bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Mg2+ in the structure and activity of maize (Zea mays L.) isocitrate lyase: indications for hysteretic behaviour
Authors: BEECKMANS Sonia , KHAN A. Salam , VAN DRIESSCHE Edilbert 
Published: August 2015

The role of Mg2+ in the structure and activity of maize isocitrate lyase has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal concentrations. From CD and trypsinolysis experiments, the existence of high-affinity binding sites for Mg2+ was demonstrated, and a KdME of 200 μM was determined. Both free enzyme (E) and enzyme molecules with high-affinity sites occupied (ME) are catalytically competent, the former showing 40% of the activity of the latter. Mg2+ thus acts as a non-essential activator. A second Mg2+-binding site with a KdMEM of 6 mM was revealed from protection experiments by increasing Mg2+ concentrations against inactivation. From activity measurements at different Mg2+ concentrations, the affinity of the enzyme for the Mg2+–isocitrate complex (MI) was determined to be KdE(MI) = 9 μM. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affinity binding sites with Mg2+ induces a conformational change in the high-affinity binding sites resulting in an even higher affinity for Mg2+ (KdME* = 40 μM). On lowering the Mg2+ concentration again, the enzyme only responds slowly: the time needed for all enzyme molecules to return to the conformation at which KdME is 200 μM was found to be 60 min. Finally it was shown that the high-affinity binding site for Mg2+ is not formed at low (4 °C) temperature.
Hydroxylated naphthoquinones as substrates for Escherichia coli anaerobic reductases
Authors: ROTHERY Richard A. , CHATTERJEE Indraneal , KIEMA Gregory , McDERMOTT Mark T. , WEINER Joel H. 
Published: August 2015

We have used two hydroxylated naphthoquinol menaquinol analogues, reduced plumbagin (PBH2, 5-hydroxy-2-methyl-1,4-naphthoquinol) and reduced lapachol [LPCH2, 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol], as substrates for Escherichia colianaerobic reductases. These compounds have optical, solubility and redox properties that make them suitable for use in studies of the enzymology of menaquinol oxidation. Oxidized plumbagin and oxidized lapachol have well resolved absorbances at 419 nm (ε = 3.95 mM-1·cm-1) and 481 nm (ε = 2.66 mM-1·cm-1) respectively (in Mops/KOH buffer, pH 7.0). PBH2 is a good substrate for nitrate reductase A (Km = 282±28 µM, kcat = 120±6 s-1) and fumarate reductase (Km = 155±24 µM, kcat = 30±2 s-1), but not for DMSO reductase. LPCH2 is a good substrate for nitrate reductase A (Km = 57±35 µM, kcat = 68±13 s-1), fumarate reductase (Km = 85±27 µM, kcat = 74±6 s-1) and DMSO reductase (Km = 238±30 µM, kcat = 191±21 s-1). The sensitivity of enzymic LPCH2 and PBH2 oxidation to 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition is consistent with their oxidation occurring at sites of physiological quinol binding.
Overexpression of myristoylated alanine-rich C-kinase substrate enhances activation of phospholipase D by protein kinase C in SK-N-MC human neuroblastoma cells
Authors: MORASH Sherry C. , ROSÉ Sergio D. , BYERS David M. , RIDGWAY Neale D. , COOK Harold W. 
Published: August 2015

Signal transduction can involve the activation of protein kinase C (PKC) and the subsequent phosphorylation of protein substrates, including myristoylated alanine-rich C kinase substrate (MARCKS). Previously we showed that stimulation of phosphatidylcholine (PtdCho) synthesis by PMA in SK-N-MC human neuroblastoma cells required overexpression of MARCKS, whereas PKCα alone was insufficient. We have now investigated the role of MARCKS in PMA-stimulated PtdCho hydrolysis by phospholipase D (PLD). Overexpression of MARCKS enhanced PLD activity 1.3–2.5-fold compared with vector controls in unstimulated cells, and 3–4-fold in cells stimulated with 100 nM PMA. PMA-stimulated PLD activity was blocked by the PKC inhibitor bisindolylmaleimide. Activation of PLD by PMA was linear with time to 60 min, whereas stimulation of PtdCho synthesis by PMA in clones overexpressing MARCKS was observed after a 15 min time lag, suggesting that the hydrolysis of PtdCho by PLD preceded synthesis. The formation of phosphatidylbutanol by PLD was greatest when PtdCho was the predominantly labelled phospholipid, indicating that PtdCho was the preferred, but not the only, phospholipid substrate for PLD. Cells overexpressing MARCKS had 2-fold higher levels of PKCα than in vector control cells analysed by Western blot analysis; levels of PKCβ and PLD were similar in all clones. The loss of both MARCKS and PKCα expression at higher subcultures of the clones was paralleled by the loss of stimulation of PLD activity and PtdCho synthesis by PMA. Our results show that MARCKS is an essential link in the PKC-mediated activation of PtdCho-specific PLD in these cells and that the stimulation of PtdCho synthesis by PMA is a secondary response.
Identification and purification of the reconstitutively active glutamine carrier from rat kidney mitochondria
Authors: INDIVERI Cesare , ABRUZZO Giuseppe , STIPANI Italo , PALMIERI Ferdinando 
Published: August 2015

The glutamine carrier from rat kidney mitochondria, solubilized in dodecyl octaoxyethylene ether (C12E8) and partly purified on hydroxyapatite, was identified and completely purified by Celite chromatography. On SDS/PAGE, the purified glutamine carrier consisted of a single protein band with an apparent molecular mass of 41.5 kDa. When reconstituted into liposomes, the glutamine carrier catalysed both the unidirectional flux of glutamine and the glutamine/glutamine countertransport, which were completely inhibitable by a mixture of pyridoxal 5´-phosphate and N-ethylmaleimide. The carrier protein was purified 474-fold with a recovery of 58% and a protein yield of 0.12% with respect to the mitochondrial extract. The glutamine carrier-mediated transport is quite specific for l-glutamine. l-Asparagine is the only other amino acid that is efficiently transported by the reconstituted carrier protein. d-Glutamine, l-glutamate and l-aspartate are very poor substrates. The transport activity was inhibited by several thiol-group and amino-group reagents.
Cloning of the gene and cDNA for hamster chymase 2, and expression of chymase 1, chymase 2 and angiotensin-converting enzyme in the terminal stage of cardiomyopathic hearts
Authors: SHIOTA Naotaka , FUKAMIZU Akiyoshi , OKUNISHI Hideki , TAKAI Shinji , MURAKAMI Kazuo , MIYAZAKI Mizuo 
Published: August 2015

Chymase is responsible for the formation of angiotensin II, which plays crucial roles in the pathogenesis of cardiovascular diseases. In the present study we determined the gene organization of a novel hamster chymase (hamster chymase 2) and analysed the expression of chymase 1, chymase 2 and angiotensin-converting enzyme (ACE) in hamster hearts at the terminal stage of cardiomyopathy. The gene encoding hamster chymase 2 is 3.2 kb in length and has five exons and four intervening sequences. The overall organization of this gene is similar to that of several other serine proteases. The deduced amino acid sequence revealed the existence of a preproenzyme composed of a signal peptide with 19 amino acids, a propeptide with two amino acids and a catalytic domain with 226 amino acids. The predicted full sequence of the catalytic domain was revealed to be very similar to the sequences of mouse mast-cell protease 5 (86%), rat mast-cell protease III (85%) and human chymase (70%) and less similar to hamster chymase 1 (56%). The expression of chymase 1 in heart was higher than that of chymase 2. The cardiac chymase-like activity, as well as the mRNA levels of chymase 1 and 2 of BIO 14.6 cardiomyopathic hamsters at the age of 60 weeks were increased 3.4-, 2.8- and 5.1-fold respectively compared with age-matched BIO F1B control hamsters. The cardiac ACE activity and the ACE mRNA level of cardiomyopathic hamsters were also increased 4.1- and 2.4-fold compared with those of age-matched controls. These results suggest that up-regulation of both ACE and chymases participates in the pathophysiology of the terminal stage of cardiomyopathy.
The nucleotide sequence data reported will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AB007622.
Biosynthesis of mucins in bovine trachea: identification of the major radiolabelled species
Authors: SVITACHEVA Naila , HOVENBERG Hans W. , DAVIES Julia R. 
Published: August 2015

Bovine trachea in organ culture secretes mucus containing a ‘high-density ’ (1.46 g/ml) and a ‘low-density ’ (1.37 g/ml) mucin similar to those identified previously in bovine respiratory secretions [Hovenberg, Carlstedt and Davies (1997) Biochem. J. 321, 117–123]. After pulse-labelling, autoradiography showed uptake of [35S]sulphate by both epithelial goblet cells and submucosal glands, while [3H]proline was mainly incorporated into the ciliated surface epithelial cells. After 24 h of radiolabelling, neither the high- nor the low-density mucin in the secreted mucus gel was heavily radiolabelled with the precursors. In contrast, a population of molecules banding at 1.50 g/ml was heavily radiolabelled with [35S]sulphate. This component was smaller than the high-density mucin from the mucus gel and was insensitive to reduction or digestion with chondroitin ABC lyase or heparan sulphate lyase. The molecules yielded two populations of high-Mr glycopeptides upon trypsin digestion, were sensitive to keratanase and endo-β-galactosidase digestion and contained O-linked glycans. Extracts of the surface epithelium and submucosal tissue after radiolabelling showed that the high- and low-density mucins in the tissue were also poorly radiolabelled. Thus, under these conditions, the radiolabelled precursors were not effectively incorporated into the large oligomeric mucins but into a high-Mr monomeric species. This study suggests that data obtained in investigations where mucins are radiolabelled and studied without further separation into distinct components may rather reflect the turnover of this ‘novel ’ monomeric species than the large oligomeric mucins.
Evaluation of the stoichiometry and energetics of carbohydrate binding to Ricinus communis agglutinin: a calorimetric study
Authors: SHARMA Shalini , BHARADWAJ Satish , SUROLIA Avadhesha , PODDER Sunil Kumar 
Published: August 2015

High-sensitivity isothermal titration calorimetry has been used to investigate the thermodynamics of binding of Ricinus communis agglutinin to galactose, lactose and their derivatives in the temperature range 280.5–298 K. The present study unequivocally establishes the carbohydrate-binding stoichiometry of the tetrameric agglutinin from castor bean as two, i.e. the (As–sB)2-type tetramer of the agglutinin has two equivalent sites that are non-interacting and independent. The site binding constants range from 2.2×103 M-1 at 282 K for galactose to 4.84×104 M-1 at 281 K for N-acetyl-lactosamine. The binding enthalpies range from -21.9 kJ·mol-1 at 293 K for 4-methylumbelliferyl-β-galactoside to -50.2 kJ·mol-1 at 292.9 K for thiodigalactoside. The observation of limited entropy–enthalpy compensation for binding of the sugars to the lectin indicates that reorganization of water molecules plays an important role in binding. As the slope of the compensation plot is greater than unity, the reactions are largely enthalpically driven. These studies show that the stronger binding of N-acetyl-lactosamine than lactose is due to a favourable interaction between the acetamido group of the reducing-end N-acetylglucosamine of the former and the corresponding loci in the agglutinin molecule. Preferential binding of methyl-β-galactoside over methyl-α-galactoside also indicates the apolar nature of the interaction with the methyl group of the former sugar.
Expression and phosphorylation of fibroblast-growth-factor-inducible kinase (Fnk) during cell-cycle progression
Authors: CHASE Dan , FENG Yang , HANSHEW Brian , WINKLES Jeffrey A. , LONGO Dan L. , FERRIS Douglas K. 
Published: August 2015

Fnk is a member of the polo family of cell-cycle-regulated serine/threonine kinases. We report here that it is present in serum-starved quiescent cells and that mitogenic stimulation of quiescent cells with calf serum results in the modification of a significant fraction of the Fnk pool. This modification results in a slower migrating form when analysed by SDS/PAGE. The modification is transient and by 9 h after stimulation all of the Fnk is again present as the faster migrating form. We also show that the Fnk protein increases in abundance as cells progress from G1 to mitosis and is post-translationally modified as cells enter and exit mitosis. The Fnk modification is again manifested as a slower migrating species by SDS/PAGE and is due to phosphorylation of the protein. The mitotic-specific phosphorylation of Fnk correlates with an increase in its kinase activity, and this activity is dramatically reduced by phosphatase treatment of mitotic Fnk immunoprecipitates. During the later stages of mitosis, Fnk is dephosphorylated such that, by the time the cells enter G1, it is all present as the dephosphorylated form. These results suggest that Fnk has two functions, one during the entry of cells into the cell cycle and a second during mitosis of cycling cells.
Precise determination of RNA–protein contact sites in the 50 S ribosomal subunit of Escherichia coli
Authors: THIEDE Bernd , URLAUB Henning , NEUBAUER Helga , GRELLE Gerlinde , WITTMANN-LIEBOLD Brigitte 
Published: August 2015

RNA–protein cross-linked complexes were isolated and purified to obtain precise data about RNA–protein contact sites in the 50 S ribosomal subunit of Escherichia coli. N-terminal microsequencing and matrix-assisted laser desorption ionization MS were used to identify the cross-linking sites at the amino acid and nucleotide levels. In this manner the following contact sites of five ribosomal proteins with the 23 S rRNA were established: Lys-67 of L2 to U-1963, Tyr-35 of L4 to U-615, Lys-97 of L21 to U-546, Lys-49 of L23 to U-139 or C-140 and Lys-71 and Lys-74 of L27 to U-2334.
Microsomal epoxide hydrolase of rat liver is a subunit of theanti-oestrogen-binding site
Authors: MÉSANGE Fabienne , SEBBAR Mohamed , KEDJOUAR Blandine , CAPDEVIELLE Joël , GUILLEMOT Jean-Claude , FERRARA Pascual , BAYARD Francis , DELARUE Frédéric , FAYE Jean-Charles , POIROT Marc 
Published: August 2015

A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039–17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.
Luminal Ca2+ regulates passive Ca2+ efflux from the intracellular stores of hepatocytes
Authors: BEECROFT Mike D. , TAYLOR Colin W. 
Published: August 2015

Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either ionomycin or thapsigargin, although both were required for complete inhibition. Unidirectional efflux of 45Ca2+ after removal of ATP from cells loaded to steady state (1.60±0.12 nmol/106 cells) was monoexponential and occurred with a half-time of 103±10 s. However, the 45Ca2+ content of the stores did not return to their pre-ATP level, but reached a plateau at 0.12±0.04 nmol/106 cells. A similar amount of Ca2+ was trapped within the stores when Ca2+ uptake was prevented by thapsigargin and chelation of Ca2+; at all temperatures between 2 °C and 37 °C; and after stores had first been loaded with unlabelled Ca2+. Simultaneous addition of inositol 1,4,5-trisphosphate (InsP3) and inhibition of Ca2+ uptake reduced the amount of trapped Ca2+ to a level consistent with InsP3 rapidly and more completely emptying a fraction of the stores that could be only partially emptied by the passive leak. After dilution of the specific activity of the 45Ca2+ under conditions that maintained the steady-state activities of the pumps and leaks, the stores rapidly lost their entire 45Ca2+ content. We conclude that the channel responsible for mediating the leak of Ca2+ abruptly closes when the luminal [Ca2+] of the intracellular stores falls below a critical threshold corresponding to about 7% of their steady-state loading. Whereas InsP3 is capable of completely emptying a fraction of the stores, regulation of the passive leak by luminal [Ca2+] is likely to prevent it from completely emptying them; such regulation may ensure that the many other functions of Ca2+ within the endoplasmic reticulum are not compromised.
Synthesis in Escherichia coli of two smaller enzymically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene
Authors: MO Yin-yuan , SESHU J. , WANG Dong , MALLAVIA Louis P. 
Published: August 2015

FK506-binding proteins (FKBPs) have been identified in a variety of eukaryotic and prokaryotic organisms. Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Coxiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (PPIase) activity. Further characterization of the cbmip gene has identified two additional proteins with molecular masses of 15.5 and 15.0 kDa that are synthesized, in addition to the 23.5 kDa CbMip, when expressed in Escherichia coli. Amino acid sequencing at the N-terminus combined with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation. When the internal methionines serving as start sites were replaced with lysine by site-directed mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was abolished even though the synthesis of 23.5 kDa CbMip was intact. This confirmed that the 15.5 and 15.0 kDa proteins are indeed generated by translational reinitiation and are not degradation products of the 23.5 kDa protein. Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit PPIase activity. Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15.0 kDa proteins are designated as C. burnetiiFKBP (Cb-FKBP) analogues I and II, respectively. TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indicating that structural variations could allow for different subcellular compartmentalization of similar proteins. Western-blot analysis of lysates of purified C. burnetii using a CbMip-specific monoclonal antibody revealed the presence of a protein migrating at ≈ 15 kDa, indicating the presence of smaller Cb-FKBP analogue(s) in C. burnetii, although at much lower levels compared with 23.5 kDa CbMip. This unique gene organization seen with cbmip may provide the organism with a mechanism of efficient use of its limited genetic information to synthesize proteins that are structurally different yet functionally similar.
Cobalamin (vitamin B12) biosynthesis: identification and characterization of a Bacillus megaterium cobI operon
Authors: RAUX Evelyne , LANOIS Anne , WARREN Martin J. , RAMBACH Alain , THERMES Claude 
Published: August 2015

A 16 kb DNA fragment has been isolated from a Bacillus megaterium genomic library and fully sequenced. The fragment contains 15 open reading frames, 14 of which are thought to constitute a B. megaterium cobalamin biosynthetic (cob) operon. Within the operon, 11 genes display similarity to previously identified Salmonella typhimurium cobalamin biosynthetic genes (cbiH60, -J, -C, -D, -ET, -L, -F, -G, -A, cysGA and btuR), whereas three do not (cbiW, -X and -Y). The genes of the B. megaterium cob operon were compared with the cobalamin biosynthetic genes of Pseudomonas denitrificans, Methanococcus jannaschii and Synechocystis sp. Taking into account the presence of cbiD and cbiG, the absence of a cobF, cobG and cobN, -S and -T, it was concluded that B. megaterium, M. jannaschii and Synechocystis sp., like S. typhimurium, synthesize cobalamin by an anaerobic pathway, in which cobalt is added at an early stage and molecular oxygen is not required.
Effect of nutritional state on the formation of a complex involving insulin receptor IRS-1, the 52 kDa Src homology/collagen protein (Shc) isoform and phosphatidylinositol 3′-kinase activity
Authors: DUPONT Joëlle , DEROUET Michel , SIMON Jean , TAOUIS Mohammed 
Published: August 2015

The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3´-kinase (PI 3´-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by αIRS-1(mostly the 46 kDa isoform). IR–Shc association was decreased by fasting and restored by refeeding. In liver, αShc immunoprecipitated the three forms of regulatory subunits of PI 3´-kinase and a PI 3´-kinase activity which was decreased by fasting. In muscle, αShc immunoprecipitated only the p85 isoform; the associated PI 3´-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (αIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3´-kinase and an equal PI 3´-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3´-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3´-kinase activity, is suggested.