bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polypeptide Synthesis in Toluene‐Treated Lymphocytes
Authors: BURRONE Oscar R. 
Published: March 2005

Normal human lymphocytes stimulated by phytohemagglutinin P, and other mammalian cells were rendered permeable to macromolecules such as poly(U) and proteins, by treatment with a low concentration of toluene. Under this condition, poly(U) translation was more efficient in the permeabilized cells than in 10000 ×g extracts. Such a process occurs inside the treated cells as demonstrated by the fact that [14C]uridine‐labelled ribosomes remain associated with the toluene‐treated lymphocytes even after incubation at 37 °C.A nuclease from Staphylococcus aureus was able to penetrate the permeabilized cells and to break the polysome‐bound endogenous messenger RNA. However, the protein‐synthesizing machinery inside the toluene‐treated lymphocytes was unaffected by the nuclease, as demonstrated by the unimpairement of polyphenylalanine synthesis when poly(U) was added after the preincubation with the enzyme. These results suggest that the toluene treatment can be considered as an important tool for the study of the synthesis of macromolecules and its regulation in eukaryotic cells.
Covalent Binding of an NAD Analogue to Liver Alcohol Dehydrogenase Resulting in an Enzyme‐Coenzyme Complex not Requiring Exogenous Coenzyme for Activity
Authors: MÅNSSON Mats‐Olle , LARSSON Per‐Olof , MOSBACH Klaus 
Published: March 2005

The NAD analogue, N6‐[N‐(6‐aminohexyl)carbamoylmethyl]‐NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide‐mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled.
The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3–1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess.
It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40000 cycles/h in a coupled substrate assay.
The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20000 times as high a concentration of coenzyme.
Dual Action of Double‐Stranded RNA in Inhibiting Protein Synthesis in Extracts of Interferon‐Treated Mouse L Cells
Authors: LEWIS John A. , FALCOFF Ernesto , FALCOFF Rebecca 
Published: March 2005

We have investigated the mechanism by which double‐stranded RNA (dsRNA) inhibits protein synthesis in cell‐free extracts prepared from mouse L cells which had been pre‐treated with purified interferon. Preincubation of such extracts for a determined period of time causes inactivation of tRNA species and hence an inhibition of protein synthesis with either Mengo RNA or poly(U,C) as mRNA. This inhibition can be overcome by addition of tRNA. We show that dsRNA inhibits the translation of Mengo RNA irrespective of the period of preincubation and that this inhibition is not overcome by tRNA. Translation of poly(U,C) is unaffected by dsRNA. Thus dsRNA does not act at the level of peptide chain elongation and does not cause inactivation of tRNA species, indicating that dsRNA inhibits protein synthesis in extracts of interferon‐treated cells by a mechanism which is different from that responsible for the inhibition observed in the absence of dsRNA.The addition of dsRNA to extracts prepared from interferon‐treated cells causes two modifications which are expected to inhibit protein synthesis. (a) An enhancement of the level of nuclease activity, causing Mengo [3H]RNA to be degraded more rapidly in interferon‐treated cell extracts than control extracts, even when association of Mengo RNA with ribosomes is inhibited by the addition of pactamycin. (b) An impairment in the formation of methionyl‐tRNA · 40‐S‐ribosomal‐subunit complexes. The reduced binding of methionyl‐tRNA to 40‐S subunits can be overcome by the addition of a crude initiation factor preparation suggesting that dsRNA causes inactivation of an endogenous initiation factor(s).In the early stages of translation of Mengo RNA (10–15 min) dsRNA causes a reduction in the incorporation of [35S]methionine into the N‐terminal position of polypeptides suggesting that initiation of protein synthesis is impaired, presumably as a result of the block of methionyl‐tRNA · 40‐S‐subunit complex formation. At a later stage (30–45 min) Mengo RNA is almost completely destroyed under the normal conditions of protein synthesis.
The Effect of Fatty Acids on the Regulation of Pyruvate Dehydrogenase in Perfused Rat Liver
Authors: SCHOLZ Roland , OLSON Merle S. , SCHWAB Andreas J. , SCHWABE Ursula , NOELL Christiane , BRAUN Wolfgang 
Published: March 2005

The effect of fatty acids on the rate of pyruvate decarboxylation was studied in perfused livers from fed rats. The production of 14CO2 from infused [1‐14C]pyruvate was employed as a monitor of the flux through the pyruvate dehydrogenase reaction. A correction for other decarboxylation reactions was made using kinetic analyses. Fatty acid (octanoate or oleate) infusion caused a stimulation of pyruvate decarboxylation at pyruvate concentrations in the perfusate below 1 mM (up to 3‐fold at 0.05 mM pyruvate) but decreased the rate to one‐third of control rates at pyruvate concentrations near 5 mM. These effects were half‐maximal at fatty acid concentrations below 0.1 mM. Infusion of 3‐hydroxybutyrate also caused a marked stimulation of pyruvate decarboxylation at low pyruvate concentrations.The data suggest that the mechanism by which fatty acids stimulate the flux through the pyruvate dehydrogenase reaction in perfused liver at low (limiting) pyruvate concentrations involves an acceleration of pyruvate transport into the mitochondrial compartment due to an exchange with acetoacetate. Furthermore, it is proposed that a relationship exists between ketogenesis and the regulation of pyruvate oxidation at pyruvate concentrations approximating conditions in vivo.
Cycloheximide Causes Increased Accumulation of Translatable mRNA for Tyrosine Aminotransferase and Tryptophan Oxygenase in Livers of Cortisol‐Treated Rats
Authors: HOFER Erhard , SEKERIS Constantin E. 
Published: March 2005

Messenger RNA activities for two cortisol‐inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated.Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five‐fold and two‐fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still severalfold higher than in control animals. Application of α‐amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs.The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.
Mechanistic Aspects of the Transfer of Nascent Periplasmic Proteins across the Cytoplasmic Membrane in Escherichia coli
Authors: PAGÈS Jean Marie , PIOVANT Michel , VARENNE Stanislas , LAZDUNSKI Claude 
Published: March 2005

Spheroplasts, prepared according to an efficient procedure which is described, secrete a smaller protein fraction of the total cell content than that secreted by intact cells. However, more proteins are found associated to the cell envelopes derived from these spheroplasts than to those derived from intact cells.This result was confirmed when we measured, by specific immunoprecipitation, the amount of secreted and unsecreted alkaline phosphatase. Whereas at any time, at 30 °C, in intact cells 99.6% of the alkaline phosphatase produced is secreted and found in the periplasm [see A. Torriani (1968) J. Bacteriol. 96, 1200–1207], only 40% was secreted by our spheroplast preparation. We suggest that partial depletion of outer‐membrane‐bound protease (or proteases), involved in processing of precursors of secreted protein, is responsible for the significant defect of secretion of precursors which then remain membrane‐bound. Such an experimental protocol has been carried out at various temperatures and the secretion of alkaline phosphatase was assayed. This secretion was found to slightly vary above 22 °C whereas it dramatically decreased below 22 °C. Practically no phosphatase was secreted at 13 °C. However, the synthesis of alkaline phosphatase was not differentially altered at low temperatures compared to total proteins. The inactive presumed precursor of this enzyme which was then not secreted was found associated to the cell envelope. Similar results have been observed in intact cells above 22°C where only the energy of protein synthesis (ΔE= 79 kJ), which causes the elongation of nascent chains, is used for protein secretion; below 22 °C, in the temperature range of the disorder‐order transition of the lipid paraffin chains associated to membrane, a ΔE of 125 kJ is required for secretion of alkaline phosphatase. A ΔE of 146 kJ was found for this process to occur in the same temperature range in spheroplasts. When oleate was incorporated into the membrane of strain K 1059, an unsaturated fatty acid auxotroph, the same result as with wild‐type strains was observed, but a decrease in secretion at high temperatures was induced by this modification of fatty acid composition of the membrane. When elaidate instead of oleate was incorporated into K1059 membrane, the fall in alkaline phosphatase secretion occurred at 33 °C instead of 22 °C, which indicates that the physical state of the lipid phase is really involved in the secretion process. A hypothetical model for secretion of nascent periplasmic proteins, based on the data reported, is presented.
Dependence upon pH of Steady‐State Parameters for the β‐Galactosidase‐Catalysed Hydrolyses of β‐D‐Galactopyranosyl Derivatives of Different Chemical Types
Authors: WITHERS Stephen G. , SINNOTT Michael L. , JULLIEN Magali , YON Jeanine M. , VIRATELLE Odile M. 
Published: March 2005

The effect of pH upon the β‐glactosidase‐catalyzed hydrolyses of aryl galactosides is essentially similar for each of the three steps of their hydrolysis. It differs markedly from that on the hydrolysis of galactosyl pyridinium salts; these proceed through a ‘non‐bottleneck’ pathway.While pH increase abolishes the rate of every step of the reaction for aryl galactosides, it favors the first step of hydrolysis of the galactosyl pyridinium salts, which supports the hypothesis that catalysis of these compounds originates largely in non‐covalent interactions.
Probable Internal Homology in Thyroglobulin Peptide Chain
Authors: MARRIQ Claudine , ROLLAND Marcel , STEIN Anne , LISSITZKY Serge 
Published: March 2005

The time course of digestion of porcine thyroglobulin with subtilopeptidase at an enzyme to substrate ratio of 1:200 was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis after reduction with dithiothreitol. Two fragments of Mr 155000and 135000 were formed which showed a precursor‐product relationship, the 135000‐Mr species accumulating between 1 and 4 h of digestion. After purification by elution from electrophoretic gels, these fragments were treeated by CNBr and analyzed by dodecylsulfat/polyacrylamide gel electrophoresis. Both mixtures of CNBr peptides showed a closely similar electrophoretic band pattern. These results suggest the presence in the thyroglobulin chain (Mr about 140000 associated throught the continuity of the chain to a less structured fragment of Mr about 20000.A 15000‐Mr peptide was purified by Sephadex G‐200 gel filtration from CNBr‐treated thyroglobulin. Its uniqueness was demonstrated by N‐terminal sequence analysis and by tryptic fingerprinting. Using densitometer recording of stained electrophoretic gels in sodium dodecylsulfate, the molar ratio of this peptide was estimated to be 2 mol/mol of 12‐S thyroglobulin chain.These results are consistent with a protein structure in which two domains of similar size and structure are linked to form a single chain. Thus, the possibility exists that the structural gene for thyroglobulin might have evolved from duplication and fusion of an half‐sized ancestral gene.
Polyamine‐Induced Actin Polymerization
Authors: ORIOL‐AUDIT Christine 
Published: March 2005

Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain lingth of the polyamine. Aming the biological polyamines tested, spermidine and spermine are the most efficient.The polymerization of actin can also be induced by the corresponding mono of diguanidine derivatives of these polyamines but monoamines of amino acids are inactive at the same concencentration.The transformation of actin from a globular form upon addition of spermidine is also demonstrated by the changes in the near‐ultraviolet circular dichroic spectrum of this protein.Moreover, the polyamine‐induced F‐actin exhibits the same properties as the salt‐induced F‐actin: it strongly activates the Mg2+‐ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.
Subcellular Metabolite Concentrations
Authors: SOBOLL Sibylle , SHOLZ Roland , HELDT Hans W. 
Published: March 2005

Mitochondrial and cytosolic contents of adenine nucleotides and phosphate were measured in perfused rat livers empolying a technique of fractionation of freeze‐fixated tissue in non‐aqueous solvents. From the subcellular contents the mitochondrial and cytosolic concentrations of ATP. ADP, AMP and phosphate and the phosphorylation potentials of the subcellular ATP systems were calculated. An attempt was made to elucidate the relationship between mitochnodrial and cytosolic adenine nucleoide systmes and the dependency on the metabolic state of the liver.The following results were obtaines.

Under all mtabolic conditions studied the mitochnodrial ATP/ADP ratios were considerably lower than the cytosolic ratios (mitochondria: 0.1–0.7); cytosol: 2–11).
The ATP/ADP ratios calculated from overall tissue contents refflect mainly the cytosolic rations.
An inverse relationship was found between mitochondrial and cytosilic ATP/ADP ratios, i.e. when the mitchondrial ratios tended to increase, the cytosolic ratios decrease and vice versa.
The phosphorylation potentials calculated from the subcellular concertratopms were higher in the cytosol that in the mitochondria. The potential difference varied between 11 and 3 kJ/mol in livers from fed and starved rats, respectively.
In the presence of mitochondrial inhibitors, i.e. amytal, dinitrophenol and carboxyatractyloside the potential difference between the subcellular ATP systems decreased predominantly due to an increase in the mitocnodrial ATP/ADP ratios.
A correlation between mitochondrial ATP/ADP ratios and the respiratory rates was not obsrved, but the subcellular ratios appeared to correlate with the rate of glycolysi. When the rate of lactate + pyruvate production was increased, the cytosolic ATP/ ADP ratios were increased, too, wheres the mitochondrial ratios tended to decrease.
The adenine nucleotides in the cytosol appear to be in near equilibrium catalysed by the adenylate kinase. In the mitochondria, the AMP concentration is much lowe than to be expected under equilibrium conditions.

These results were discussed with respect to rate control of processes involved in ATP generation, i.e. Oxidative phosphorylation, adenine nucleotide translocation and glycolysis.
Dissociation and Oxygen‐Binding Behaviour of β‐Hemocyanin from Helox pomatia
Authors: ZOLOLA Lello , KUIPER Harry A. , VECCHINI Paola , ANTONINI Eraldo , BRUNORI Maurizio 
Published: March 2005

The dissociation behaviour of Helix pomatiaβ‐ hemocyanin as influenced by pH, calcium‐iopn concentraion and ionic strength, and the oxygen vinging properiotes were investigated. The dissociation behaviour of β‐hemocyuanin and α‐hemocyanin differs distinctly in a number of properties: (a) they show a different response to high salt concentration, (b) the stabilizing influence of calcium ions on the whole molecules of β‐hemocyanin is smaller than that on α‐hemocyanin, (c) half molecules of β‐hemocyanina are stablized to a large extent in teh presence of calcium ions and (d) at low ionic strength no peak separation between one‐tenth and one‐twentieth molecules of β‐hemocyanin is observed in the ultracentrifuge, which indicates an equilibrium between the species.The oxygen binding behaviour of β‐hemocyanin shows the typical features of proteins with a large hnumber of binding sites. The Hill plot is characterized by a high value of the Hill coefficient (maxm nH=7), coupled to a low value for the interatrion free energy per site. The oxygen binding behaviour can bacisally be described by a mocdified tewo‐state allosteric model with the variation, imposed by teh expeimental data, taht KT is pH‐dependent (negative Bohr effect), while KR is pH‐indepenmdent. Regulation of the cooperativity by teh alloosteric effector H+ is mainly due to changes in the allosteric equilibrium constant.In order to describe the cooperativity as a function of pH, the minimum number of interacting binding sites has to be varied, indicating that the size of the ‘functional constellation’ is variavle. This finding may have important implications on the relationship between the so‐called ‘structural’ and ‘functional constellation’.A comparison fo the oxygen binding properties of β‐and α‐hemocyanin shows that these proteins are complementary in their behaviour, with respect to the responses to changes in H+ anmd Ca2+ concentrations. This may be of importance for the animal in order ito meet specific physiological and enviromental requirements.
Chemical Structure and Biochemical Activity of Cord Factor Analogs. A Comparative Study of Esters of Methyl Glcoside and Non‐Hydroxylated Fatty Acids
Authors: KATO Masahiko , TAMURA Toshihide , SILVE GAby , ASSELINEAU Jean 
Published: March 2005

6‐Monoesters of methyl α‐D‐glucopyranoside and the even‐numbered normal chain fatty acids C14toC22 or 2‐tetradecyloctadecanoic acid (DL‐deoxy‐corynomycolic acid) have been prepared. All the normal chain fatty acid esters were devoid of toxicity for mice at the dose of 2 mg per animal, whereas the branched chain ester (deoxy‐corynomycolate) exhibited a low, bnut characteristic, toxicity.Preincubation of rat liver mitochondria with aqueous suspensions of these synthetic esters gave the following results. The shorter chain (C14 to C20) esters affected the mitochondrial respiration and coupled phosphorylation in both succinate and NADH pathways at both coupling sites II and III. The same effects were obtained by using methyl 2‐hexad3ecanoyl‐α‐D‐glucopyranoside or 6‐hexadecanoyl‐l‐asorbic acid, showing that the position of the ester linkage on the sugar residue or the conformation of the sugar moiety are not essential. Deoxycorynomyucolate induced an impairmant of mitochondrial oxidative phosphoryl;ation mainly localized at couplilng site II. Methyl 6‐do‐cosanoyl and 2,6‐sisoxoanoyl α‐D‐glucopyranosides affected only coupling site II. Inbtroductionof a double bnonmd in the fattyacid chain (docos‐13‐cis‐enote) gave results similar to those obtained with shorter chain esters.Two classes of glucoside esrters can thus be distinguished: those with a shosrtchain fattyacid part (C14 to C20), devopid of toxicitgy and affectomg pxodfatove phosphorylzation at both coupling II and III;ester of the C32‐branched chain acid exhibniting toxicity in vivo and specific inhibnition of oxidative phosphorylation at coupling site IIin vitro. Mono and diesters of docosanoic acid have an intermediate position (no toxicity, but a specific effect on mitochondria)The absence of toxicity of the short cahin ester (C14 to C22)seems to be expplained by their neutralization bty serum albumin.
Regulation of Mammary Gland Metabolism
Authors: GREENBAUM A. Leslie , SALAM Aziza , SOCHOR Milena , MCLEAN Patricia 
Published: March 2005

A method for the opreparation of isolated mammary gland cells of fthe rat is described.
The procedure involves disaggregation of the tissue in a collagenase‐hyaluronidase mixture and subsequent purification of the heterogeneous population of cells bycentrifugation in discontinuous Ficoll‐400 gradients; the preparation takes 60 minutes. The yield of cells is approximagtely 14%.
The cell as prepared have high rates of metabolism and synthetic capacity and exhibitmetabolic characteristics comparable to intact tissue.
Measurements of the co9ntent of metabnolic intermediates show cells to have, and retain, outstandingly hjigh levels of ATP and to have an energycharge close tgo 0.9. Levels of other intermediatesapproximate to those found in the int6acttissu, The level of glycolytic intermedites kbelow the triose phosphate stage indicatethe highly aerobic state of the cells.
The patytern and scale of glucose utilization, measusred using specifically labelled glucose incorporation into 14CO2 and 14C‐labelled lipid production, approximates closelyhin isolated cells at 5 and 20mM glucose and in tissue slices at 20mM glucose concentration. Mammary gland slices incubated with 5 mM glucose have a considerablylower rate of metabolism. Isolated cells exhibnit a higher proportionate rate of glucose utilization by way of the pentose phosphate pathway.
The isolated cells are hormone responsive. Insulin increases the oxication of glucose by the pentose phosphate pathway and stimulates lipid synthesis. Addition ofprogesterone and cortisonein vitro (10μM) leads to a marked and rapid decreasein the rate of glucose oxisdation and conversion to lipid.
Purification of Aspartate Transcabamylase from Drosophila melanogaster
Authors: JARRY Bruno P. 
Published: March 2005

A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of DDrosophila melanogaster as part of a high‐molecular‐weight enzyme complex. The complex is shown to contain several polypeptides.An antiserum directed against the complex enzyme nihibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihyro‐orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220000‐molecular‐weight protein in addition to the polypeptides present in the complex.A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro‐orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high‐molecular‐weight complex of cultured cells.