bluuebunny/crossref_metadata_2025_split huggingface dataset:
β subunit of mitochondrial F1‐ATPase from the fission yeast
Authors: FALSON Pierre , LETERME Serge , CAPIAU Carine , BOUTRY Marc 
Published: March 2005

The Schizosaccharomyces pombe nuclear gene, atp2, encoding the β subunit of the mitochondrial ATP synthase, was sequenced and found to contain a 1575‐bp open reading frame. Two adjacent transcription‐initiation sites were found at positions 34 and 44 nucleotides upstream of the translation‐initiation codon. The deduced polypeptide sequence was composed of 525 amino acid residues (molecular mass = 56875 Da). The mature polypeptide starts at residue 45 (molecular mass = 51685 Da), indicating the presence of a presequence of 44 residues, presumably involved in mitochondrial targeting.The atp2 mutant B59‐1 [Boutry, M. & Goffeau, A. (1982) Eur. J. Biochem. 125, 471–477] and its related revertant allele R4‐3 [Jault, J. M., Di Pietro, A., Falson, P., Gautheron, D. C., Boutry, M. & Goffeau, A. (1989) Biochem. Biophys. Res. Commun. 158, 392–399] were also cloned and sequenced. A single nonsense mutation, CAG (Gln170) → TAG (stop) in mutant B59‐1, became a missense mutation, TAG (stop) → TAC (Tyr) in revertant R4‐3. Gln170 is located between the first and second elements belonging to the nucleotide‐binding site. Its substitution by a tyrosine residue increases the enzyme affinity towards ADP, the amount of endogenous nucleotides and the apparent negative cooperativity for ATPase activity.
Binding of proteins of HeLa S3 cell extract to oligonucleotides containing the consensus interferon‐response sequence (IRS) and to the IRS‐containing fragment of the human c‐myc gene
Authors: TURPAEV Kirill T. , ITKES Alexander V. , ALEXANDROVA Nadja M. , POKROVSKAYA Olga V. , IMAMOVA Laele R. , CHERNOV Boris K. , KISSELEV Lev L. 
Published: March 2005

The human c‐myc proto‐oncogene was recently found to contain a regulatory sequence similar to the consensus interferon‐response sequence (IRS) of interferon‐activating genes. Binding of regulatory protein(s) to this sequence of cloned fragment of c‐myc, lacking the main part of 5′‐nontranscribing region, regulates in vitro transcription from I1/I2 initiation sites located in the first intron of the gene.Here, we have shown that HeLa S3 nuclear extract contains different protein factors, at least two, that bind preferentially to the IRS sequence of either the c‐myc gene or the interferon‐dependent 6–16 gene. Moreover, each of these factors ‘cross‐binds’ to the region of the other gene, although affinity of this interaction is lower. Binding constants of these proteins to oligonucleotide fragments of c‐myc and 6–16 genes were determined. In vitro transcription of the human full‐length c‐myc gene (i.e. the gene containing the complete 5′ ‐noncoding region) initiated from I1/I2 sites, that is controlled by the IRS region, was demonstrated to be blocked. A possible physiological role for the mechanisms described is discussed.
Complementation of soluble phosphofructokinase activity in yeast mutants
Authors: GAYATRI Archana G. , MAITRA Pabitra K. 
Published: March 2005

We describe here the genetic and biochemical analyses of two classes of mutations in the soluble phosphofructokinase (PFK I) of Saccharomyces cerevisiae: those leading to the loss of activity and those giving rise to a kinetically altered enzyme. Complementation and allele‐testing between these two classes of mutants show that loss of enzyme activity in vitro can come about not only by mutations in the catalytic subunit but also in the regulatory subunit. Also, a mutation in the catalytic subunit can give rise to an enzyme altered in its kinetic properties in a manner phenomenologically similar to that caused by a mutation in the regulatory subunit.The results of the complementation studies in diploids suggest that, in spite of their distinct functions, both the subunits are essential for activity to be detected in vitro. This is confirmed by the reconstitution of an active PFK I enzyme by mixing cell‐free extracts of two complementing parents, each of which lacks the enzyme activity. PFK activity appears in the mixture, reaching a maximum value of 60–100% of that of the diploid in 15–30 min at 24°C. Unlike the catalytic subunit which exists in various multimeric states in cell‐free extracts of the mutant bearing only this subunit, the regulatory subunit exists largely as a monomer in a mutant devoid of the catalytic subunit. The reconstituted enzyme, however, is indistinguishable from that of the wild type, as analysed by sedimentation studies and Western blot analysis, demonstrating that only the heteromeric complex of the two subunits is active, while neither of the individual subunits displays activity in vitro.
Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase
Authors: DE MEIS Leopoldo , SUZANO Valdecir A. , CALDEIRA Teresa , MINTZ Elisabeth , GUILLAIN Florent 
Published: March 2005

The first step towards ATP synthesis by the Ca2‐ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35°C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10‐fold, whereas it had no effect on the apparent affinity for Mg2+.In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations.Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+.The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.
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.
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.
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.
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.
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.
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.