bluuebunny/crossref_metadata_2025 huggingface dataset:
The devapamil‐binding site of the purified skeletal muscle receptor for organic‐calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+
Authors: SCHNEIDER Toni , REGULLA Stefan , HOFMANN Franz
Published: March 2005

The interaction of 2,7‐dimethyl‐3‐(3,4‐dimethoxyphenyl)‐3‐cyan‐7‐aza‐9‐(3‐methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of {3‐[2‐(3,4‐dimethoxyphenyl)ethyl]‐methylaminopropyl‐3,4‐dimethyoxy‐(1‐methylethyl)benzeneacetonitrile (verapamil), with the purfied skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4°C and 30°C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium‐channel α1 subunit, with an apparent Kd of 13 ± 2.6 nM at 4°C in the presence of 0.4 μM Ca2+. The affinity, and not the density, of the devapamil‐binding site was decreased by lowering the pH from 8.5–6.5, or by increasing the Ca2+ concentration from 0.4 μM to 100 mM. The same results were obtained at 30°C, although the ligand · receptor complex was not stable at Ca2+ concentrations below 10 μM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo‐first‐order and a second‐order reactions were identical and yielded fourfold lower k‐1/k+1 (KD) values than the equilbrium experiments performed using 1 nM and 0.4 μM Ca2+, but the same values using 1 mM Ca2+· 1 mM Ca2+ increased the k‐1/k+1 (KD) by decreasing 10‐fold the association rate at 4°C. The dissociation rate was increased about 10‐fold by 5 μM devapamil or 100 μM d‐cis‐diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low‐affinity site.

Incubation of the CaCB receptor in the absence of Ca2+ and devapamil at 30°C, but not at 4°C, resulted in an apparent loss of devapamil‐binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 μM. The apparent half‐maximal inactivation times of the devepamil‐binding site were 90 s and 12 min in the presence of 1 nM and 0.4 μM Ca2+, respectively.

These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high‐affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low‐affinity state of the devapamil‐binding site on a stable CaCB receptor.
Thermodynamics of interaction of a fluorescent DNA oligomer with the anti‐tumour drug netropsin
Authors: PATEL Naina , BERGLUND Helena , NILSSON Lennart , RIGLER Rudolf , McLAUGHLIN Larry W. , GRÄSLUND Astrid
Published: March 2005

Fluorescence spectroscopy was used to study the interaction between the minor‐groove‐binding drug netropsin and the self‐complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2‐aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2‐aminopurine fluorescence, observed by steady‐state as well as time‐resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 105 M−1 at 25°C, 3–4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as ΔG= ‐29 kJ/mol, ΔH= ‐12 kJ/mol and ΔS=+55 J. mol−1. K−1 at 25°C. These parameters resemble those of the interaction of poly[(dG‐dC). (dG‐dC)] with netropsin, indicating a mainly entropy‐driven reaction. The amino group of 2‐aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.
Localization of two interchain disulfide bridges in dimers of bovine αs2‐casein
Authors: RASMUSSEN Lone K. , HØJRUP Peter , PETERSEN Torben E.
Published: March 2005

Carboxymethylation of bovine skimmed milk with 14C‐labelled iodoacetic acid followed by purification of the αs2‐casein dimer showed that all four cysteine residues in the protein are engaged in disulfide linkages. Mass spectrometry and sequence analysis of cystine‐containing tryptic peptides revealed the presence of two interchain disulfide bridges in the protein. Sequence analysis of disulfide‐linked peptides resulting from an enzymatic cleavage between the bridges demonstrated that the individual chains in the dimers are either aligned in an antiparallel or a parallel orientation. The identity of some of the disulfide‐linked peptides was further verified by performic acid oxidation followed by sequence analysis of the resulting peptides.
Studies on synthetic pathway of xylose‐containing N‐linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.)
Authors: TEZUKA Katsunari , HAYASHI Makoto , ISHIHARA Hideko , AKAZAWA Takashi , TAKAHASHI Noriko
Published: March 2005

We measured the activities of α‐1,3‐mannosyl‐glycoprotein β‐1, 2‐N‐acetylglucosaminyltrans‐ferase, α‐1,6‐mannosyl‐glycoprotein β‐1,2‐N‐acetylglucosaminyltransferase, β‐1,4‐mannosyl‐glyco‐protein β‐1,2‐xylosyltransferase and glycoprotein 3‐α‐l‐fucosyltransferase in the Golgi fraction of suspension‐cultured cells of sycamore (Acer pseudoplatanus L.) using fluorescence‐labelled oligosaccharides as acceptor substrates for these transferase reactions. The structures of the pyridylaminated oligosaccharides produced by these reactions were analyzed by two‐dimensional sugar mapping using high‐performance liquid chromatography. We demonstrated that

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produce by these in vitro reactions. On the basis of these results, we discuss a biosynthetic pathway for xylose containing N‐linked oligosaccharides in plant glycoproteins.
Isolation and characterization of a novel glycosyl‐phosphatidylinositol‐anchored glycoconjugate expressed by developing neurons
Authors: NEDELEC Joël , PIERRES Michel , MOREAU Hervé , BARBET Jacques , NAQUET Philippe , FAIVRE‐SARRAILH Catherine , ROUGON Geneviève
Published: March 2005

In search of new markers for studying thymic and nervous system ontogeny, we raised rat monoclonal antibodies against glycosyl‐phosphatidylinositol‐anchored molecules among which larger groupings have been shown to be ectoenzymes and adhesion molecules. Two of these monoclonal antibodies (H193–4 and H194–563, IgG) were found to recognize glycosyl‐phosphatidylinositol‐anchored glycoconjugates of 28–33 kDa (P31) and 50–70 kDa in developing mouse brain and thymus respectively, when these tissues were analysed by immunoblot experiments. P31 antigen was found to be transiently expressed by neurons in neural primary cultures [Rougon, G., Alterman, L., Dennis, K., Guo, X. J. & Kinnon, K. (1991) Eur. J. Immunol. 21, 1397–1402]. We show in this report that, in developing mouse brain, a maximal expression occurred between embryonic day 17 and post‐natal day 5, a period that corresponds to the formation of neuronal networks.

P31 antigen was immunopurified and found to possess the following properties: (a) it was soluble in alkaline solvents; (b) it bound to DEAE‐cellulose and was eluted by a salt gradient of 0–1 M NaCl; (c) it was sensitive to endoglycosidase F digestion; (d) it was insensitive to heparinase, hyaluronidase, chondroitinase ABC, endo‐β‐galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoractivity; (f) it contained phosphate; (g) it lost its immunoreactivity after treatment with phosphatidylinositol phospholipase C and treatment.

These characteristics combine to suggest that P31 is an anionic glycoconjugate sharing similarities with Leishmania donovani lipophosphoglycan and with the heat‐stable antigen recognized by J11d antibody on murine hematopoïetic cells. This last hypothesis was further confirmed by the observation that oligonucleotide probes derived from the heat‐stable antigen‐encoding cDNA detect, in developing brain, a 1.8‐kb mRNA species similar in size to that reported for the heat‐stable antigen mRNA and following the same developmental expression as P31 antigen.
Three‐dimensional solution structure of apo‐neocarzinostatin from Streptomyces carzinostaticus determined by NMR spectroscopy
Authors: ADJADJ Élisabeth , QUINIOU Éric , MISPELTER Joël , FAVAUDON Vincent , LHOSTE Jean‐Marc
Published: March 2005

The three‐dimensional solution structure of apo‐neocarzinostatin has been resolved from nuclear magnetic resonance spectroscopy data. Up to 1034 constraints were used to generate an initial set of 45 structures using a distance geometry algorithm (DSPACE). From this set, ten structures were subjected to refinement by restrained energy minimization and molecular dynamics. The average atomic root mean square deviations between the final ten structures and the mean structure obtained by averaging their coordinates run from 0.085 nm for the best defined β‐sheet regions of the protein to 0.227 nm for the side chains of the most flexible loops. The solution structure of aponeocarzinostatin is closely similar to that of the related proteins, macromomycin and actinoxanthin. It contains a seven‐stranded antiparallel β‐barrel which forms, together with two external loops, a deep cavity that is the chromophore binding site. It is noteworthy that aromatic side chains extend into the binding cleft. They may be responsible for the stabilization of the holo‐protein complex and for the chromophore specificity within the antitumoral family.
Chemical modification of cholecystokinin‐A receptors in rat pancreatic membranes
Authors: IJZERMAN Adriaan P. , MELMAN Caroline T. M.
Published: March 2005

Chemical modification of amino acids was used to probe the molecular structure of the cholecystokinin‐A (CCK‐A) receptor on rat pancreatic membranes. Radioligand binding studies with [3H]N‐(2,3‐dihydro‐l‐methyl‐2‐oxo‐5‐phenyl‐lH‐l,4‐benzodiaz epin‐3‐yl)1H‐indole‐2‐carboxamide[(±)‐[3H]L‐364,718], a tritiated highly potent CCK‐A receptor antagonist, enabled the evaluation of the effects caused by the modifying reagents. The apparent fragility of the receptor protein necessitated the development of a modification procedure without wash and centrifugation steps. Treatment of a concentrated membrane preparation with the group‐specific agents N‐ethylmaleimide, phenylglyoxal and diethylpyrocarbonate, subsequent dilution and incubation at lower temperatures (20°C instead of the more generally used 37°C) proved successful. All modifiers affected the binding characteristics for both agonists and antagonists considerably. CCK‐A receptor coupling to guanosine‐nucleotide‐binding proteins was substantially diminished upon modification with N‐ethylmaleimide and diethylpyrocarbonate, as could be concluded from the effects on the (±)‐[3H]L‐364,718 displacement by the cholecystokinin C‐terminal octapeptide (CCK‐8). The ligand‐binding site was affected by all three reagents, as could be inferred from the specific protection obtained with the CCK‐A receptor antagonist, lorglumide. It therefore appears that sulfhydryl, arginyl, and histidyl residues form an essential part of the ligand‐binding domain on the CCK‐A receptor and that sulfhydryl and histidyl residues are also involved in the signal‐transduction pathway.
Principles for designing enzyme‐like catalysts based on the rate‐acceleration mechanisms of semisynthetic oxidases
Authors: YOMO Tetsuya , URABE Itaru , OKADA Hirosuke
Published: March 2005

Combinations of substrate‐binding sites and catalytic groups constitute various kinds of enzyme‐like catalysts. The design of such catalysts can be evaluated by the enhancement of the overall catalytic activity by combining these parts into one catalyst. For a catalyst having one substrate‐binding site and one catalytic group, an equation was obtained which shows the relationship between the rate‐acceleration due to the combination, the affinity of the site (1/Kd), intrinsic effective concentration (kin/kex) and substrate concentration ([S]). The intrinsic effective concentration is the ratio of the first‐order rate constant (kin) of the intramolecular reaction between the catalytic group and the bound substrate and the second‐order rate constant (kex) of the intermolecular reaction between the catalytic group and the free substrate; the value depends on the method of linking the catalytic group and the binding site. This equation provides the following principles for designing catalysts of this type with a considerable grade of rate‐acceleration: [S]≦kin/kex and (1/10)[S]≦kd≦kin/kex. To increase kin/kex, the structure of the binding site is required not to reduce the reactivity of the bound substrate, and the linker connecting the binding site and the catalytic group is required to be flexible and to have an appropriate length. A subunit structure is also found to be effective to improve the catalytic activity: the activity of an n‐mer is at most n2 times as high as that of the monomer. As for the substrate‐binding sites, the sites of natural enzymes and antibodies are good candidates because various kinds of binding sites with high affinity and specificity to the corresponding substrates are available. In addition, the equation relating the rate‐acceleration with kd, kin/kex, and [S] is used for explaining the catalytic efficiency of enzymes energetically.

The principle for designing a multifunctional catalyst having several kinds of binding sites for its substrates and intermediates and several kinds of catalytic groups was then investigated. In this case, the diffusion of the intermediates strongly affects the activity of the multifunctional catalyst, and such a diffusion process was also analyzed. On the basis of these analyses, the following principles were obtained. For a multistep reaction by which a substrate is converted to intermediates successively and finally to a product (linear coupling), the ratio of the dissociation rate constant and the intramolecular rate constant (including the transfer step to the next site) for each intermediate should be made as small as possible; but in practice, it seems very difficult. For a multistep reaction in which reaction steps are coupled by the recycling of the intermediates (cyclic coupling), covalent linking of the intermediates to the catalyst is the best way, and the number of the linked intermediates should be more than that of the binding sites.
Dissection of the ribonuclease T1 subsite
Authors: STEYAERT Jan , HAIKAL Abdel F. , STANSSENS Patrick , WYNS Lode
Published: March 2005

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661–8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3′‐(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666–8670]. The kinetics of the Asn36Ala, Asn98Ala and wild‐type catalyzed transesterification of guanosine 3′‐(5′‐d‐ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by d‐ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.
Extended type‐1 chain glycosphingolipid antigens
Authors: STROUD Mark R. , LEVERY Steven B. , SALYAN Mary Ellen K. , ROBERTS Carl E. , HAKOMORI S.
Published: March 2005

A Lewis‐b‐active glycosphingolipid containing a repetitive type‐1 chain carbohydrate core was isolated from human colonic adenocarcinoma cell line Colo205. This glycosphingolipid was purified by HPLC and preparative high‐performance thin‐layer chromatography and its structure elucidated by positive‐ion fast‐atom‐bombardment mass spectrometry with collision‐induced disassociation, 1H‐NMR spectroscopy and methylation analysis. The glycosphingolipid was found to be a trifucosylated derivative of this novel carbohydrate core, having the following structure:

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Primary structure and import pathway of the rotenone‐insensitive NADH‐ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae
Authors: DE VRIES Simon , VAN WITZENBURG Richard , GRIVELL Leslie A. , MARRES Carla A. M.
Published: March 2005

The gene encoding the yeast mitochondrial rotenone‐insensitive internal NADH: ubiquinone‐6 oxidoreductase has been sequenced. The DNA sequence indicates the presence of an open reading frame of 1539 bp predicted to encode a protein of 513 amino acid residues (57.2 kDa). The NADH dehydrogenase is synthesized as a precursor protein containing a signal sequence of 26 residues. In vitro import experiments show that the precursor NADH dehydrogenase is cleaved to the mature size by the matrix processing peptidase. Both cleavage and translocation across the mitochondrial membrane(s) are dependent on the membrane potential component of the proton‐motive force. Comparison of the protein sequence of the yeast NADH dehydrogenase with the data bank indicates that the enzyme from yeast is homologous to the NADH dehydrogenase of Escherichia coli (22.2% identical residues). Both NADH dehydrogenases contain in the central part of the protein a sequence predicted to fold into a βαβ structure involved in the binding of NADH or FAD(H2). Various aspects of the protein structure are discussed.
Carnitine biosynthesis in hepatic peroxisomes
Authors: PAUL Harbhajan S. , SEKAS Gail , ADIBI Siamak A.
Published: March 2005

We have investigated whether hepatic peroxisomes are capable of synthesizing carnitine. When purified peroxisomes were incubated with γ‐butyrobetaine, a precursor of carnitine, formation of carnitine was observed. These results indicate that peroxisomes contain γ‐butyrobetaine hydroxylase, the enzyme which catalyzes the final step in the biosynthesis of carnitine. This enzyme was previously believed to be present only in the cytosol. γ‐Butyrobetaine hydroxylase activity in peroxisomes was not due to cytosolic contamination as evaluated by marker enzyme analysis. When proliferation of peroxisomes was induced by clofibrate treatment, γ‐butyrobetaine hydroxylase/mass liver increased by 7.6‐fold and the specific activity by 2.5‐fold. We conclude that hepatic peroxisomes synthesize carnitine and this synthesis becomes substantial under conditions of peroxisomal proliferation.
Developmental changes in the activity of membrane‐bound γ‐glutamyl transpeptidase and in the sialylation of synaptosomal membranes from the chick embryonic brain
Authors: DVOŘÁKOVÁ Lenka , LISÝA Václav , S̆T̆ASTNÝ Frantiˇek
Published: March 2005

γ‐Glutamyl transpeptidase (GGT) is a membrane‐bound sialoglycoprotein. The developmental changes in GGT activity and in sialic acid content were determined in a crude synaptosomal membrane fraction from the cerebral hemispheres of the chick embryo between days 11 and 19 of incubation. The GGT activity increased almost eightfold during the examined developmental period, while sialic acid content rose significantly only between days 11 and 15. Cortisol administered on day 13 significantly increased GGT activity. On the other hand, the content of membrane bound sialic acid was not substantially affected. The value of the GGT apparent Michaelis constant (Kmapp) for γ‐glutamyl‐p‐nitroanilide in the presence of 20 mmol · 1−1 glycylglycine was 1.5 mmol · l−1 and cortisol did not influence it. However, Vmax was increased by this hormone. The affinity of GGT to concanavalin A (ConA) did not change during development. Neither the administration of cortisol nor neuroaminidase treatment had any effect on the interaction of GGT with ConA. Desialylation of crude synaptosomal fraction did not change GGT activity. The results presented here suggest no developmental nor functional relationship between the activity of GGT and the level of sialylation in synaptosomal membranes from the cerebral hemispheres of the chick embryo.
Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12
Authors: ANDERSEN Paal Skytt , SMITH John M. , MYGIND Bente
Published: March 2005

The upp gene coding for uracil phosphoribosyltransferase was subcloned on a 5‐kb EcoRI restriction fragment along with the purMN operon. By a combination of complementation, deletion and minicell analyses, the upp gene was located adjacent to and divergently transcribed from the purMN operon. All three gene products could be identified in minicell extracts. The cloned upp gene shows an elevated expression upon uracil starvation. The nucleotide sequence and transcription start of the gene were determined. The sequence yields an open reading frame of 624 nucleotides encoding a protein of 22.5 kDa which is in agreement with the previously determined subunit Mr of the purified enzyme. A putative 5‐phosphoribosyl‐α‐1‐diphosphate (PRPP) binding site has been identified which is similar to the PRPP binding site of the yeast uracil phosphoribosyltransferase.
Anthracycline binding to DNA
Authors: LEONARD Gordon A. , BROWN Tom , HUNTER William N.
Published: March 2005

Crystallographic methods have been applied to determine the high‐resolution structure of the complex formed between the self‐complementary oligonucleotide d(TGTACA) and the anthracycline antibiotic 4′‐epiadriamycin. The complex crystallises in the tetragonal system, space group P41212 with a= 2.802 nm and c= 5.293 nm, and an asymmetric unit consisting of a single DNA strand, one drug molecule and 34 solvent molecules. The refinement converged with an R factor of 0.17 for the 2381 reflections with F≥3σF in the resolution range 0.70–0.14 nm. Two asymmetric units associate such that a distorted B‐DNA‐type hexanucleotide duplex is formed incorporating two drug molecules that are intercalated at the TpG steps. The amino sugar of 4′‐epiadriamycin binds in the minor groove of the duplex and displays different interactions from those observed in previously determined structures. Interactions between the hydrophilic groups of the amino sugar and the oligonucleotide are all mediated by solvent molecules. Ultraviolet melting measurements and comparison with other anthracycline‐DNA complexes suggest that these indirect interactions have a powerful stabilising effect on the complex.
The amino acid sequence of glutamate decarboxylase from Escherichia coli
Authors: MARAS Bruno , SWEENEY Glen , BARRA Donatella , BOSSA Francesco , JOHN Robert A.
Published: March 2005

The amino acid sequence of glutamate decarboxylase from Escherichia coli was solved by a combination of automated Edman degradation of peptide fragments derived by proteolytic and chemical cleavage and sequencing of DNA. Correct alignment of three peptides, for which no peptide overlaps were available, was achieved by sequencing a 1.1‐kbp fragment of DNA produced by a polymerase‐chain reaction using primers corresponding to sequences known to be in amino‐terminal and carboxy‐terminal regions of the protein. Sequence similarity (24% identity) with mammalian glutamate decarboxylase was found to be limited to a 55‐residue sequence around the lysine residue that with binds the coenzyme. Stronger similarity (38% identity), again confined to the same region, is seen with bacterial pyridoxal‐phosphate‐dependent histidine decarboxylase.
Interaction of Prodan with tubulin
Authors: MAZUMDAR Manjari , PARRACK Pradip Kumar , BHATTACHARYYA Bhabatarak
Published: March 2005

The compound 6‐propionyl‐2‐(N,N‐dimethyl)‐aminonaphthalene (Prodan), an efficient fluorescent probe for proteins, is shown to bind to tubulin. Detailed experiments on fluorescence enhancement, anisotropy and energy transfer were carried out to unravel the nature of Prodan‐tubulin interaction and the Prodan‐binding site on tubulin. It was found that Prodan binds to tubulin at a rigid site, with a stoichiometry of 1:1 and a dissociation constant of 20 μM. Competition experiments using 1,8‐[3H]anilinonaphthalenesulfonate ([3H]ANS) indicated that the three aminonaphthalenes, ANS, bis(1,8‐anilinonaphthalenesulfonate) and Prodan probably bind to a common region on the tubulin molecule.
Studies on the variants of the protein toxins ricin and abrin
Authors: HEGDE Ramesh , PODDER Sunil K.
Published: March 2005

This study elucidates some structural and biological features of galactose‐binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl‐Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl‐Sepharose CL‐6B ion‐exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross‐reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two‐dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post‐translational differential proteolytic processing, a secondary event in the production of abrin variants.
A comparative study on the catalytic properties of guanyl‐specific ribonucleases
Authors: YAKOVLEV Gennady I. , MOISEYEV Gennady P. , BEZBORODOVA Svetlana I. , BOTH Valentin , SEVCIK Jozef
Published: March 2005

The kinetic parameters of reactions catalyzed by four guanyl‐specific RNases T1, Pb1, Th1 and Sa were studied comparatively using three types of substrates; guanosine‐2′,3′‐cyclophosphates, GpN dinucleoside phosphates and synthetic polyribonucleotides. The kinetic parameters were shown to be similar in spite of considerable differences in primary structures of these RNases, including amino acid residues of the active sites. Therefore, primary structures of guanyl RNases allow for a considerable number of substitutions (both in the ‘recognising’ and catalytical parts of the active site) without changes in the catalytical parameters.
Cytoplasmic high‐level expression of a soluble, enzymatically active form of the Escherichia coli penicillin‐binding protein 5 and purification by dye chromatography
Authors: van der LINDEN Mark P. G. , MOTTL Harald , KECK Wolfgang
Published: March 2005

High‐level expression of a soluble form of penicillin‐binding protein 5 (PBP5), called PBP5s, and translocation across the cytoplasmic membrane results in lysis of Escherichia coli cells. The detrimental effect of increased amounts of this D,D‐carboxypeptidase on the stability of the murein polymer can be avoided by accumulation of the overexpressed protein in the cytoplasm. The signal peptide of the structural gene dacAs, coding for PBP5s was deleted by creating a BamHI site at the site of processing, and the truncated gene dacAsc was cloned under the control of the lambda PR promoter. Temperature induction resulted in a 200‐fold overproduction of the mature PBP5s in the cytosol (PBP5sc) which is no longer harmful to the cells. PBP5sc could quantitatively be recovered in the soluble fraction after disrupting the cells. The protein retained full enzymatic activity as measured by the release of D‐alanine from bisacetyl‐L‐Lys‐D‐Ala‐D‐Ala and formation of [14C]penicillin‐protein complex at a 1:1 stoichiometry. A one‐step purification procedure using the immobilized dye Procion rubine MX‐B resulted in homogeneous preparations of both wild‐type and mutated forms of PBP5sc.
Use of influenza C virus for detection of 9‐O‐acetylated sialic acids on immobilized glycoconjugates by esterase activity
Authors: ZIMMER Gert , REUTER Gerd , SCHAUER Roland
Published: March 2005

An overlay and a solid‐phase assay are presented which allow the specific detection of 9‐O‐acetylated sialic acids on sialoglycoconjugates immobilized on microtiter plates, nitrocellulose or separated on thin‐layer chromatograms. The assay takes advantage of two different biological properties of influenza C virus, its high‐affinity binding to 9‐O‐acetylated sialic acids and its sialate 9‐O‐acetylesterase that is used for detection of bound virus with fluorogenic or chromogenic substrates. Though simple and rapid, the assay is highly sensitive with a detection limit of 65 fmol 9‐O‐acetylated sialic acid in 9‐O‐acetylated ganglioside GD1a.

Influenza C virus is able to bind to a wide spectrum of sialoglycoconjugates like mucins, serum glycoproteins or gangliosides containing naturally or synthetically O‐acetylated sialic acids. 9‐O‐Acetyl‐N‐glycoloylneuraminic acid can also function as a high‐affinity receptor determinant for influenza C virus. While the acetyl ester at the 9 position is essential for virus binding in all cases, a 4‐O‐acetyl group is not recognized. In addition to α(2,3) or α(2,6) bonds, 9‐O‐acetyl‐N‐acetylneuraminic acid in α(2,8) linkage to N‐acetylneuraminic acid is also functionally active.
Structural analysis of a novel sialic‐acid‐containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide
Authors: KRAUSS Jürgen Hinrich , HIMMELSPACH Karl , REUTER Gerd , SCHAUER Roland , MAYER Hubert
Published: March 2005

Sialic‐acid‐containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S‐form lipopolysaccharide), KB‐1 (R‐type lipopolysaccharide) and Sp 18 (deep R‐type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards acid and enzymic hydrolysis indicated a non‐reducing terminal location of sialic acids in the R‐type lipopolysaccharide of strain Sp 18, whereas an internal, chain‐linked location of sialic acids was found in the lipopolysaccharides of strains 37b4 and KB‐1. For these latter strains, methylation analysis revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4 and 7 for strain KB‐1.

In accordance with the chain‐linked position of sialic acids, mild hydrolysis of R. capsulatus 37b4 lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus. Structural investigation of this trisaccharide by methylation analysis, 1H‐and 13C‐NMR spectroscopy revealed the presence of the disaccharide Gal1–6Glc at the non‐reducing end, probably with an α‐anomeric configuration of the galactose residue, i.e. melibiose, β‐glycosidically linked to position 7 of sialic acid. Therefore the structure Galα1–6Glcβ1–7Neu5Ac is proposed for this core oligosaccharide from R. capsulatus 37b4 lipopolysaccharide.
Structure of the capsular polysaccharide of Actinobacillus pleuropneumoniae serotype 5b
Authors: ALTMAN Eleonora , BRISSON Jean‐Robert , GAGNÉ Stéphane M. , PERRY Malcolm B.
Published: March 2005

The capsular polysaccharide of Actinobacillus pleuropneumoniae serotype 5b (strain L20) was found to be a high molecular mass polymer composed of 2‐acetamido‐2‐deoxy‐D‐glucose, D‐glucose, and 3‐deoxy‐D‐manno‐octulosonic acid (KDO). Methylation analysis, partial hydrolysis and a combination of homonuclear and 1H‐detected heteronuclear shift‐correlated nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a trisaccharide repeating unit, having the structure:

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Membrane protein acylation
Authors: NYSTRÖM Susanne , WALLBRANDT Pia , WIESLANDER Åke
Published: March 2005

Mycoplasmas are small bacteria without a cell wall, often found as surface parasites on eukaryotic cells. Of the more than 200 membrane proteins from Acholeplasma laidlawii resolved by two‐dimensional PAGE, 23 were covalently modified with acyl chains. These acyl proteins had lower pI values than average and were all labelled by different exogenously supplied radioactive fatty acids attached by O‐ester bonds. The fatty acids were selectively incorporated in the order myristic acid (14:0) > palmitic acid (16:0) > stearic acid (18:0) > oleic acid (18:1). However, endogenously synthesised saturated fatty acids, most of which were 16:0, were preferred over the supplied ones. A fraction of the exogenous 14:0 was elongated to 16:0. Absence of saturated fatty acids increased the incorporation of 18:1. The maximum extent of modification was one acyl chain for protein T2, on the exterior surface and two acyl chains for protein D12, spanning the membrane. Exogenously supplied fatty acids were incorporated into membrane lipids in proportion to their occurrence. However, the acylated proteins always contained 8–10 times more saturated chains than did the lipids. When exogenously supplied, all A. laidlawii polar membrane lipids could donate acyl chains to the acylated proteins but the neutral fraction (fatty acids and diacylglycerol) was most efficient. An incorporation into the acylated proteins of labelled cysteine, but not glucose or glycerol, was observed. Acylated proteins with different chains interacted similarily with a Triton X‐114 detergent phase, and no full‐size proteins (or acylated fragments) were released from cells by proteolytic enzymes. The results indicate an anchoring with peptide segments in addition to the acyl chains. Both 14:0 and 16:0 were attached at one end of both T2 and D12, but the N‐terminal methionine of T2 was not acylated. The extent of modification and preference for saturated chains in the A. laidlawii membrane acylated proteins is more similar to eukaryotic than to eubacterial proteins.
Channelling can decrease pool size
Authors: MENDES Pedro , KELL Douglas B. , WESTERHOFF Hans V.
Published: March 2005

It is widely considered that a possible advantage of metabolite channelling, in which a product of an enzyme is transferred to the next enzyme in a metabolic pathway without being released to the ‘bulk’ solution, is that channelling can decrease the steady‐state concentrations of ‘pool’ intermediates. This then spares the limited solvent capacity of the cell, and reduces the loss of pathway flux due to leakage or instability of the free intermediate. Recently, however, based on simulations of a particular model of a ‘dynamic’ channel, Cornish‐Bowden [“Failure of channelling to maintain low concentrations of metabolic intermediates” (1991) Eur. J. Biochem. 195, 103–108] has argued that this is not in fact the case; his simulations indicated that the channel was rather ineffective at decreasing the concentration of the pool intermediate, and in some cases actually increased it. However, although his simulations were restricted to very specific thermodynamic and kinetic parameters, he generalised his conclusions, arguing that “channelling has no effect on the free concentration of a channelled intermediate in a pathway”.

By showing that, for a number of kinetic cases, the concentration of the pool intermediate did decrease substantially with increased channelling, we demonstrate here that the conclusion of Cornish‐Bowden is not correct. In particular, if the reaction catalysed by the enzymes forming the channel has an equilibrium constant K higher than 1, and if the enzyme removing the product of the channel reaction is kinetically competent, channelling in the model system studied by Cornish‐Bowden (1991) can decrease the steady‐state concentration of the pool by a factor of 1000, independently of the mechanism of the terminal reaction and under conditions of essentially constant overall flux. If the channel is a ‘static’ channel, the decrease in the pool can be to arbitrarily low levels. This conclusion also holds for a system in which other reactions may consume the pool intermediate. Thus, channelling can maintain metabolite concentrations at low levels.
1H‐NMR structural determination of the N‐linked carbohydrate chains on glycopeptides obtained from the bean lectin phytohemagglutinin
Authors: STURM Arnd , BERGWERFF Aldert A. , VLIEGENTHART Johannes F. G.
Published: March 2005

Phytohemagglutinin, the lectin of the common bean Phaseolus vulgaris, is a N‐linked glycoprotein with one high‐mannose‐type and one xylose‐containing oligosaccharide side chain per polypeptide. The high‐mannose‐type glycan is attached to Asn12 and the complex‐type glycan to Asn60 [Sturm, A. & Chrispeels, M. J. (1986) Plant Physiol. 81, 320–322]. The structures of the oligosaccharides were elucidated from two glycopeptides obtained from the lectin by Pronase digestion, affinity chromatography on concanavalin‐A–Sepharose and gel‐filtration chromatography on a column of BioGel P‐4. The N‐linked glycan structures were investigated by 500‐MHz 1H‐NMR spectroscopy and were established to be:

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The phosphorylated and/or sulfated structure of the carbohydrate – protein‐linkage region isolated from chondroitin sulfate in the hybrid proteoglycans of Engelbreth‐Holm‐Swarm mouse tumor
Authors: SUGAHARA Kazuyuki , MIZUNO Nanae , OKUMURA Yutaka , KAWASAKI Toshisuke
Published: March 2005

The structure of the linkage region of chondroitin sulfate chains attached to the hybrid proteoglycans of the Engelbreth‐Holm‐Swarm mouse tumor was investigated. The peptidoglycan fraction which contains oversulfated chondroitin sulfate rich in the GlcAβ1‐3GalNAc‐4,6‐diO‐sulfate unit and undersulfated heparan sulfate rich in GlcAβ1‐4GlcNAc and GlcAβ1‐4GlcN‐2N‐sulfate units was isolated after exhaustive protease digestion of the acetone powder of the tumor tissue, (GlcA, glucuronic acid; GalNAc, 2‐deoxy‐2‐N‐acetylamino‐D‐galactose). Glycosaminoglycans were released by β‐elimination using NaB3H4 and digested with chondroitinase ABC. The linkage region fraction was separated from heparan sulfate by gel filtration and fractionated by HPLC on an amine‐bound silica column. Six radiolabeled compounds (L1–L6) were obtained and structurally analyzed by co‐chromatography with authentic hexasaccharide alditols recently isolated by us from the linkage region, and by digestion using chondroitinase ACII, alkaline phosphatase and β‐galactosidase in conjugation with HPLC. These compounds shared the conventional hexasaccharide backbone structure:

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(ΔGlcA, Δ4,5‐GlcA or D‐gluco‐4‐enepyranosyluronic acid). L1 was not sulfated or phosphorylated. L2 and L4 were monosulfated at C‐6 and C‐4 of the GalNAc residue, respectively. Upon alkaline phosphatase digestion, L3, L5 and L6 were converted to L1, L2 and L4, respectively. Analysis of the periodate oxidation products indicated that the phosphate group in L3, L5 and L6 is located at C‐2 of Xyl‐ol. These results suggest that Xyl‐2‐O‐phosphate is associated with both 4‐O‐sulfated and 6‐O‐sulfated GalNAc units and does not directly determine the sulfation pattern of chondroitin sulfate.
Glutamyl‐tRNA synthetase from Thermus thermophilus HB8
Authors: NUREKI Osamu , SUZUKI Kenji , HARA‐YOKOYAMA Miki , KOHNO Toshiyuki , MATSUZAWA Hiroshi , OHTA Takahisa , SHIMIZU Toshiyuki , MORIKAWA Kosuke , MIYAZAWA Tatsuo , YOKOYAMA Shigeyuki
Published: March 2005

The gene for the Glu‐tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N‐terminal amino acid sequence of Glu‐tRNA synthetase. Nucleotide‐sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53901). Codon usage in the T. thermophilus Glu‐tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu‐tRNA synthetase showed high similarity with bacterial Glu‐tRNA synthetases (35–45% identity); the sequences of the binding sites for ATP and for the 3′ terminus of tRNAGlu are highly conserved. The Glu‐tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu‐tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three‐step column chromatography. Single crystals of T. thermophilus Glu‐tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor‐diffusion technique. The crystals diffract X‐rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P212121, with unit‐cell parameters of a= 8.64 nm, b= 8.86 nm and c= 8.49 nm.
Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.
Authors: FACCHIANO Francesco , RAGONE Raffaele , PORCELLI Marina , CACCIAPUOTI Giovanna , COLONNA Giovanni
Published: March 2005

The effect of temperature on the molecular structure of propylamine transferase from Sulfolobus solfataricus has been investigated. Sulfolobus solfataricus is an extreme thermophilic archaebacterium with an optimum living condition at 90°C. The enzyme is an oligomeric (trimer) protein of molecular mass 112 kDa. The frictional ratio for the native protein suggests an irregularly shaped compact globular structure. The protein matrix is well organized as suggested by far ultraviolet circular dichroism at 25°C (18%α helix, 43%β structure, 19%β bends and 20% unordered: root mean square = 7). Structural effects of temperature were investigated over 25–85°C. The protein retains its quaternary structure in this temperature range. A highly reversible subtle conformational transition was detected by numerous structure‐dependent techniques over 40–50°C, with a midpoint centered at 45°C. Functional data also support this view. In fact, two enzyme forms, characterized by different catalytic properties, are present in solution. The Arrhenius plot suggests the occurrence of two different activation‐energy‐dependent processes, one at a temperature higher and one at a temperature lower than 45°C. The transition has been considered as a molecular switch between two protein populations at equilibrium with different functional and structural properties, temperature modulated. A physiological role for the molecular switch has also been postulated. The protein also shows some subtle and reversible spectroscopic changes around 75°C. The molecular basis of the thermophilic nature of this enzyme seems to reside in its capability to dynamically couple catalytic and structural events to the thermal properties of the ambient medium.
The nucleotide and partial amino acid sequences of rat fetuin
Authors: RAUTH Günther , PÖSCHKE Oliver , FINK Edwin , EULITZ Manfred , TIPPMER Stefanie , KELLERER Monika , HÄRING Hans‐Ulrich , NAWRATIL Peter , HAASEMANN Martina , JAHNEN‐DECHENT Willi , MÜLLER‐ESTERL Werner
Published: March 2005

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full‐length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor.
Changes in myosin heavy‐chain isoform synthesis of chronically stimulated rat fast‐twitch muscle
Authors: TERMIN Angelika , PETTE Dirk
Published: March 2005

Chronic low‐frequency stimulation was used for studying the adaptive potential of rat fast‐twitch muscle to increased neuromuscular activity. The sequential exchange of myosin heavy chain isoforms HCIIb with HCIId and HCIIa was studied at the translational level using an in‐vivo‐labeling technique with [35S]methionine.

Alterations in heavy chain isoform synthesis, i.e. a decrease in the labeling of HCIIb concomitant with an enhanced labeling of HCIId/IIa, were detectable already two days after the onset of stimulation. This time course corresponds to the previously observed alterations in the amounts of HCIIb and HCIIa mRNAs. However, significant changes in the relative protein amounts of HCIIb and HCIId/IIa were recorded only after an 8‐day stimulation period. This delay at the protein level was interpreted to relate to the slow turnover of HCIIb which was estimated from its decay in long‐term stimulated muscles with an approximate value of 14.7 days. Therefore, protein degradation seems to be an important post‐translational regulatory step in the remodeling process of the thick filament.
The aconitase of Escherichia coli
Authors: PRODROMOU Chrisostomos , ARTYMIUK Peter J. , GUEST John R.
Published: March 2005

The nucleotide sequence of the aconitase gene (acn) of Escherichia coli was determined and used to deduce the primary structure of the enzyme. The coding region comprises 2670 bp (890 codons excluding the start and stop codons) which define a product having a relative molecular mass of 97513 and an N‐terminal amino acid sequence consistent with those determined previously for the purified enzyme. The acn gene is flanked by the cysB gene and a putative riboflavin biosynthesis gene resembling the ribA gene of Bacillus subtillis. The 1004‐bp cysB–acn intergenic region contains several potential promoter and regulatory sequences.

The amino acid sequence of the E. coli aconitase is similar to the mitochondrial aconitases (27–29% identity) and the isopropylmalate isomerases (20–21% identity) but it is most similar to the human iron‐responsive‐element‐binding protein (53% identity). The three cysteine residues involved in ligand binding to the [4Fe‐4S] centre are conserved in all of these proteins. Of the remaining 17 active‐site residues assigned for porcine aconitase, 16 are conserved in both the bacterial aconitase and the iron‐responsive‐element‐binding protein and 14 in the isopropylmalate isomerases. It is concluded that the bacterial and mitochondrial aconitases, the isopropylmalate isomerases and the iron‐responsive‐element‐binding protein form a family of structurally related proteins, which does not include the Fe‐S‐containing fumarases. These relationships raise the possibility that the iron‐responsive‐element‐binding protein may be a cytoplasmic aconitase and that the E. coli aconitase may have an iron‐responsive regulatory function.