bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of Minor‐Groove‐Binding Diamidine Ligands with an Asymmetric DNA Duplex
Authors: Conte Maria R. , Jenkins Terence C. , Lane Andrew N.
Published: March 2005

The aromatic diamidines berenil and propamidine bind reversibly to A+T‐rich sites in the minor groove of B‐form DNA duplexes. Based on extensive solution and crystallographic information we have designed a non‐self‐complementary double‐stranded DNA sequence, d(GCAATGAGCG) d(CGCTCATTGC), that should contain a near‐ideal binding site for berenil and a poorer site for the larger propamidine molecule, viz. d(AAT) d(ATT). 1H‐NMR studies show that both ligands bind with 1:1 stoichiometry to the embedded 5′‐AAT site, and induce numerous shifts of NMR resonances of DNA protons located in the minor groove. In addition, interactions with each strand can be distinguished by NOE spectroscopy due to the inherent asymmetry of the DNA. Detailed modelling based on experimental data show that no significant distortion of the B‐DNA duplex is induced by either ligand. Sufficient NOE data were obtained to determine the position of the bound ligands in each complex. These conclusions are in agreement with predictions from molecular modelling calculations that provide a microscopic energy profile for interaction with the minor groove tract. Such calculations reveal an unexpected heterogeneous 5′‐ATGA binding site that includes a spanned guanosine. This secondary binding site accounts for the extensive chemical shift perturbation induced by these ligands. The structures of the free DNA and the reversible complexes formed with each ligand molecule have been refined using an NOE‐restrained isothermal annealing procedure. These structures confirm that the introduced ligands effect minimal perturbation of the helix, with binding to the 5′‐AAT base sequence largely determined by specific non‐bonded interactions.
The Cea10 Gene Encodes A Secreted Member of the Murine Carcinoembryonic Antigen Family and is Expressed in the Placenta, Gastrointestinal Tract and Bone Marrow
Authors: Keck Ute , Nédellec Patrick , Beauchemin Nicole , Thompson John , Zimmermann Wolfgang
Published: March 2005

Although members of the carcinoembryonic antigen (CEA) family have been shown to convey cell adhesion in vitro, their location in some tissues contradicts such a function. As a basis for investigating their in vivo functions, we are characterizing the mouse CEA family. This paper describes the structure and expression of a new murine family member, cea 10. Two full‐length cDNA clones were isolated from a mouse colon library, whose deduced protein sequence comprises two immunoglobulin variable‐like N‐domains, directly followed by a short C‐terminal domain indicating that this molecule is secreted. Although this domain organization suggests a closer relationship to the murine pregnancy‐specific glycopro‐teins (PSG), which form a subgroup within the CEA family, sequence comparisons place Cea 10 within the CEA subgroup. Overlapping cosmid clones containing the complete cea 10 locus were mapped and the exons determined. No A2‐like exon, characteristic for all other members of the murine CEA family, could be found. Sequences of the promoter and the first exon showed remarkably high similarity to the corresponding regions of bgpl and bgp2, two other members of the murine CEA subgroup. Consensus sequences for two transcription factors (USF and an AP‐2‐like factor) that bind to the human BGP gene promoter were also present in the cea10 promoter and possibly convey expression of these genes in epithelial cells. RNase protection assays revealed transcriptional activity of cea10 in the colon and early placenta (10.5–12.5‐day embryos) and to a lower extent in the small intestine, cecum, stomach, salivary glands and bone marrow. As some other CEA family members are deregulated in tumors, we quantified the expression levels of Cea10 transcripts in colonic mucosa and in adenomatous polyps of Minl+ mice. No differences in the steady‐state levels of Cea10 mRNA could be found, suggesting that the Cea10 protein does not play a role in early tumor development. Taken together, Cea10 combines characteristic features of both CEA and PSG subgroup members in its structure and expression pattern.
Interactions with Retinol and Retinoids of Bovine Cellular Retinol‐Binding Protein
Authors: Malpeli Giorgio , Stoppinf Monica , Zapponi Maria Carla , Folli Claudia , Berni Rodolfo
Published: March 2005

The interactions with retinol and retinol analogs of bovine cellular retinol‐binding protein (CRBP) have been investigated, by means of fluorescence titrations, to obtain more information on the structural features of retinoids that may be required for their interaction with the binding protein. An approximately stoichiometric binding of retinol to bovine CRBP (K′d∼2 nM) has been found in direct binding assays. Although retinal exhibited relatively high binding affinity to bovine CRBP (K′d∼ 30 nM), a large excess of the retinoid could not compete with retinol for the carrier protein. On the assumption that retinol and retinal interact with the same binding site, this result indicates that the above‐mentioned apparent dissociation constant for retinol CRBP may be an overestimate and that its value may be as low as 0.1 nM. The finding of an exceedingly tight binding of retinol to CRBP provides further support for the possible role of CRBP‐bound retinol, rather than its uncomplexed labile form, as substrate of enzymes involved in the metabolism of the vitamin. The results of these and previous studies indicate that CRBP is particularly sensitive to modifications of the retinol hydroxyl end group. Axerophthene, a retinol analog bearing a hydrogen atom in place of the hydroxyl end group, and β‐ionone exhibit rather low binding affinities for CRBP (Kd∼ 0.2 μM and ∼ 4 μM, respectively), suggesting that the hydroxyl group and isoprene tail moieties contribute substantially to the retinol‐binding affinity and specificity. These findings are consistent with the indications emerging from the three‐dimensional structure determination of retinol CRBP [Cowan, S. W., Newcomer, M. E. & Jones, T. A. (1993) J. Mol. Biol. 230, 1225–1246]. Additionally, the bulky end groups of fenretinide and N‐ethyl retinamide replacing the retinol hydroxyl group have been found to prevent retinoid binding to CRBP. The primary structure of bovine CRBP has been determined and is highly similar to the structures of both human and rat CRBP (97% and 95% identical, respectively).
Transketolase a of Escherichia coli K12
Authors: Sprenger Georg A. , Schörken Ulrich , Sprenger Gerda , Sahm Hermann
Published: March 2005

Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19‐derived plasmid. These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to ≤0.1 U/mg in wild‐type cells. Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion‐exchange chromatography steps (Q‐Sepharose FF, Fractogel EMD‐DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE. The enzyme, both in its active and apoform, had a molecular mass of 145000 Da (±10000 Da), judged by gel‐filtration chromatography. Subunits of 73000 Da (±2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer. N‐terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA. The specific activity of the purified enzyme, determined at 30°C with the substrates xylulose 5‐phosphate (donor of C2 compound) and ribose 5‐phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg. Km values for the substrates xylulose 5‐phosphate and ribose 5‐phosphate were 160μM and 1.4mM, respectively. Km values for the other physiological substrates of transketolase A were 90 μM for erythrose 4‐phosphate (best acceptor substrate), 2.1 mM for D,L‐glyceraldehyde 3‐phosphate, 1.1 mM for fructose 6‐phosphate, and 4 mM for sedoheptulose 7‐phos‐phate. Hydroxypyruvate served as alternative donor (Km=18 mM). Unphosphorylated acceptor compounds were formaldehyde (Km= 31 mM), glycolaldehyde (14 mM), D,L‐glyceraldehyde (10 mM) and d‐erythrose (150 mM). The enzyme was competitively inhibited by d‐arabinose 5‐phosphate (Ki= 6 mM at a concentration of 2.5 mM d‐arabinose 5‐phosphate) or by the chelating agent EDTA. The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations. The reconstitution of the apoenzyme pro‐ceded faster in the presence of manganese ions (Kd= 7 μM at 10 μM thiamine diphosphate) than with other divalent cations.
Evidence for a Catalytic Role of Tyrosine 383 in the Peptidase Reaction of Leukotriene A4 Hydrolase
Authors: Blomster Martina , Wetterholm Anders , Mueller Martin J. , Haeggström Jesper Z.
Published: March 2005

Leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4 and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA4 hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site‐directed mutagenesis. The mutated cDNAs were expressed in Escherichia coli and the resulting recombinant proteins, named [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA4 into leukotriene B4, and the peptidase activity.

None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3% that of the wild‐type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LTA4 hydrolase, [Y383H]LTA4 hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10‐fold increase in Km for leukotriene A4 compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with Vmax values of 600 nmol · mg−1· min−1 and 1000 nmol · mg−1· min−1 for [Y383Q]LTA4 hydrolase and the wild‐type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA4 hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA4 into LTB4.
The Preparation of Catalytically Active Human Cathepsin B from Its Precursor Expressed in Escherichia coli in the Form of Inclusion Bodies
Authors: Kuhelj Robert , Dolinar Marko , Pungerčar Jože , Turk Vito
Published: March 2005

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/1 E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three‐amino‐acid extension at its N‐terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.
Recombinant and Chemical Derivatives of Apamin
Authors: Devaux Christiane , Knibiehler Martine , Defendini Maria‐Léria , Mabrouk Kamel , Rochat Hervé , Van Rietschoten Jurphaas , Baty Daniel , Granier Claude
Published: March 2005

The use of the colicin A lysis protein to direct the extracellular release of a fusion protein from Escherichia coli was investigated as an approach for the preparation of recombinant animal toxins. Apamin, a bee venom neurotoxin, was used as the model toxin. It is reticulated by two disulfide bridges and interacts with small conductance Ca2+‐activated K+ channels. Substantial amounts of free recombinant apamin were obtained by CNBr cleavage of the fusion protein [col‐(1–171)‐apa] and HPLC purification. It was recognized by conformation‐dependent monoclonal antibodies with a K0.5 value close to that for natural apamin, indicating that folding was correct. In toxicity and binding experiments, the recombinant apamin displayed low activity. The recombinant and natural molecules differed by the amidation of the C‐terminal histidine residue. Previous structure/activity relationship studies do not implicate this C‐terminal residue in activity but the role of its amidation was not investigated. An apamin analog with a non‐amidated C‐terminal residue was then chemically synthesized. The biological properties of both recombinant and chemical molecules were determined. Amidation of the C‐terminal α‐carboxyl of apamin appears to be essential for full expression of its biological activity.
Top‐Down Control Analysis of Systems with More than one Common Intermediate
Authors: Ainscow Edward K. , Brand Martin D.
Published: March 2005

The analysis of the control of complex metabolic systems can be greatly simplified by application of the top‐down approach of metabolic control analysis, in which the reactions of the system are grouped together into a small number of blocks connected by a common intermediate. The experimental application of the top‐down approach has so far been limited to systems that have only a single intermediate. In this study, we demonstrate that the connectivity and summation theorems of metabolic control analysis hold with any number of intermediates between the metabolic blocks, and in doing so show that top‐down analysis is valid for systems with multiple intermediates and so can be applied to most metabolic systems regardless of their complexity; an example of such an application is provided. Top‐down control analysis has successfully described the control of mitochondrial respiration by dividing the system into three blocks, the respiratory chain, phosphorylation system and proton leak, all linked by a single intermediate, proton motive force. Here, we subdivide the respiratory chain into succinate consumers and cytochrome oxidase so that a second intermediate, cytochrome c redox state, is generated. Despite the fact that the redox state of cytochrome c is not measured, we solve the control over the system fluxes. In common with previous studies, we find that under conditions where there is no ATP turnover (state 4), respiration is largely controlled by proton leak, while at maximal ATP turnover (state 3) respiration is controlled by the respiratory chain and the phosphorylating system. In state 4, 85% of the control by the respiratory chain resides with cytochrome oxidase. As ATP turnover increases, the respiration rate increases, and the control by the respiratory chain shifts from cytochrome oxidase to the succinate consumers, so that in state 3 83% of the control by the respiratory chain lies in the reactions between succinate and cytochrome c and only 17% resides with cytochrome oxidase.
Phosphatidylinositol Hydrolysis and an Increase in Ca2+Concentration in the Signal‐Transduction Process Triggered by Murine FcγRIII are not Required for Protein Kinase C Translocation
Authors: Altrichter Jens , Guse Andreas H. , Resch Klaus , Brock Josef , Daëron Marc , Hückel Christian
Published: March 2005

Murine class III receptors for IgG (mFcγRIII) are composed of an IgG‐binding α chain associated with a γ subunit dimer. These receptors have been shown to trigger the release of serotonin and tumor necrosis factor‐α [Daëron, M., Latour, S., Hückel, C., Bonnerot, C. & Fridman, W. H. (1992)Immunobiology 185, 159–174], and are involved in endocytosis and phagocytosis [Daëron, M., Malbec, O., Bonnerot, C., Latour, S., Segal, D. M. & Fridman, W. H. (1994)J. Immunol. 152, 783–792]. Using a transfection model where the cDNA encoding mFcγRIII was stably transfected into the rat basophilic leukemia cell line RBL‐2H3, we found that the functional efficiency of mFcγRIII is correlated with its ability to increase the intracellular Ca2+concentration and to stimulate inositol phosphate metabolism.

The deletion of intracellular sequences of the α subunit did not alter the ability of mFcγRIII to trigger the Ca2+and phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] response. After substitution of the intracellular domain of mFcγRII for that of mFcγRIIIγ, but not that of mFcγRIIIα, the chimeric receptor was also able to trigger Ca2+and PtdIns(4,5)P2responses.

In contrast, all transfected receptors induced protein kinase C translocation. Furthermore, dimerization of the receptor was sufficient for the initiation of this protein kinase C translocation while a further cross‐linking was necessary for the induction of the Ca2+and PtdIns(4,5)P2responses.

Protein kinase C translocation therefore can be dissociated from Ca2+mobilization, PtdIns(4,5)P2turnover and mast cell secretory responses induced by murine FcγRIII.
Native Dimer Stabilizes the Subunit Tertiary Structure of Porcine Class pi Glutathione S‐transferase
Authors: Erhardt Julija , Dirr Heini
Published: March 2005

Solvent‐induced unfolding of porcine class pi glutathione S‐transferase (pGST P1‐1), a homodimeric protein, was monitored under equilibrium conditions using different physicochemical parameters (tryptophan fluorescence, anisotropy, degree of tyrosine exposure, binding of 8‐anilino‐1‐naphthalenesulphonic acid, size‐exclusion HPLC). The coincidence of unfolding curves obtained with functional (enzyme activity) and structural probes (anisotropy), the absence of thermodynamically stable intermediates such as a folded monomer (determined by binding of 8‐anilino‐1‐naphthalenesulphonic acid and size‐exclusion HPLC), and the dependence of pGST P1‐1 stability upon protein concentration (measured with structural and functional probes), indicate a cooperative and concerted two‐state unfolding transition between native dimeric pGST P1‐1 and unfolded monomeric enzyme.
Cloning, Expression and Characterization of Two Putative Calcium‐binding Sites in Human Non‐Erythroid α‐Spectrin
Authors: Lundberg Susanne , Björk Johanna , Löfvenberg Lars , Backman Lars
Published: March 2005

The C‐terminus of α‐spectrins contains two putative calcium‐binding sites or EF‐hands. To characterize the binding, we have isolated clones from a human fetal liver cDNA library and expressed several fragments comprising either one or both of these sites. When the isolated clones were sequenced, we found that three consecutive nucleotides differed compared to the published sequence. The discrepancy affected two codons in the first of the two putative calcium sites. These codons translated into glutamate and phenylalanine, which are identical to the residues present at the same position in other α‐spectrins.

In the presence of magnesium, only recombinant peptides comprising the second putative site bound calcium as determined by a calcium overlay assay. Although the first putative EF‐hand appeared to bind some calcium in the absence of magnesium, no binding could be detected under stringent conditions. Therefore, it is likely that the second EF‐hand constitutes the only functional calcium‐binding site in the C‐terminus of human non‐erythroid α‐spectrin. Since peptides comprising the second EF‐hand bound calcium nearly as well as intact spectrin, it is also apparent that the second EF‐hand constitutes the major binding site for calcium in spectrin. The relative change in negative ellipticity, induced by the binding of calcium, indicates a dissociation constant of approximately 120 μM.
The Complete Primary Structure of Glycosylated Porcine Platelet Factor 4
Authors: Proudfoot Amanda E. I. , Magnenat Edith , Haley Terry M. , Maione Theodore E. , Wells Timothy N. C.
Published: March 2005

We have purified platelet factor 4 from porcine platelets and shown that it is glycosylated. The purified protein migrated as a broad band at approximately 14000 Da, characteristic of glycoproteins. Electrospray mass spectroscopy of the intact protein gave a predominant mass of 11 111 Da, with a minor component of 10804 Da. Sialidase digestion reduces both forms to a single mass of 10497 Da. Upon Edman degradation, the amino terminus was found to be blocked by the presence of a pyroglutamate residue. We have determined the complete primary structure of platelet factor 4 by peptide mapping and Edman degradation, thereby completing information on the amino‐terminal and carboxy‐terminal regions which is missing in the previously published partial sequence. Sequencing of the intact and deglycosylated protein show that the glycosylation site is at Thr8. The amino acid composition accounts for a mass of 9623 Da, and the carbohydrate moeity was found to contribute 1490 Da. The biological activity of the porcine protein has been compared to recombinant human platelet factor 4 in an endothelial cell proliferation assay; both inhibit at a concentration giving half the maximal inhibition of 0.1 μM. Removal of the 19 amino‐terminal residues carrying the carbohydrate moiety results in no change in the biological activity.
Comparison of the Specificity of Bacterially Expressed Cytoplasmic Protein‐Tyrosine Phosphatases SHP and SH‐PTP2 Towards Synthetic Phosphopeptide Substrates
Authors: Dechert Ute , Affolter Michael , Harder Kenneth W. , Matthews James , Owen Philip , Clark‐Lewis Ian , Thomas Matthew L. , Aebersold Ruedi , Jirik Frank R.
Published: March 2005

SHP and SH‐PTP2 are related cytoplasmic protein‐tyrosine phosphatases having two tandem amino‐terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH‐PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy‐terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH‐PTP2 demonstrated a similar specificity pattern. Similar to SH‐PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over ≈40–1.6×103 M−1 s−1 towards other substrates. In contrast, the PTP‐1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH‐PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.
Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH
Authors: Pineda Teresa , Osei Yaa Difie , Churchich Jorge E.
Published: March 2005

4‐Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time‐dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, фobs= 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL≈ 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements.

The fluorescent probe 1‐anilinonaphthalene‐8‐sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped‐flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped‐flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4‐aminobutyrate aminotransferase in the cell.
cDNA Cloning and Tissue‐specific Regulation of Expression of Rat Calcium‐binding Protein 65/67
Authors: Fan Hua , Josić Djuro , Lim Yow‐Pin , Reutter Werner
Published: March 2005

We isolated a cDNA encoding the rat membrane‐associated 65/67‐kDa calcium‐binding protein, CBP 65/67, from a λ ZAP II cDNA‐expression library of rat liver by immunoscreening using monospecific polyclonal anti‐(CBP 65/67) antibodies and monoclonal anti‐(CBP 65/67) IgG. The product of this cDNA expressed in Escherichia coli was confirmed as CBP 65/67 both by immunostaining and by comparison of the molecular mass with the CBP 65/67 isolated from rat liver by SDS/PAGE. The cDNA sequence and the deduced amino acid sequence of CBP 65/67 both show a high degree of identity to human p68 and human calelectrin, which belong to a family of calcium‐dependent, membrane‐associated, phospholipid‐binding proteins, called annexins. This means that CBP 65/67 is a homolog of the two human proteins just mentioned above. We are not aware that a rat annexin VI has previously been isolated and sequenced.

The mRNA expression of CBP 65/67 in different rat organs during development was investigated by Northern blot analysis. In adult tissues, high mRNA levels of CBP 65/67 were found in lung, heart, muscle, spleen and especially in thymus and pancreas, whereas in liver, kidney, intestine, stomach and brain only low levels of CBP 65/67 mRNA could be detected. The amount of mRNA during tissue development in kidney, stomach and muscle showed only slight changes. In contrast, a significant increase of CBP 65/67 expression was observed in liver, lung, heart and brain. In most of the organs investigated, the level of mRNA correlated closely with the level of protein expression, indicating that the expression of CBP 65/67 in most organs is controlled primarily at the transcriptional level.
Effects of Novel Analogues of D‐Glucose on Glycogen Phosphorylase Activities in Crude Extracts of Liver and Skeletal Muscle
Authors: Board Mary , Hadwen Moira , Johnson Louise N.
Published: March 2005

The inhibitory properties of a series of both N‐linked and C‐linked C1‐substituted glucose derivatives towards glycogen phosphorylase (GP) activity from crude extracts of rat liver and muscle have been measured. The most effective inhibitor was N‐acetyl‐β‐D‐glucopyranosylamine, which has KiS of 51 μM (muscle GPa), 30 μM (muscle GPb), 2.7 mM (liver GPa) and 4 mM (liver GPb). All analogues tested inhibit muscle GP more potently than liver GP, highlighting some differences between the two isoen‐zymes, which are nearly 80% similar. The human liver GP enzyme has been modelled on the basis of the rabbit muscle structure and, together with comparison of structures of muscle GPa and GPb, has provided some insights into possible explanations for the different properties of the two isoenzymes. Maximal activities of GP have also been measured in tissues from diabetic (dbldb) and wild‐type (db/+) mice. Liver GP from db/db mice exhibits higher activity [132% (a)‐67% (b)] than from db/+ controls, although similar activities were observed for muscle GP from both db/db and db/+ animals.
Carbohydrate‐deficient Glycoprotein Syndrome Type II
Authors: Charuk Jeffrey H. M. , Tan Jenny , Bernardini Marcus , Haddad Sheila , Reithmeier Reinhart A. F. , Jaeken Jaak , Schachter Harry
Published: March 2005

Carbohydrate‐deficient glycoprotein syndromes (CDGS) are a family of multisystemic congenital diseases resulting in underglycosylated glycoproteins, suggesting defective N‐glycan assembly. Fibroblast extracts from two patients with a recently described variant of this disease (CDGS type II) have previously been shown to have over 98% reduced activity of UDP‐GlcNAc:α‐6–d‐mannoside β‐1,2‐N‐acetylglucos‐aminyltransferase II [GlcNAc‐TII; Jaeken, J., Schachter, H., Carchon, H., De Cock, P., Coddeville, B. & Spik, G. (1994) Arch. Dis. Childhood 71, 123–127]. We show in this paper that mononuclear cell extracts from one of these CDGS type‐II patients have no detectable GlcNAc‐TII activity and that similar extracts from 12 blood relatives of the patient, including his father, mother and brother, have GlcNAc‐TII levels 32–67% that of normal levels (average 50.1%±10.7% SD), consistent with an autosomal recessive disease. The poly(N‐acetyllactosamine) content of erythrocyte membrane glycoproteins bands 3 and 4.5 of this CDGS patient were estimated, by tomato lectin blotting, to be reduced by 50% relative to samples obtained from blood relatives and normal controls. Similar to patients with hereditary erythroblastic multinuclearity with a positive acidified‐serum lysis test (HEMPAS), erythrocyte membrane glycoproteins in the CDGS patient have increased reactivities with concanavalin A, demonstrating the presence of hybrid or oligomannose carbohydrate structures. However, bands 3 and 4.5 in HEMPAS erythrocytes have almost complete lack of poly(N‐acetyllactosamine). Furthermore, CDGS type‐II patients have a totally different clinical presentation and their erythrocytes do not show the serology typical of HEMPAS, suggesting that the genetic lesions responsible for these two diseases are possibly different.
Structural Elucidation of the O‐Antigen Lipopolysaccharide from two Strains of Plesiomonas Shigelloides that Share a Type‐Specific Antigen with Shigella Flexneri 6, and the Common Group 1 Antigen with Shigella Flexneri spp and Shigella Dysenteriae 1
Authors: Linnerborg Malin , Widmalm Göran , Weintraub Andrej , Albert M. John
Published: March 2005

Sugar and methylation analyses of native polysaccharides together with one‐dimensional 1H‐ and 13C‐NMR spectroscopy revealed that the two polysaccharides from strains 22074 and 12254 of Plesiomonas shigelloides are identical. The structure of the polysaccharide from strain 22074 was deduced from a uronic acid degradation and by NMR spectroscopy where heteronuclear multiple bond connectivitiy and two‐dimensional nuclear Overhauser effect spectroscopy experiments established the pentasaccharide repeating unit as →4)‐α‐D‐Galp A‐(1→3)‐α‐D‐Glcp NAc‐(1→3)‐α‐L‐Rhap ‐(1→2)‐α‐L‐Rhap ‐(1→2)‐α‐L‐Rhap ‐(1→.
Characterisation of Taurochenodeoxycholic Acid 6α‐Hydroxylase from Pig Liver Microsomes
Authors: Araya Zufan , Hellman Ulf , Hansson Ronnie
Published: March 2005

A fraction of cytochrome P ‐450 catalysing an efficient 6α‐hydroxylation of taurine‐conjugated 3α,7α‐dihydroxy‐5β‐cholanoic acid (taurochenodeoxycholic acid) was partially purified from pig liver microsomes. The specific content of cytochrome P ‐450 was 6 nmol/mg protein and the preparation showed two major protein bands upon SDS/PAGE. These two bands were isolated after SDS/PAGE and protein blotting. The protein band with a molecular mass of 53 kDa had an N‐terminal amino acid sequence and internal sequences resembling that of the cytochrome P ‐450 4A subfamily (CYP 4A). Polyclonal antibodies raised against this protein were able to, after SDS/PAGE and immunoblotting, detect the protein in microsomal fractions as well as in the purified cytochrome P ‐450 fraction. Furthermore, addition of these antibodies to a reconstituted system containing the cytochrome P ‐450 fraction, inhibited 6α‐hydroxylation of taurochenodeoxycholic acid by up to 90%. Experiments with irrelevant antibodies did not show inhibition of 6α‐hydroxylation. The purified cytochrome P‐450 fraction catalysed in addition ω‐ and ω‐1 hydroxylation of lauric acid and 6α‐hydroxylation of 3α‐hydroxy‐5β‐cholanoic acid (lithocholic acid). However, these hydroxylase activities were rather low compared to 6α‐hydroxylation of taurochenodeoxycholic acid. The enzyme fraction did not show hydroxylase activities towards cholesterol and 5β‐choles‐tane‐3α,7α‐diol. These results indicate that 6α‐hydroxylation of taurochenodeoxycholic acid is catalysed by a specific species of cytochrome P ‐450 that, according to N‐terminal amino acid sequence as well as catalytic properties, could be a member of the CYP 4A subfamily.
Characterization of Blood‐Group‐ABO(H)‐Active Glycosphingolipids in Type‐AB Human Erythrocytes
Authors: Kushi Yasunori , Tsunoda Atsunobu , Komatsuzaki Atsushi , Watanabe Kiyohiro , Kasama Takeshi , Handa Shizuo
Published: March 2005

Neutral glycolipids in Folch's upper phase were isolated from human erythrocyte membranes of 22 individuals with blood type AB. On immunostaining by TLC with anti‐A IgG, all reactive glycolipids in type A corresponded to reactive glycolipids in type‐AB erythrocytes. With anti‐B IgM, all reactive glycolipids in type‐B erythrocytes also corresponded to reactive glycolipids in type‐AB erythrocytes. By comparison of the reactivity to that of the anti‐A and anti‐B antibodies, it was found that, in type‐AB erythrocytes, all glycolipids reactive with either one of the anti‐A or anti‐B antibodies were detected in both type‐A and type‐B erythrocytes, and that A‐active glycolipids had higher Rf values than B‐active glycolipids on TLC plates. A series of glycolipids reactive with both antibodies were purified from the Folch's upper neutral glycolipid fraction of erythrocyte membranes by column chromatography, and was characterized by TLC‐immunostaining and negative secondary‐ion mass spectrometry. The results strongly suggested that A‐active and B‐active carbohydrate chain epitopes existed separately as glycolipid molecules in blood‐type‐AB erythrocytes. It was also confirmed that these phenotypes observed in erythrocyte membranes were exhibited by blood‐group‐active glycosphingolipids in the small intestine of blood‐type‐AB individuals.

Furthermore, upon treatment of fractions obtained from silicic acid column chromatography with α‐N ‐acetylhexosaminidase or α‐galactosidase, a branched hybrid‐type molecule with both A and B determinants was not detected.
The Effect of Amino Acids on the Metabolic Fate of 15NH4Cl in Isolated Sheep Hepatocytes
Authors: Luo Q. J. , Maltby Susan A. , Lobley Gerald E. , Calder A. Graham , Lomax Michael A.
Published: March 2005

Ruminants characteristically absorb a large proportion of dietary nitrogen across the portal‐drained viscera as ammonia nitrogen which is detoxified by conversion to urea in the liver. In theory, ammonia can supply both nitrogen atoms of the urea molecule via mitochondrial (carbamoyl phosphate) and cyto‐plasmic (aspartate) precursor pathways of the ornithine cycle but the effect of amino acids on the flux of nitrogen from ammonia to each of the two urea nitrogen atoms has not been determined. We report a study designed to determine the distribution of [15N] ammonia between [15N1]urea and [15N2]urea in sheep hepatocytes in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in the presence or absence of amino acids. In the absence of amino acids, the enrichment of [15N2]urea rose more rapidly during incubations than [15N1]urea and attained enrichments of 66–88% within 5 min of incubation. At the end of 2.5 h of incubation, [15N2]urea represented 60% and 90% of the total urea molecules at low and high ammonia concentrations, respectively. The enrichments of glutamate and aspartate were similar to [15N1]urea in the cells at the end of the incubations, even in the presence of unlabelled amino acids, supporting the concept of mitochondrial ammonia being in equilibrium with cytosolic aspartate formation. In the presence of amino acids basal urea synthesis increased but ammonia uptake and 15NH4Cl conversion to urea was less than in the absence of amino acids. The rate of formation of [15N1]urea was greater in incubations containing amino acids but when ammonia concentration in the media was raised only [15N2]urea flux increased with no change in either [15N1]urea or the unlabelled species. Measurement of media amino acid concentrations after 2.5 h of incubation in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of aspartate, threo‐nine, serine, glutamate, and the branched chain amino acids. However, less than 12% of the 15N transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the presence of amino acids could be numerically accounted as the sum of arginine and glutamine removal from incubations. It is concluded that in sheep hepatocytes 15NH4Cl removal leads to quantitative formation of [15N2]urea, even in the presence of a physiological mixture of amino acids. The increase in the formation of the [15N1]urea in the presence of amino acids can be explained by the preferential utilisation of the amide nitrogen of glutamine for urea synthesis.
Primary Structure, Sequence‐Specific 1H‐NMR Assignments and Secondary Structure in Solution of Bromelain Inhibitor VI from Pineapple Stem
Authors: Hatano Ken‐ichi , Kojima Masaki , Tanokura Masaru , Takahashi Kenji
Published: March 2005

One of the bromelain inhibitors, isoinhibitor VI (BI‐VI), was purified from pineapple stem powder and its complete amino acid sequence was determined by conventional protein sequencing. These results revealed that the protein consists of an 11‐residue light chain and a 41‐residue heavy chain, cross‐linked to each other by disulfide bonds to form the native inhibitor of 52 residues (Mr= 5888). The secondary structure of BI‐VI was analyzed based on the sequence‐specific 1H resonance assignment of its two‐dimensional NMR spectra. BI‐VI was shown to be composed of two domains (A and B) which are formed by antiparallel β‐sheets, but has no α‐helix. These results were consistent with the CD spectra of BI‐VI. Residues Lys27‐Ile29 (heavy chain) form a triple‐stranded antiparallel β‐sheet with residues Asp9‐Tyr11 and Lys22–Glu24 (heavy chain) in the A domain and residues Cys5–Cys7 (heavy chain) form another triple‐stranded β‐sheet with residues Cys6–Cys8 (light chain) and Asp32–Ile34 (heavy chain) in the B domain. The secondary structure as well as the primary structure of BI‐VI was distinctly different from that of the other cysteine protease inhibitor, cystatin, and from that of basic pancreatic trypsin inhibitor.
Nitric‐Oxide‐Mediated Activation of Iron‐Regulatory Protein Controls Hepatic Iron Metabolism During Acute Inflammation
Authors: Cairo Gaetano , Pietrangelo Antonello
Published: March 2005

The molecular regulation of intracellular iron metabolism has been studied in the livers of rats undergoing an acute inflammatory reaction following turpentine injection. Treatment induced an increase in the steady‐state level of the transferrin receptor (TfR) mRNA, peaking 18 h after treatment and returning to control levels 24 h after treatment, with no change in TfR gene transcription. RNA band‐shift assays documented an activation of the cytoplasmic RNA‐binding protein called the iron‐regulatory protein (IRP), in parallel with a rise in the amount of TfR transcripts. A 2–3‐fold increase in the amount of H and L ferritin subunit mRNAs was found 12–18 h after turpentine treatment. Surprisingly, higher accumulation of ferritin mRNAs did not result in appreciable differences in the liver ferritin content. This might be due to the concomitant rise of IRP activity, which is known to prevent ferritin mRNA translation. The absence of significant changes in the total iron and ferritin contents prompted us to investigate the role of nitric oxide (NO), an inflammatory mediator which is also known to modulate the activity of IRP. Northern‐blot analysis showed a marked enhancement in the expression of the inducible form of nitric oxide synthase mRNA in turpentine‐treated rats. Furthermore, the activation of IRP and the increase of the TfR mRNA content that occur in turpentine‐treated rats were abolished by treatment withN5‐nitro‐l‐arginine, a specific nitric oxide synthase inhibitor. The present data suggest that NO‐mediated activation of IRP regulates alterations of hepatic iron homeostasis that occur in acute inflammation.
Characterization of the Prismane Protein from Desulfovibrio vulgaris (Hildenborough) by Low‐Temperature Magnetic Circular Dichroic Spectroscopy
Authors: Marritt Sophie J. , Farrar Jaqui A. , Breton Jacques L. J. , Hagen Wilfred R. , Thomson Andrew J.
Published: March 2005

The prismane protein of Desulfovibrio vulgaris (Hildenborough) contains a putative [6Fe‐6S] cluster. This novel iron‐sulfur cluster has been characterized here by magnetic circular dichroism (MCD) spectroscopy. Three paramagnetic redox states of the cluster, [6Fe‐6S]5+, [6Fe‐6S]4+ and [6Fe‐6S]3+, each show a distinctive low‐temperature MCD spectrum which is unlike that observed for any other iron‐sulfur clusters. Magnetization data for the prismane protein in these three redox states indicate ground state spins that are in accordance with previous EPR assignments. For the protein as isolated, with the [6Fe‐6S]5+ form of the cluster, magnetizations show an exceptionally steep initial slope that can be fit to a ground state of spin S= 9/2. For the semi‐reduced protein, the cluster in the [6Fe‐6S]4+ form, magnetizations show an initial slope characteristic for a ground state of spin S= 4. For the dithionite‐reduced protein, with the [6Fe‐6S]3+ form of the cluster, magnetizations are typical for a ground state of spin S= 1/2.
Refined Solution Structure of the Tyr41→His Mutant of the M13 Gene V Protein
Authors: Prompers Jeanine J. , Folmer Rutger H. A. , Nilges Michael , Folkers Paul J. M. , Konings Ruud N. H. , Hilbers Cornelis W.
Published: March 2005

The three‐dimensional solution structure of mutant Tyr41→His of the single‐stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been refined in two stages. The first stage involved the collection of additional NOE‐based distance constraints, which were then used in eight cycles of back‐calculations and structure calculations. The structures of the gene V protein dimers were calculated using simulated annealing, employing restrained molecular dynamics with a geometric force field. In the second stage of the refinement procedure, solvent was explicitly included during the dynamic calculations. A total of 30 structures was calculated for the protein, representing its solution structure in water. The first calculation step significantly improved the convergence of the structures, whereas the subsequent simulations in water made the structures physically more realistic. This is, for instance, illustrated by the number of hydrogen bonds formed in the molecule, which increased considerably upon going to aqueous solution. It is shown that the solution structure of the mutant gene V protein is nearly identical to the crystal structure of the wild‐type molecule, except for the DNA‐binding loop (residues 16–28). This antiparallel β‐hairpin is twisted and partially folded back towards the core of the protein in the NMR structure, whereas it is more extended and points away from the rest of the molecule in the X‐ray structure. Unrestrained molecular dynamics calculations suggest that this latter conformation is energetically unstable in solution.
Phytanic Acid α‐oxidation in Rat Liver Peroxisomes
Authors: Mihalik Stephanie J. , Rainville Anne M. , Watkins Paul A.
Published: March 2005

Patients with generalized peroxisomal disorders, rhizomelic chondrodysplasia punctata, and Refsum disease are all unable to α‐oxidize 3,7,11,15‐tetramethylhexadecanoic (phytanic) acid. The exact cause of the oxidation defect in these patients is not well characterized, in part because there is only limited knowledge of the biochemical pathway. In 1969, the α‐oxidation of phytanic acid was reported [Tsai, S.‐C., Avigan, J. & Steinberg, D. (1969) Studies on the α‐oxidation of phytanic acid by rat liver mitochondria, J. Biol. Chem. 244, 2682–2692] to involve the formation of an α‐hydroxyphytanic acid intermediate prior to removal of the α carbon. Subsequently, most researchers have had difficulty detecting this intermediate. In the present study, cofactors known to form hydroxy intermediates by both monooxygen‐ase and dioxygenase reaction mechanisms were incubated with purified rat liver peroxisomes and either [2,3‐3H]phytanic acid or [1‐14C]phytanic acid. Reaction products were separated by reverse‐phase HPLC. A single reaction product, identified as α‐hydroxyphytanoyl‐CoA rather than the free fatty acid, was detected when 2‐oxoglutarate/Fe+2/ascorbate, cofactors associated with a dioxygenase reaction mechanism, were present. Concomitant with α‐hydroxyphytanoyl‐CoA production, there was an increased accumulation of formate and CO2. This increase in α‐oxidation products is evidence that α‐hydroxyphytanoyl‐CoA is a true pathway intermediate and that the entire pathway functions in peroxisomes. In contrast, α‐hydroxyphytanoyl‐CoA was not formed in any quantity in mitochondria. These studies suggest that the α‐hydroxylation step of phytanic acid oxidation, which has been shown to be defective in Refsum disease, is located in peroxisomes.
Macromolecules Increase the Channeling of ADP from Externally Associated Hexokinase to the Matrix of Mitochondria
Authors: Veer Fanny D. Later , Gellerich Frank N. , Nicolay Klaas
Published: March 2005

Macromolecules can restore the morphological changes in the outer mitochondrial compartment that occur upon isolation of the organelle. They decrease the volume of the intermembrane space and increase the number of intermembrane contact sites. In this study, we investigated the effects of macromolecules on one of the processes occurring in the mitochondrial outer compartment and for which the native structure might be important, i.e. the ADP supply from outer‐membrane‐bound hexokinase‐I to oxidative phosphorylation. With the use of a reconstituted system in which rat liver mitochondria and extramitochondrial pyruvate kinase compete for ADP generated by hexokinase, it was shown that (a) part of the ADP generated by mitochondrially associated hexokinase is not accessible to pyruvate kinase and is channeled into the mitochondrion, (b) in the presence of 10% (mass/vol.) macromolecules (i.e. dextran M20 or BSA) the pyruvate kinase inaccessible fraction increases from 19% to 31 % of the ADP produced by hexokinase, (c) the ADP channeling is a characteristic property of bound hexokinase, and (d) the increased channeling induced by macromolecules can neither be explained by direct effects of these macromolecules on the basic respiratory properties of rat liver mitochondria, nor by direct effects on the kinetic properties of hexokinase‐I. ATP and ADP determinations were performed in hexokinase/mitochondria incubation mixtures in the presence of macromolecules. These determinations showed that an important consequence of the channeling capacity of bound hexokinase is that lower extramitochondrial ADP levels and consequently higher extramitochondrial ATP/ADP ratios are maintained than when hexokinase is not bound. The experimental data demonstrate that the ADP channeling activity associated with bound hexokinase leads to the formation of two ADP concentration gradients, one across the outer membrane and one between bound hexokinase and the bulk phase.