from single‐channel conductance experiments, this porin forms trimeric pores that show no ion selectivity in potassium chloride solution, which indicates that the charges within or near the channel are balanced. Based on peptide fragment sequence, the gene porG, which codes for this general pore protein, was cloned and analyzed. Its primary translation product contains a 20‐residue signal sequence, followed by the 295 amino acids of the mature protein with a molecular mass of 31.9 kDa. Sequence alignments with porins from Rhodopseudomonas blastica and Rhodobacter capsulatus and secondary structure predictions suggest a typical rigid barrel structure consisting of 16 antiparallel β‐strands.
Some DNA Targets of the Yeast CYP1 Transcriptional Activator are Functionally Asymmetric
Authors: Naït‐Kaoudjt Rachida , Williams Roy , Guiard Bernard , Gervais Michel
Published: July 2004

CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA‐binding domain. It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence. Notably, this ideal sequence is however not found in wild‐type CYP1 target sites.

In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1‐A/CYCI, UAS1‐A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1‐(1‐ 200)‐peptide were analyzed. Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding, (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1. (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1. (d) CYP1‐(1 ‐ 200)‐peptide is usually a monomer in solution but binds DNA as a dimer.

Finally, we found evidence for the presence of two half‐sites with different measured affinities in the asymmetric sequences of some CYP1 targets.
Proteinase A, A Storage‐Globulin‐Degrading Endopeptidase of Vetch (Vicia Sativa L.) Seeds, is not Involved in Early Steps of Storage‐Protein Mobilization
Authors: Becker Claudia , Senyuk Vitalyi I. , Shutov Andrei D. , Van Nong Hai , Fischer Jürgen , Horstmann Christian , Müntz Klaus
Published: July 2004

Proteinase A is a papain‐like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage‐globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin‐containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein‐body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N‐terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C‐terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydrylendopeptidase (SH‐EP), a cysteine endopeptidase from germinating Vigna tnungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1‐day‐germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29‐kDa bands of mature proteinase A were detected in cotyledon extracts of 6‐day‐germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage‐globulin breakdown during germination.
Differential Stabilization of Topoisomerase‐II–DNA Cleavable Complexes by Doxorubicin and Etoposide in Doxorubicin‐Resistant Rat Glioblastoma Cells
Authors: Montaudon Danièle , Pourquier Philippe , Denois Françoise , De Tinguy‐Moreaud Éliane , Lagarde Philippe , Robert Jacques
Published: July 2004

Using the technique of alkaline filter elution, we have evaluated the DNA damage induced by doxorubicin and etoposide in a rat glioblastoma cell line, C6, and its doxorubicin‐selected resistant variant, C6 0.5. DNA damage paralleled ding‐induced cytotoxicity, but it appeared that the same DNA damage generated much less cytotoxicity in resistant cells than in sensitive ones, resistant cells being able to tolerate more DNA damage than sensitive cells.

We have then quantified the doxorubicin‐ and etoposide‐induced complexes between topoisomerase II (topoll) DNA with the technique of SDS/KCl precipitation. Etoposide produced a concentration‐dependent increase in topoII‐DNA complexes, which was higher in resistant cells at equitoxicity, just as was DNA damage. In contrast, doxorubicin‐induced topoII‐DNA complexes, which were much less abundant than those induced by etoposide, were not differently produced in sensitive and resistant cells. This indicates that the DNA damage occurring in resistant cells at high doxorubicin concentrations might originate from source other than topoII‐DNA complex formation.

When verapamil was added during drug exposure, it restored doxorubicin intracellular accumulation to the level reached in sensitive cells, partially reversed both doxorubicin and etoposide resistance, increased the formation of etoposide‐induced topoII‐DNA complexes, but not those induced by doxorubicin. Immunoblot analysis of topoll as well as the measure of its catalytic activity in nuclear extracts revealed a quantitative defect of this enzyme in the resistant line. When inhibiting this activity by doxorubicin and etoposide, we observed that the concentrations of etoposide required for a given inhibition of kinetoplast DNA decatenation are much higher that those of doxorubicin. The topoII extracted from both cell lines is, therefore, much more sensitive to doxorubicin than to etoposide, but no difference in drug sensitivity was evident between sensitive and resistant cells, indicating that no qualitative alteration in topoll catalytic activity was likely to occur.
Comparative In‐Vivo and In‐Vitro99Mo‐Time‐Differential‐Perturbed‐Angular‐Correlation Studies on the Nitrogenase Mofe Protein and on Other Mo Species of Different N2‐Fixing Bacteria
Authors: Müller Achim , Suer Waltraud , Pohlmann Carsten , Schneider Klaus , Thies Wolf‐Gerolf , Appel Helmut
Published: July 2004

Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing 99MoO2‐4. The distribution of 99Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion‐exchange (DEAE‐Sephacel) chromatography. Cells of K. pneumoniae took up MoO2‐4 only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kp1) as the predominant Mo‐containing species. In the case of A. vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Av1). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo‐storage protein. In the presence of 20 mM NH4+, conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo‐storage protein exclusively. This protein proved to be unstable towards DEAE‐Sephacel, apparently releasing all the molybdenum in form of MoO2‐4 during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rc1), significant amounts of other not‐yet‐identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase.

The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process 99Mo(β−)99Tc using time differential perturbed angular correlation spectroscopy. The quadrupole coupling constant (VQ) determined for the Mo center in MoFe proteins was consistently in the range 66–81 MHz. The values of the coupling constants determined with intact cells and with the isolated, partially purified, MoFe proteins were in very good agreement. For the Mo‐storage protein of A. vinelandii, a quadrupole coupling constant of approximately 180 MHz was determined by measurements performed with nitrogenase‐repressed cells as well as with gel‐filtered cell‐free extracts. Our work proves that the relevant study of hyperfine interactions allows the identification of the MoFe protein and also other Mo proteins in vivo as well as in vitro.
Polymorphic Curvature of Satellite DNA in Three Subspecies of the Beetle Pimelia sparsa
Authors: Barceló Francisca , Pons Joan , Petitpierre Eduard , Barjau Ignasi , Portugal José
Published: July 2004

The curvature of the monomeric repeats of satellite DNAs from three subspecies of the beetle Pimelia sparsa (Coleoptera, Tenebrionidae) has been analysed. Evidence of curvature was inferred from their retarded migration in native polyacrylamide gels, which was confirmed by direct electron microscopy visualisation. Sequence‐comparison analysis, which included sequence alignments and modelling studies, was used to reveal the patterns of local bending and curvature. The effects of the minor‐groove‐binding drugs distamycin and berenil on the curvature were assayed, and analysed with respect to their (A+T)‐rich sequence preferences. Since our study deals with satellite DNAs from closely related organisms (three subspecies), we correlated the differences in sequence, and also the high similarity conserved in the (A+T)‐rich regions, with the changes in the patterns of curvature.
Comparison of the Diphtheria Mutant Toxin, Crm197, with a Haemophilus Influenzae Type‐b Polysaccharide‐Crm197 Conjugate by Optical Spectroscopy
Authors: Crane Dennis T. , Bolgiano Barbara , Jones Christopher
Published: July 2004

A Haemophilus influenzae type‐b capsular polysaccharide‐CRM 197 protein conjugate vaccine was compared with unconjugated CRM197 and diphtheria toxin, its parent molecule. Using CD and fluorescence spectroscopy, it has been possible to observe differences in structure and stability to pH and temperature due to the G52→ mutation in CRM197 and the ‘glycosylation’ of CRM197 in the conjugate. CRM197 resembles the ‘open’ conformation of diphtheria toxin [Blewitt, M. G., Chung, L. A. & London, E. (1985) Biochemistry 24, 5458–5464] and the attachment of poly(ribosyl‐ribitol phosphate) carbohydrate chains results in a still ‘more open’ state, although only a small decrease in the amount of ordered structure was observed. Fluorescence spectra of gel‐filtration column fractions of the conjugate suggest that material of higher apparent molecular size is in the ‘more open’ conformation. Conjugated CRM197 begins unfolding at slightly lower temperatures (25–35°C) than native material (> 35°C). In the conjugate, tryptophan residues are more accessible to the non‐ionic flourescence quencher acrylamide at 35°C. The eonformational change observed at pH4–6 for diphtheria toxin is also observed for CRM197, but in the conjugate begins at higher pH. This may result from the presence of charged oligosaccharide residues on the surface or the conjugation methods used. The consequences of these changes in conformation and solution behaviour of the carrier protein in terms of its ability to induce a protective, T‐cell‐dependent response to H. influenzae polysaccharide remain to be determined.
Multiheme Cytochromes from the Sulfur‐Reducing Bacterium Desulfuromonas Acetoxidans
Authors: Pereira Inês A. C. , Pacheco Isabel , Liu Ming‐Y. , Legall Jean , Xavier António V. , Teixeira Miguel
Published: July 2004

Two new multiheme cytochromes were isolated from the anaerobic sulfur reducing bacterium Desulfuromonas acetoxidans. They have monomeric molecular masses of 50 and 65 kDa and contain six and eight hemes, respectively. Visible and EPR spectroscopies, in the as‐isolated (oxidised) cytochromes, show the presence of only low‐spin hemes in the 50‐kDa cytochrome, and of high‐spin and low‐spin hemes in the 65‐kDa cytochrome. The EPR spectra of the native 65‐kDa cytochrome indicate multiple heme–heme interactions, including integer‐spin systems as judged by parallel‐mode EPR. The 50‐kDa cytochrome has a complex redox pattern, as shown by EPR redox titrations, and contains one heme with unusual characteristics. Both cytochromes cover an extremely wide range of reduction potentials, which go from +100 mV to ‐375 mV for the 50‐kDa cytochrome, and +185 mV to ‐235 mV for the 65‐kDa cytochrome.

The two cytochromes were tested for hydroxylamine oxidoreductase activity and polysulfide reductase activity, but neither displayed any activity. In contrast, it was found for the first time that the previously characterised cytochrome c551.5, from the same bacterium is very active in the reduction of polysulfide, which suggests that it acts as a terminal reductase in D. acetoxidans.
The Ordered Phosphorylation of Cardiac Troponin I by the cAMP‐Dependent Protein Kinase Structural Consequences and Functional Implications
Authors: Keane Noeleen E. , Quirk Philip G. , Gao Yuan , Patchell Valerie B. , Perry S. Victor , Levine Barry A.
Published: July 2004

The pattern of phosphorylation of adjacent serine residues in several peptides based on the N‐terminal region of human cardiac troponin I has been analysed by PAGE and 'H NMR spectroscopy to identify the products. With cAMP‐dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild‐type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N‐terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two‐dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N‐terminal pep‐tide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N‐terminal to the serine pair and stabilisation of the structure in the region of residues 25–29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.
Protection of Mice Against a Lethal Influenza Challenge by Immunization with Yeast‐Derived Recombinant Influenza Neuraminidase
Authors: Martinet Wim , Saelens Xavier , Deroo Tom , Neirynck Sabine , Contreras Roland , Min Jou Willy , Fiers Walter
Published: July 2004

The head domain of recombinant neuraminidase of A/Victoria/3/75 influenza virus was produced in a secreted form in the methylotrophic yeast Pichia pastoris using the P. pastoris alcohol oxidase 1 promoter and the Saccharomyces cerevisiaeα‐mating‐factor signal sequence. Cultures in shake flasks provided expression levels of approximately 2.5–3 mg/1. Recombinant neuraminidase was purified from the culture medium to over 99% homogeneity. Although P. pastoris‐secreted products are believed to carry shorter N‐linked carbohydrate side chains than glycoproteins of S. cerevisiae, secreted neuraminidase was hyperglycosylated, with N‐glycans of the high‐mannose type containing up to 30–40 mannose residues. N‐glycans were phosphorylated and only partially sensitive to α‐mannosidase treatment. Balb/c mice immunized three times with 2 μg purified recombinant neuraminidase were 50% protected against a lethal challenge of mouse‐adapted homologous virus; removal of glycosylation at the top of neuraminidase resulted in improved protection. The results provide a system for the production of an effective recombinant influenza vaccine that can easily be scaled up.
Concentration of, and SDS Removal from Proteins Isolated from Multiple two‐Dimensional Electrophoresis Gels
Authors: Hatt Paola Dainese , Quadroni Manfredo , Staudenmann Werner , James Peter
Published: July 2004

We have developed a gel electrophoresis system that can concentrate proteins from spots cut out of up to 50 two‐dimensional electrophoresis gels. During protein concentration, SDS is substituted with a non‐ionic detergent (octyl β‐glucopyranoside) which allows digestion and MS analysis of the protein directly extracted from the gel without fixation or staining. The system avoids the problems associated with the digestion of dilute protein in multiple bands by (a) greatly reducing the gel volume for digestion and thus the amount of protease required, hence lowering contamination by autodigestion products, (b) reducing the volume of solvent required for extraction of protein from the gel, thus minimising loss of material to container surfaces, and (c) removing SDS which interferes with subsequent MS or HPLC analysis.

The efficiency of protein recovery ranges between an average of 80% for proteins from silver stained two‐dimensional gels to 90% for fluorescence and Coomassie‐blue‐stained gels. The method is compatible with MS analysis of very low amounts of protein from any staining system, but appears not to be useful for Edman sequencing of silver‐stained or fluorescent‐stained proteins since the amount of N‐terminal blockage appears to increase as the amount of protein isolated from the two‐dimensional gel decreases.
Studies on the NusB Protein of Escherichia Coli Expression and Determination of Secondary‐Structure Elements by Multinuclear NMR Spectroscopy
Authors: Berglechner Fabian , Richter Gerald , Fischer Markus , Backer Adelbert , Gschwind Ruth M. , Huenges Martin , Gemmecker Gerd , Kessler Horst
Published: July 2004

The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial and bacteriophage λ genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion‐exchange chromatography followed by gel‐permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with 15N, 13C and 2H in various combinations. Heteronuclear three‐dimensional triple‐resonance NMR experiments combined with a semi‐automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on experimentally derived scalar couplings, chemical‐shift values, amide‐exchange data, and a semiquantitative interpretation of NOE data, the secondary structure of NusB has classified as α helical, comprising seven α helices.
Analysis of Backbone Dynamics in Cytochrome b5 using 15N‐NMR Relaxation Measurements
Authors: Kelly Geoffrey P. , Muskett Frederick W. , Whitford David
Published: July 2004

Determination of 15N‐NMR relaxation rates has allowed the characterisation of the dynamic properties of 55 1H‐15N bond vectors in a soluble haem‐binding domain of bovine microsomal ferricytochrome b5 consisting of the first 104 amino acid residues. Measurements of heteronuclear {1H}‐15N‐NMR nuclear Overhauser effects, and 15N‐NMR longitudinal and transverse relaxation rates have been analysed using both model‐free and reduced spectral density mapping approaches, demonstrating the application of these methods to a paramagnetic system. Analysis reveals that, for those regions which have been assessed, the polypeptide backbone of ferricytochrome b5 is largely rigid (average S2= 0.80), and that minimal internal backbone motion occurs on the sub‐nanosecond timescale. In contrast, motions on the microsecond to millisecond timescale, especially pronounced for the loop region extending from residues 28 to 31, and which may be of biological relevance, are indicated.
Three‐Dimensional Structure in Solution of the N‐Terminal Lipoyl Domain of the Pyruvate Dehydrogenase Complex from Azotobacter vinelandii
Authors: Berg Axel , Vervoort Jacques , De Kok Aart
Published: July 2004

The three‐dimensional structure of the N‐terminal lipoyl domain of the acetyltransferase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been determined using hetero‐nuclear multidimensional NMR spectroscopy and dynamical simulated annealing. The structure is compared with the solution structure of the lipoyl domain of the A. vinelandii 2‐oxoglutarate dehydrogenase complex. The overall fold of the two structures, described as a β‐barrel‐sandwich hybrid, is very similar. This agrees well with the high similarity of NMR‐derived parameters, e.g. chemical shifts, between the two lipoyl domains. The main structural differences between the two lipoyl domains occur in a solvent‐exposed loop close in space to the lipoylation site. Despite their high structural similarity, these lipoyl domains show a high preference for being reductively acylated by their parent 2‐oxo acid dehydrogenase. Potential residues of the lipoyl domain involved in this process of molecular recognition are discussed.
Steady‐State and Picosecond‐Time‐Resolved Fluorenscence Studies on the Recombinant Heme Domain of Bacillus megaterium Cytochrome P‐450
Authors: Khan Kishore Kumar , Mazumdar Shyamalava , Modi Sandeep , Sutcliffe Mike , Roberts Gordon C. K. , Mitra Samaresh
Published: July 2004

The conformational changes associated with the interaction of sodium laurate with the recombinant heme domain for cytochrome P‐450BM3 have been investigated by steady‐state and picosecond‐time‐resolved fluorescence spectroscopy. The steady‐state quenching experiments show that while all the five tryptophan residues are accessible to acrylamide in the free enzyme as well as the enzyme. substrate complex, the number of tryptophan residues accessible to ionic quenchers decreases on interaction of the substrate with the enzyme. This indicates that some of the tryptophan residues move towards the core of the protein on interaction with the substrate. The number of tryptophan residues accessible to the solvent as determined by the calculation of the solvent‐accessible area for the free enzyme agrees with the values obtained by the quenching experiments.

The time‐resolved fluorescence studies carried out by means of the time‐correlated single‐photon‐counting technique show that the fluorescence‐decay curve is best fitted to a three‐exponential model (0.2, 1.0 and 5.4 ns). Lifetime distributions, as recovered by the maximum‐entropy method, agree with the discrete exponential model. The binding of the substrate does not lead to any significant change in the lifetime components of the enzyme, indicating that the tryptophan residues are possibly away from the substrate‐binding domain. The decay‐associated emission spectra and the magnitudes of amplitude of different lifetimes indicate that the shortest lifetime component (τ1) originates from the three tryptophan residues that are completely or partially accessible to the solvent, and τ2 originates from the tryptophan residues that are buried in the core of the enzyme and not accessible to the solvent. X‐ray crystallographic data and solvent‐acessible‐area calculations have been used to identify these residues.
Site‐Directed Metagenesis and Halophilicity of Dihydrolipoamide Dehydrogenase from the Halophilic Archaeon, Haloferax Volcanii
Authors: Jolley Keith A. , Russell Rupert J. M. , Hough David W. , Danson Michael J.
Published: July 2004

A homology‐modelled structure of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluorescens. Analysis of the halophilic enzyme structure identified a potential K+‐binding site comprising four co‐ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein. Whilst E426 is conserved throughout non‐halophilic dihydrolipoamide dehydrogenases, E423 is only present in the halophilic enzyme. Four site‐directed mutations of the Haloferax dihydrolipoamide dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinant mutants expressed and characterised. From an analysis of their kinetic properties, salt‐dependent activities and thermal stabilities, it is concluded that this site has an important influence on the halophilicity of the enzyme. The findings support the view that the arrangement and interaction of the negatively charged amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations.
Properties of Chloride‐Conductive Pathways in Rat Kidney Cortical and Outer‐Medulla Brush‐Border Membranes
Authors: Benharouga Mohamed , Fritsch Janine , Banting George , Edelman Aleksander
Published: July 2004

The activity of the Cl− ‐conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush‐border‐membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin‐induced microsomal swelling by light scattering in the presence of an inward Cl− gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl− ‐conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion‐transport inhibitors were tested {N‐phenylanthranilic acid (PhNHPhCOOH), 5‐nitro‐2‐(3‐phenyl‐propylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4‐acetamido‐4′‐isothiocyanato‐stil‐bene‐2,2′‐disulfonic acid}. Ph2COOH and 4‐acetamido‐4′‐isothiocyanato‐stilbene‐2,2′‐disulfonic acid inhibited the basal Cl− ‐conductive pathways, while PKA‐treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl− pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl− channel. Two anti‐CFTR mAbs inhibited PKA‐activated valinomycin‐induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170‐kDa protein. The results thus indicate the presence of a PKA‐activated Cl− ‐conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA‐activated Cl− fluxes in these vesicles.
The Primary Structure of Rhodoferax fermentans High‐Potential Iron‐Sulfur Protein, an Electron Donor to the Photosynthetic Reaction Center
Authors: Van Driessche Gonzalez , Ciurli Stefano , Hochkoeppler Alejandro , Van Beeumen Jozef J.
Published: July 2004

The complete amino acid sequence of Rhodoferax fermentans high‐potential iron‐sulfur protein (Hipip), which is known to be an efficient electron donor to the photosynthetic reaction center, has been determined using both N‐terminal and C‐terminal analyses. The sequence contains 75 residues, with 11 positive charges, 10 negative charges, and one histidine residue. The molecular mass of apo‐Hipip, determined by electrospray ionization mass spectrometry, is 7849.64 Da. Multiple sequence alignment, based both on primary and tertiary structure information, reveals conservation of Tyrl9 and Gly75 (Chromatium vinosum numbering) in addition to the four [Fe4S4]‐bound cysteines. The Hipip from Rf. fermentans is most similar (57% similarity) to the Hipip from Rubrivivax gelatinosus, a photosynthetic bacterium belonging to the β‐1 subgroup of the proteobacteria.