bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular cloning of the gene encoding CDPdiacylglycerol–inositol 3‐phosphatidyl transferase in Saccharomyces cerevisiae
Authors: NIKAWA Jùn‐ichi , YAMASHITA Satoshi 
Published: March 2005

The structure gene encoding CDPdiacylglycerol – inositol 3‐phosphatidyltransferase was isolated by complementation in a Saccharomyces cerevisiae mutant after transformation with a comprehensive gene library containing Sau3AI partial restriction fragments of wild‐type yeast DNA inserted into the YEp13 shuttle vector.
Introduction of the cloned plasmid into the mutant restored both growth of cells and CDPdiacylglycerol – inositol 3‐phosphatidyltransferase activity.
A subcloning study indicated that the complementing gene was contained within a 1700‐base‐pair fragment of DNA. Expression of the cloned sequence was independent of its orientation in the vector.
CDPdiacylglycerol – inositol 3‐phosphatidyhransferase activity in the transformant was eight fold higher than that in the wild‐type strain. Although the mutant enzyme had a greatly increased Km for myo‐inositol, the enzyme in the transformant had an apparent Km for myo‐inositol equal to that of the wild‐type enzyme, indicating the presence of the wild‐type structure gene on the recombinant plasmid.
The elevated level of enzyme activity in the transformant did not lead to an increase in the content of phosphatidylinositol. In contrast, supplementation of the culture medium with increasing concentrations of myo‐inositol resulted in an increase in the phosphatidylinositol content. Thus, the availability of myo‐inositol is a critical regulatory factor in yeast phosphatidylinositol synthesis.
The interaction of skeletal myosin subfragment 1 with the polyanion, heparin
Authors: LABBÉ Jean‐Pierre , BERTRAND Raoul , AUDEMARD Etienne , KASSAB Ridha , WALZTHÖNY Doris , WALLIMANN Theo 
Published: March 2005

The association between chymotryptic skeletal muscle myosin subfragment 1 (S1) and the polyanion, heparin, was investigated as an experimental approach in probing the functional importance of the cationic sites on S1 and their involvement in ionic interactions within the myosin head during energy transduction. The direct binding of heparin, used at micromolar concentrations, and its influence on the structural and functional properties of S1 were followed by gel chromatography, electron microscopy, chemical cross‐linking techniques and limited digestion studies.

The limited tryptic digestion of S1 showed that the presence of heparin, as well as of the homopolymer, poly‐(L‐glutamic acid) causes a specific structural change in the 50‐kDa heavy chain region of S1 and accelerates the breakdown of this segment into a 45‐kDa species by a proteolytic cleavage restricted to its COOH‐terminal portion. Under similar experimental conditions, the binding of MgATP and MgADP to S1 led also to the 50‐kDa → 45‐kDa conversion, suggesting that the S1‐nucleotide interactions exhibit some resemblances to the polyanion‐S1 interactions; a distortion of the COOH‐terminal peptide stretch of the 50‐kDa fragment seems to result from the binding of polyanionic ligands to S1. This particular area is adjacent to the actin site containing the 45‐kDa and 20‐kDa segments of the S1 heavy chain. On the other hand, the polyanions as well as nucleotides induced changes in the interface between the heavy chain and the alkali light chains.
Moreover, the binding of heparin to S1 resulted in the self‐association of the enzyme and the production of stable small S1 oligomers, most likely dimers, which were demonstrated by the alteration of the size of the S1 particles examined by electron microscopy and their freezing by chemical cross‐linking agents. These findings are relevant to the recently reported property of skeletal chymotryptic S1 to form dimers under convenient ionic conditions, in particular in the presence of Mg‐nucleotides. The interaction of cationic sites on S1 and possibly on the 50‐kDa region of the heavy chain with polyanions promotes the dimerization of the S1 molecules. The binding of S1 to F‐actin abolished S1 aggregation.
Nucleotide pyrophosphatase from potato tubers
Authors: BARTKIEWICZ Marcjanna , SIERAKOWSKA Halina , SHUGAR David 
Published: March 2005

Purification of potato tuber nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800‐fold and homogeneous in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The Mr of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an Mr for the subunit of 74000. Analytical isoelectrofocusing reveals a broad isoelectric range of pH 8.3–8.7. The enzyme is a glycoprotein.The purified enzyme exhibits the previously reported activities versus pyrophosphate linkages located at either the 5′‐OH or 3′‐OH of nucleosides, and phosphodiester linkages in: (a) aryl esters of nucleoside 3′‐ and 5′‐phosphates, p‐nitrophenylphosphate and orthophosphate, and (b) nucleoside cyclic 2′,3′‐phosphates. However, the relative rates of activity towards these substrates, and the corresponding V values, differ significantly. The enzyme exhibits additional novel activities, including ability to cleave dinucleoside polyphosphates such as A(5′)p2(5′)A– A(5′)p5(5′)A, and aryl phosphonates. Contrary to previous reports, there is no activity towards nucleoside cyclic 3′,5′‐phosphates.The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m7GMP from the 5′‐terminal m7G(5′)p3(5′)Gm of intact reovirus mRNA.NAD+ was found to be the most effective inhibitor of enzyme activity versus thymidine 5′‐p‐nitro‐phenylphosphate, with a Ki= 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2′,3′‐cAMP and thymidine 3′‐β‐nitrophenylphosphate inhibit hydrolysis of NAD+ non‐competitively and vice‐versa.
Interaction of calmodulin with muscle phosphofructokinase
Authors: MAYR Georg W. 
Published: March 2005

The hysteretic calmodulin‐induced inactivation of muscle phosphofructokinase and the calmodulin‐mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg. ATP, fructose 6‐phosphate, adenosine 5′‐[β, γ‐imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6‐bisphosphate, fructose 2,6‐bisphosphate and glucose 1,6‐bisphosphate was studied by two techniques, (a) A two‐step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions.Dominating in the interplay of calmodulin with metabolic effectors is the competitive‐like action of calmodulin on Mg · ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg · ATP. In the allosteric assay it counteracts the ATP‐induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a miner degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6‐phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control of metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.
Measurements of electron spin resonance with the pyruvate dehydrogenase complex from Escherichia coli
Authors: SCHRENK Dieter F. , BISSWANGER Hans 
Published: March 2005

Binding of the feedback inhibitor acetyl‐coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin‐labelled acetyl‐CoA analogue 3‐carboxy‐2,2,5,5‐tetramethylpyrrolidine‐1‐oxyl‐CoA‐thioester. The spin‐labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl‐CoA, while CoA has no effect. AMP and fructose 1, 6‐bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin‐labelled acetyl‐CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl‐CoA. The binding site for these activators seems to overlap with the acetyl‐CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8‐anilino‐1‐naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component.The spin‐labelled acetyl‐CoA analogue binds also to the product binding site of acetyl‐CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl‐CoA as well as by CoA.
Permissive action of thyroid hormones in the cAMP‐mediated induction of phosphoenol pyruvate carboxykinase in hepatocytes in culture
Authors: SÜSSMUTH Werner , HÖPPNER Wolfgang , SEITZ Hans J. , LUDA Dagmar , HARNEIT Angelika 
Published: March 2005

Thyroid hormones are known to stimulate in vivo the synthesis of several liver proteins. In order to determine their role in the regulation of important hepatic proteins at the cell level, the effect of T3 on the induction of the glucoregulatory enzyme phosphoenol pyruvate carboxykinase (PEPck) was investigated in cultured hepatocytes. The cells were isolated from adult hypothyroid rats and exposed to a chemically defined medium, devoid of serum supplement and hormone‐free over a period of 48 h.(a) Addition of T3 to the medium had only a minor effect on PEPck induction, (b) Dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP) or epinephrine provoked significant increase in PEPck synthesis within 4–6h. (c) Simultaneous addition of T3 was found significantly to promote the Bt2cAMP‐mediated enzyme induction, (d) Physiological concentrations of T3 (10 pM to 1 nM) were found to be effective in enhancing this cAMP‐mediated signal, (e) Exposure of the cells to T3 for only 90 min, 16 h prior to addition of Bt2cAMP was nearly as effective as continuous exposure to T3 in enhancing the cAMP‐effect on PEPck synthesis, (f) In contrast, no effect of T3 on the Bt2cAMP‐induced tyrosine‐aminotransferase activity could be detected.It is concluded that the role of T3 on the cAMP‐elicited PEPck synthesis is selective, rapid, primarily permissive and significant within the physiological range of the hormone. In addition, these data indicate that such hepatocyte cultures prepared from hypothyroid rats are ideally suited to the analysis of the regulatory role of thyroid hormones on specific gene expression.
Primary structure of Vicia angustifolia proteinase inhibitor
Authors: SHIMOKAWA Yukiko , KUROMIZU Kenji , ARAKI Tomiko , OHATA Junko , ABE Okitoshi 
Published: March 2005

The complete amino acid sequence (72 amino acid residues) of a double‐headed proteinase inhibitor from seeds of Vicia angustifolia L. var. segetalis Koch has been determined and compared with those of other double‐headed inhibitors of known structure. Sequencing was performed by conventional methods with the aid of the fragments produced by reduction and S‐carboxymethylation of the enzymatically modified inhibitors, and also using tryptic and chymotryptic peptides.The positions of the 14 half‐cystine residues agreed among all the reported primary structures of the legume double‐headed inhibitors. However, V. angustifolia inhibitor possessed extensive amino acid differences compared to the others. The phylogenetic relationship among these inhibitors was established using the unweighted pair‐group method and revealed that the V. angustifolia inhibitor and the peanut inhibitor B‐III had diverged at a relatively earlier stage compared to the other inhibitors.
Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes
Authors: BELLOMO Giorgio , NICOTERA Pierluigi , ORRENIUS Sten 
Published: March 2005

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert‐butyl hydroperoxide metabolism, prevents the ATP‐dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3‐hydroxybutyrate in the medium, prevents this hydroperoxide‐induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum.Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+‐extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.
Studies on the nature of transition‐metal‐ion‐mediated binding of triazine dyes to enzymes
Authors: HUGHES Peter , SHERWOOD Roger F. , LOWE Christopher R. 
Published: March 2005

Several reactive azoic dichlorotriazinyl dyes specifically and irreversibly inactivate the folate‐degrading enzyme carboxypeptidase G‐2 at a site competitive with the enzyme substrates methotrexate (4‐amino‐N10‐methylfolic acid) and p‐aminobenzoyl‐l‐glutamate. Although the less reactive monochlorotriazinyl dye, Procion red H‐8BN, is unable to inactivate the enzyme, it is capable of marked inhibition of inactivation by dichlorotriazinyl dyes in the presence of Zn2+. Zinc ions and, to a lesser extent other first row transition metal ions, significantly enhance the affinity of Procion red H‐8BN and its analogues Procion red MX‐8B and Procion red MX‐2B, for carboxypeptidase G‐2. It is proposed that this effect is mediated through the formation of a specific tetracoordinate Zn2+ complex between the azo linkage and adjacent sulphonate and hydroxyl functions of the dye and an appropriate ligand on the protein.Carboxypeptidase G‐2 quantitatively inactivated with the dichlorotriazinyl dye, Procion red MX‐8B, contains approximately 1 mol dye/mol subunit of Mr 42000. Proteolytic cleavage of the labelled enzyme and resolution of the peptides by reverse‐phase high‐performance liquid chromatography yields a principal red peptide which on amino acid sequence analysis results in the identification of the dye binding domain. The affinity label, Procion red MX‐8B, is believed to be attached to the hydroxylic side chain of Thr‐279.
Na+‐dependent regulation of extramitochondrial Ca2+ by rat‐liver mitochondria
Authors: NEDERGAARD Jan 
Published: March 2005

The presence and significance of Na+‐induced Ca2+ release from rat liver mitochondria was investigated by the arsenazo technique.Under the experimental conditions used, the mitochondria, as expected, avidly extracted Ca2+ from the medium. However, when the uptake pathway was blocked with ruthenium red, only a small rate of ‘basal’ release of Ca2+ was seen (0.3 nmol Ca2+× min−1× mg−1), in marked contrast to earlier reports on a rapid loss of sequestered Ca2+ from rat liver mitochondria.The addition of Na+ in ‘cytosolic’ levels (20 mM) led to an increase in the release rate by about 1 nmol Ca2+× min−1× mg−1. This effect was specific for Na+.The significance of this Na+‐induced Ca2+ release, in relation to the Ca2+ uptake mechanism, was investigated (in the absence of uptake inhibitors) by following the change in the extramitochondrial Ca2+ steady‐state level (set point) induced by Na+. A five‐fold increase in this level, from less than 0.2 μM to more than 1 μM, was induced by less than 20 mM Na+. The presence of K+ increased the sensitivity of the Ca2+ homeostat to Na+. The effect of Na+ on the extramitochondrial level was equally well observed in an K+/organic‐anion buffer as in a sucrose buffer.Liver mitochondria incubated under these circumstances actively counteracted a Ca2+ or EGTA challenge by taking up or releasing Ca2+, so that the initial level, as well as the Na+‐controlled level, was regained.It was concluded that liver mitochondria should be considered Na+ ‐sensitive, that the capacity of the Na+ ‐induced efflux pathway was of sufficient magnitude to enable it to influence the extramitochondrial Ca2+ level biochemically and probably also physiologically, and that the mitochondria have the potential to act as active, Na+ ‐dependent regulators of extramitochondrial (‘cytosolic’) Ca2+. It is suggested that changes of cytosolic Na+ could be a mediator between certain hormonal signals (notably α1‐adrenergic) and changes in this extramitochondrial (‘cytosolic’) Ca2+ steady state level.
The refined 2.2‐Å (0.22‐nm) X‐ray crystal structure of the ternary complex formed by bovine trypsinogen, valine‐valine and the Arg15 analogue of bovine pancreatic trypsin inhibitor
Authors: BODE Wolfram , WALTER Jochen , HUBER Robert , WENZEL Herbert R. , TSCHESCHE Harald 
Published: March 2005

Large orthorhombic crystals of the complex formed by bovine trypsinogen and a semisynthetic homologous bovine pancreatic trypsin inhibitor with the reactive‐site lysine residue replaced by an arginine residue ([Arg15]PTI) have been obtained which are isomorphous with the crystals of PTI‐trypsinogen [Bode, W., Schwager, P. and Huber, R. (1978) J. Mol. Biol. 118, 99–112]. The X‐ray crystal structure of the ternary complex of trypsinogen‐[Arg15]PTI with the dipeptide Val‐Val has been determined by X‐ray data to 2.2‐Å (0.22‐nm) resolution by means of difference Fourier methods and has been crystallographically refined to a final R‐value of 0.17.Replacement of the reactive‐site Lys15 by an arginine residue is accompanied in the complex by small movements of polar side groups of trypsin and enclosed solvent molecules within the specificity pocket. Only solvent molecule 414 OH which mediates the hydrogen bond interactions between Lys15 NZ and Asp189carboxylate is expelled, thus allowing the bulkier guanidyl group to approach this carboxylate. The dipeptide Val‐Val binds in the packet accepting the Ile‐Val N‐terminus in trypsin. The cavity left by the CD‐methyl group of He16 upon replacement by a valine residue is only partially filled by slight rearrangements of neighbouring peptide side chains. Part of the positive free energy change observed upon replacement of Ile‐Val may allow for the maintenance of this cavity.
Macromolecular syntheses in the cell cycle mutant cdc25 of budding yeast
Authors: MARTEGANI Enzo , VANONI Marco , BARONI Maurizio 
Published: March 2005

A major control point of the cell cycle in Saccharomyces cerevisiae is a G1 event called ‘start’. At start a yeast cell integrates external and internal signals and decides to progress toward mitosis or to choose alternative pathways such as sporulation, conjugation etc. cdc25 is a class II temperature‐sensitive start mutant that blocks at restrictive temperature in G1 as round unbudded cells.The arrest of the cell cycle appears to be independent of the carbon and nitrogen sources, and the cell wall of cdc25‐arrested cells shows changes similar to those found in cells undergoing entry in to the stationary phase.After a shift to 36 °C the increase in cell number of cdc25 cultures is gradually inhibited. The nuclear division cycle appears to be inhibited immediately after the shift and the percentage of budded cells decreases, while cytoplasmic growth, monitored either as increase of adsorbance at 450 nm or as protein accumulation, continues for many hours leading to a progressive increase of mean cell volume and mean protein content per cell.The stable RNA accumulation instead is immediately inhibited and this is partially due to a 50% inhibition of ribosomal RNA synthesis, while the rate of synthesis of ds‐killer RNA is relatively unaffected.These data suggest that the CDC25 gene product could be a part of a mechanism that leads yeast cells to choose between the progression towards DNA replication and cell division or to enter into the stationary phase. This mechanism appears to turn off both rRNA accumulation and cell‐cycle progression and to activate differentiative pathways in response to environmental restriction.
Nucleoside diphosphate kinase from brain
Authors: HUITOREL Philippe , SIMON Colette , PANTALONI Dominique 
Published: March 2005

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP.We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100000 in its native state, and 17000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping‐pong bi‐bi mechanism.The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900‐fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically.Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditons. This result demonstrates that the tubulin‐GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.