bluuebunny/crossref_metadata_2025_split huggingface dataset:
A 13C Nuclear Magnetic Resonance Study of CO2/HCO−3 Exchange Catalyzed by Human Carbonic Anhydrase I
Authors: SIMONSSON Ingvar , JONSSON Bengt‐Harald , LINDSKOG Sven 
Published: March 2005

Rates of CO2/HCO−3 exchange, catalyzed by human carbonic anhydrase I (or B) at chemical equilibrium, were estimated from the nuclear magnetic resonance linewidths of 13C‐labeled substrates. The results show that the maximal exchange rate constant is independent of pH in the range 5.7–8.0, whereas the apparent substrate dissociation constant depends on pH. Exchange proceeds rapidly in the absence of added buffers, and the addition of buffers has negligible effects on exchange rates. Exchange is equally rapid with 1H2O or 2H2O as solvents. Chloride ions inhibit CO2/HCO−3 exchange competitively.The maximal exchange rates obtained with human carbonic anhydrase I are 50 times slower than those obtained with human isoenzyme II (or C). From a comparison of the exchange kinetics with the steady‐state kinetics of CO2 hydration and HCO−3 dehydration it is tentatively concluded that the transfer of H+ between active site and medium proceeds with rates of similar magnitudes in the two isoenzymes, whereas the central catalytic step, the interconversion of enzyme‐bound CO2 and HCO−3, is much slower in isoenzyme I than in isoenzyme II.
Control of Protein Synthesis in Cuboidal Rat Mammary Epithelial Cells in Culture
Authors: BARRACLOUGH Roger , DAWSON Karin J. , RUDLAND Philip S. 
Published: March 2005

The cuboidal rat mammary tumour cell line, Rama 25, spontaneously converts to elongated cells during culture. The polypeptides synthesised by the two morphological forms are compared by two‐dimensional polyacrylamide gel electrophoresis. In addition to major changes in the intermediate filament proteins, there are two abundant acidic polypeptides of molecular weights 9000 and 15 500, which are synthesised by the elongated cells but which are not detectable in the cuboidal cells.When mRNA preparations isolated from the cuboidal and elongated cells are translated in the reticulocyte lysate, the patterns of radioactive products resemble the patterns of polypeptides synthesised in the intact cells. Translation products corresponding to the 9000‐Mr and 15 500‐Mr polypeptides are synthesised when mRNA from elongated cells is translated, but are not synthesised when mRNA from cuboidal cells is translated. Evidence is presented that the appearance of these two proteins represents a change in gene expression between the two cell types and that control is exerted prior to mRNA translation.
Kinetics of Phosphatidylglycerol Synthesis in Isolated Membrane Vesicles from Escherichia coli Containing Different Amounts of Membrane‐Bound Phosphatidic Acid
Authors: DEMANT Erland J. F. 
Published: March 2005

Cytoplasmic membrane vesicles of Escherichia coli containing various amounts of phosphatidic acid ranging from 0.2% to more than 50% (mol/mol) of the total lipid has been prepared by de novo synthesis of phosphatidic acid in the isolated membranes from acyl‐CoA esters and sn‐glycerol 3‐phosphate. The kinetics of CTP‐inituted phosphatidylglycerol synthesis in the phosphatidic‐acid‐enriched membranes has been studied.Phosphatidic acid pools with high and low reactivity as substrate for phosphatidylglycerol synthesis were present in the membranes. The two pools were found identical with respect to fatty acid composition and content of molecular species. The rate of phosphatidylglycerol synthesis from the highly reactive phosphatidic acid pool was independent of the phosphatidic acid concentration in membranes containing from 0.2–30 (mol/mol) phosphatidic acid. Ca2+‐ions were found to inhibit the synthesis of phosphatidylglycerol.On the basis of the findings presented it is suggested that phosphatidic acid probably plays a minor role as a feed‐back modulator of sn‐glycerol 3‐phosphate acylation in E. coli, and that phosphatidylglycerol synthesis can occur at near maximal rate in growing cells.
Heterogeneity of Microtubule‐Associated Proteins and Brain Development
Authors: FRANCON Jacques , LENNON Ana Maria , FELLOUS Arlette , MARECK Alain , PIERRE Michel , NUNEZ Jacques 
Published: March 2005

Developmental changes in the composition of brain microtubule‐associated proteins have been studied in three species the rat and the mouse, which are characterized by post‐natal brain development, and the guinea‐pig, whose Drain is mature at birth.

At an adult stage, and whatever the species, two major microtubule‐associated proteins, which have been referred to MAP2 and τ, have been identified by polyacrylamide gel electrophoresis Rat τ is composed of four closely spaced bands; mouse τ contains only three components with one of them being present in higher proportion than the others; adult guinea‐pig τ is essentially present as a single band.
Microtubule‐associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the τ region at immature stages of development (fast τ and slow τ). However adult τ factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2.
Peptide mapping analysis of the purified T entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat T proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different.
The activities of young T proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast T and young slow T were purified and both show to be active in promoting pure tubulin polymerization.
These data demonstrate the existence of two types of heterogeneity of microtubule‐associated proteins: plurality of protein species at every stage of brain development and changes in composition ana activity dependent on development.
Structure‐Function Relations between Fatty Acid Oxidation and the Mitochondrial Inner‐Membrane — Matrix Region
Authors: OTTO David A. , ONTKO Joseph A. 
Published: March 2005

Decreasing the osmolarity of the incubation media for rat liver mitochondria, by lowering the KCl or sucrose content, greatly increased the oxidation of palmitate, palmitoyl‐CoA, palmitoyl‐(–)‐carnitine, octanoate and butyrate, but only slightly affected the oxidation of succinate, glutamate plus malate, and 3‐hydroxybutyrate. Kinetic studies indicated that the increased rate offatty acid oxidation commenced at the onset of incubation in the hypotonic media and remained relatively constant. This was accompanied by an increase in the 3‐hydroxy‐butyrate: acetoacetate ratio, a higher rate of oxygen consumption, an increase in the extramitochondrial adenine‐nucleotide phosphorylation‐state ratio, and a greater state of reduction of the respiratory chain cytochromes b. c, and oxidase. Incubation of mitochondria in the hypotonic media caused a rapid decrease in the intermembrane and intracristal spaces and concurrent expansion of the matrix, without changing the total volume of the mitochondria.These observations indicate that expansion of the inner‐membrane–matrix compartment of mitochondria in vitro specifically activates fatty acid oxidation at the site of β‐oxidation or at the site of its interaction with the respiratory chain, with a consequent increase in the states of reduction and phosphorylation of the mitochondria, Evidence is also presented which suggests that the activation of mitochondrial fatty acid oxidation by Ca2‐ is initiated by a Ca2+ ‐induced alteration of the structure of volume of the inner‐membrane–matrix region.
Transferrin Uptake by Cultured Rat Embryo Fibroblasts
Authors: OCTAVE Jean‐Noël , SCHNEIDER Yves‐Jacques , HOFFMANN Pierre , TROUET André , CRICHTON Robert R. 
Published: March 2005

The effect of different drugs which affect lysosomal function (chloroquine, methylamine) and endocytosis (colchicine) or of the iron chelators desferrioxamine B, rhodotorulic acid, bipyridyl, 2,3‐dihydroxybenzoic acid, isoniazid pyridoxal hydrazone on the uptake of 59Fe‐loaded 3H‐labelled transferrin by cultured rat embryo fibroblasts has been studied. All these substances, except isoniazid pyridoxal hydrazone reduce the uptake of iron by fibroblasts, without any significant effect on the uptake of [3H]transferrin, nor of its specific binding to the cells. Cell fractionation experiments indicate that in the presence of methylamine and chloroquine, most of the cell associated 3H label is no longer transferred into lysosomes and remains associated with subcellular compartments which have a 5'‐nucleotidase activity (a marker enzyme of plasma membrane and related structures such as endocytic vesicles).Short‐term kinetic experiments with fibroblasts incubated for 1 min at 37 °C with doubly labelled transferrin show that on reincubation in fresh medium almost all of the 59Fe initially bound remains associated with the cells whereas the 3H‐labelled transferrin is released with a t1/2 of about 1.5 min. In the presence of desferrioxamine B both 59Fe and transferrin, the former in part as ferrioxamine, are released from the cells with the same kinetics as is observed for transferrin in the control experiments. Similar studies with cells preincubated for 16 h with chloroquine or methylamine indicate that the release of transferrin from the cells is slowed down considerably. Chloroquine‐treated cells also release large amounts of 59Fe.These results are consistent with the model for iron release from transferrin proposed previously [1] involving receptor mediated endocytosis of transferrin followed by release of iron during a rapid passage of the transferrinreceptor complex in lysosomes and recycling of the complex to the plasma membrane where the iron‐depleted transferrin is released. The inhibition of iron uptake by iron chelators such as desferrioxamine is consistent with complexation of iron by the chelator subsequent to its release from transferrin inside the cells and transfer of the iron‐chelate into the extracellular medium.
Enhanced Breakdown of Arylsulfatase A in Multiple Sulfatase Deficiency
Authors: WAHEED Abdul , HASILIK Andrej , VON FIGURA Kurt 
Published: March 2005

Multiple sulfatase deficiency (mucosulfatidosis) is a lysosomal storage disorder characterized by the decrease in activities of all known sulfatases. To measure the apparent rate of synthesis and the half‐life of arylsulfatase A in multiple sulfatase deficiency, fibroblasts from patients with the disease and from controls were subjected to pulse‐chase labelling with radioactive amino acids. Arylsulfatase A and cathepsin D, a lysosomal enzyme that is not affected in multiple sulfatase deficiency, were isolated from cells and media by immunoprecipitation. The labelled polypeptides were separated by polyacrylamide gel electrophoresis, visualized by fluorography and quantified by liquid scintillation counting. Using single and double isotope techniques it was found that, as compared to cathepsin D, the apparent rate of synthesis of arylsulfatase A was 2–5‐times lower and the half‐ life 4–9‐times shorter in multiple sulfatase deficiency than in control fibroblasts. In multiple sulfatase deficiency fibroblasts the rates of endocytosis and the stabilities of endocytosed arylsulfatases A isolated from human urine and bovine testes were equal to those in metachromatic leucodystrophy fibroblasts.We postulate that in normal cells a gene product exists that affects the stability of sulfatases and that multiple sulfatase deficiency is due to a mutation in this gene.
Enzymic Synthesis of Li:gnin Precursors
Authors: SCHMID Günter , GRISEBACH Hans 
Published: March 2005

UDPglucose :coniferyl‐alcohol glucosyltransferase was isolated from cambial sap of spruce (Picea abies). An apparently homogeneous enzyme was obtained by a seven‐step procedure including dye‐ligand chromatography. The enzyme has an Mr, of about 50000 and consists of one polypeptide chain. Transferase activity is not influenced by metal ions.The enzyme shows a pronounced substrate specificity towards UDPglucose and coniferyl alcohol with Km values of respectively 220 μM and 250 μM. The only reaction product is coniferin (coniferyl alcohol 7‐O‐β‐d‐glucopyranoside). No formation of ‘isoconiferin’ (coniferyl alcohol 1 ‐O‐β‐d‐glucoside) was detected. The reversibility of the reaction was proved by formation of [3H]UDPglucose from [3H]UDP and coniferin in the presence of the transferase. The products UDP and coniferin inhibit the reaction noncompetitively. Product inhibition patterns are consistent with a mono‐iso‐ordered bibi mechanism involving two isomeric enzyme forms.
Local Anaesthetics Including Benzyl Alcohol Activate the Adenylate Cyclase in Trypanosoma brucei by a Calcium‐Dependent Mechanism
Authors: VOORHEIS H. Paul , MARTIN B. Richard 
Published: March 2005

The adenylate cyclase of Trypanosoma brucei is activated by local anaesthetics.
Activation of adenylate cyclase by the local anaesthetic, benzyl alcohol, requires Ca2+.
The kinetics of the presteady state reveal that the activation step occurs prior to and is separate from the catalytic step.
The activation step in the presence of Ca2+ can be stimulated some 200‐fold and is highly co‐operative with respect to benzyl alcohol concentration.
The mechanism by which local anaesthetics activate may be by exposing the Ca2+‐binding site of the catalytic step. Ca2+ receptor which is oriented towards the hydrophobic regions of the plasma membrane in the basal state.
The Distribution of Redox Equivalents in the Anaerobic Respiratory Chain of Paracoccus denitrificans
Authors: KUČERA Igor , DADÁK Vladimir , DOBRÝ Roman 
Published: March 2005

The branching of the electron flow to individual teminal acceptors NO3−, NO2− and N2O was investigated in denitrifying cells of Paracoccus denitrificans. It was found that the extent of electron flow to nitrate depends on the activity of electron flow through the terminal part of the respiratory chain comprising cytochrome c.NO2− and N2O either endogenously formed or exogenously added decreased the rate of nitrate reduction by oxidizing the respiratory chain via nitrite reductase and nitrous oxide reductase. The inhibitory effect of both intermediates of denitrification was released by antimycin. The rate of the first reaction of the sequence NO3−→ NO2−→ N2O → N2 is in the type of cells used under feedback redox control exerted by concentration of intermediates. This regulation can be designated as ‘inhibition by product via respiratory chain’.The reaction rate of nitrate denitrification could also be influenced by changing the portion of electron flow from substrate to cytochrome c by artificial electron acceptors and donors. By varying the concentration of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine in the presence of ascorbate, the desired redox state of cytochrome c and consequently various rates of nitrate reductase activity could be established. The basis of the enhancing effect of dithionite on the rate of nitrate reduction could also be explained by its functioning as an electron donor for the terminal part of respiratory chain.The quantitative evalution of the extent of electron flow revealed that both branches of the respiratory chain (to nitrate reductase and to cytochrome c) compete for a constant limited flow of redox equivalents supplied from dehydrogenases. A strong evidence exists that the same mechanism is implied in the inhibition of nitrate reduction brought about by oxygen.
Catalytically Active Monomer Forms of Immobilized Arginase
Authors: AGUIRRE Raúl , KASCHE Volker 
Published: March 2005

Native rat liver arginase was covalently coupled to Sepharose beads and the resulting matrix‐bound tetramer was subsequently dissociated by acid or EDTA treatment. The immobilized derivatives that remain in the matrix were renatured in the presence of the cofactor Mn2+ and analyzed in terms of recoverable activity before and after incubation with soluble enzyme. The activity of the immobilized enzyme was determined under conditions where it is directly proportional to the enzyme content. Both the acid‐treated and EDTA‐treated and renatured immobilized rat liver arginase have about one‐fourth the activity of the untreated immobilized enzyme. The activity of the renatured immobilized enzyme after reassociation with free enzyme was 85%(acid‐dissociated oligomer) and 60% (EDTA‐dissociated oligomer) of the activity of the initially immobilized oligomer. The acid‐dissociated and renatured immobilized arginase had a Km value (18 mM) that is approximately four times the value of the immobilized tetrameric enzyme (3.4 mM). After renaturation and reassociation with free enzyme the Km value of the immobilized enzyme was found to be equal to the value of the immobilized oligomer before dissociation. This indicates that the dissociated and renatured immobilized enzyme is monomeric. The Mn2+‐free buffer. The immobilized tetramer, on the other hand, does not lose Mn2+ after this procedure, and this ion seems to be bound even more tightly than in the case of the free tetramer. The reconstituted oligomer had a Km value approximating the value of the initially bound oligomer.From these results and binding curves we conclude that rat liver arginase oligomer can be dissociated into monomers, which are catalytically active after renaturation with cofactor (Mn2+) at neutral pH. Thus, for arginase the tertiary structure is sufficient for the expression of the function of this enzyme.The immobilized monomeric arginase has been used to purify crude arginase by subunit exchange (affinity) chromatography.
The Role of EF‐Tu in the Expression of tufA and tufB Genes
Authors: van der MEIDE Peter H. , VIJGENBOOM Erik , TALENS Anneke , BOSCH Leendert 
Published: March 2005

We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF‐Tu in Escherichia coli. To this aim we have ditermined the intracellular concentrations of EF‐TuA and EF‐TuB under varying growth conditions by an immunological assay in mutants of E. coli constructed for this purpose. The data show that in wild‐type cells the expression of tufA and tufB is regulated coordinately. This coordination is not restricted to steady‐state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF‐TuB from the cell (by insertion of bacteriophage Mu DNA into tufB) nor elevation of the intracellular EF‐TuB concentration (by transformation with plasmids harbouring tufB) has any effect on the expression of tufA. A specific single‐site mutation of tufA, however, rendering EF‐TuA resistant to the antibiotic kirromycin, disturbs the coordinate expression oftufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild‐type cells the EF‐Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF‐Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA‐tufB transcriptional unit as a template. They show that addition to this system of EF‐Tu in concentrations 2–5% of the endogenous amount results in strong inhibition of EF‐Tu synthesis.We hypothesize that EF‐Tu acts as an autogenous repressor, inhibiting tufB expression post‐transcriptionally.
Hyperproduction of Phosphate‐Binding Protein, phoS, and pre‐phoS Proteins in Escherichia coli Carrying a Cloned phoS Gene
Authors: MORITA Takashi , AMEMURA Mitsuko , MAKINO Kozo , SHINAGAWA Hideo , MAGOTA Koji , OTSUJI Nozomu , NAKATA Atsuo 
Published: March 2005

A large amount of phosphate‐binding protein, the phoS gene product, accumulated in the periplasmic space of the cells when an Escherichia coli strain carrying a multicopy plasmid containing a chromosomal fragment of the phoS‐phoT region (pSN507) was grown in a low‐phosphate medium. When the same strain carrying a plasmid containing only the phoS gene (pSN518 or pSN5182) was grown in low‐phosphate medium, phosphate‐binding protein accumulated in the periplasm, and in addition a larger protein accumulated in the non‐periplasmic fraction. The apparent Mr of this protein and the phosphate‐binding protein were 39000 and 35000 respectively, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This larger protein showed immunological cross‐reaction with the phosphate‐binding protein. The 39000‐Mr protein was also detected in cells carrying pSN507 when the proteins were pulse‐labeled with radioactive amino acids. From these findings, together with the fact that this protein is recovered from the membrane fraction, we conclude that this protein is an unsecreted precursor protein of the phosphate‐binding protein.Kinetics and regulation of accumulation of these proteins were studied. This system will be useful for preparation and purification of the precursor protein for biochemical studies in relation to the mechanism of protein secretion.
Protein Kinases of Rat Liver during Hepatocarcinogenesis Induced by an Azo Dye
Authors: WEBER Anne , COTTREAU Dominique , HENRY Joelle , PHAN DINH TUY Francoise , SKALA Henriette , KAHN Axel 
Published: March 2005

We studied phosphoproteins and protein kinases in livers from normal rats and rats fed a diet containing 3′‐methyl‐p‐dimethylaminoazobenzene, an azo‐dye carcinogen.Endogenous phosphophortein patterns obtained by incubation of soluble and insoluble cell fractions with [γ‐32P]ATP were different for livers from rats submitted to carcinogenic diet as soon as the 12th week of the regimen compared to the patterns of normal and regenerating liver.New Phosphorylated bands appeared during hepatocarcinogenesis in soluble as well as in particulate fractions. None of these bands was phosphorylatedon tyrosine residues and the global percentage of phosphotyrosine residues among phosphoproteins did not increase during the course of azo‐dye hepatocarcinogenesis.Electrophoresis of active enzymes showed, from the 12th week of carcinogenic diet, a new band identified as a cAMP‐dependent histone kinase. This form was absent in fetal liver and in regenerating liver following partial hepatectomy. It could be identified as a modified (or a new isozymic) form of protein kinase type II by its chromatographic properties and the nature of its photoaffinity‐labeled regulatory subunits. The ratio of the R1 to R2 regulatory subunits increased in the treated animals, but this phenomenon seemed to be due to cell proliferation rather than to malignancy as it was also observed in regenerating liver following partial hepatectomy. In hepatoma, the increase of the amount of R1 subunits was not associated with a concomitant increase of protein kinase type I activity, which seemed to indicate that, in these tissues, a pool of free R1 subunits could exist.Finally, combination of DEAE‐cellulose chromatography fractionation and cellulose acetate electrophoresis analysis allowed us to detect in hepatoma a phosvitin kinase band insensitive to cyclic nucleotides and heparin, inactive on casein, which was absent in normal liver. Hepatoma extracts also contained fast‐migrating caseinphosvitin kinase forms which could correspond to modified casein kinase II.