bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcription in vitro Using Mixtures of Ethylene Glycol and Water
Authors: Brody Edward N. , Leautey Josette 
Published: March 2005

We have analyzed transcription in vitro in mixtures of ethylene glycol and water. We find two major effects of this solvent on T4 transcription, both of which are reversible on dilution out of the solvent: (a) only very short (10% normal length) T4 chains are formed; (b) a small percentage of initiation errors are made; these include initiation on the “wrong” strand and initiation on the “wrong” site of the correct strand. The ratio of GTP to ATP initiation is also increased in this solvent and it may be that all faulty initiation is done with GTP. This faulty initiation seems to derive, Like normal initiation, from a σ‐dependent reaction on native T4 DNA templates, and is thus distinguished from that faulty initiation which derives from core synthesis on damaged or denatured templates. Analysis of the products of T4 transcription in vitro in ethylene glycol suggests that this reagent has a different effect on RNA chains synthesized from promoter sequences than it does on chains initiated from non‐promoter sequences. Those chains which start at promoters show a very strong asymmetry, while those chains which start from non‐promoter sequences are completely symmetrical despite their σ‐dependence.Transcription of poly(dG) · poly(dC) and poly(dA) · poly(dT) is relatively little changed in 30% ethylene glycol. The synthesis of short poly(G) chains with frequent terminations and re‐initiations takes place in this solvent just as it does in aqueous buffers. The selective synthesis of polypurines from these templates is not perturbed, even in 40% ethylene glycol.The inherent asymmetry of promoters in ethylene glycol is considered from the point of view of conformational changes undergone by DNA and homopolymers in ethylene glycol solutions.
The Computation of Parameters in Isotopic Tracer Systems not in a Steady‐State
Authors: Ottaway James H. , Mowbray John 
Published: March 2005

Methods of computing estimates of the variable parameters of a tracer kinetic experiment in which the chemical pools were not all in a steady‐state, by means of a function minimisation (optimisation) program coupled to a numerical integration procedure, have been discussed. It has been shown that if the transport rates depend non‐linearly on the concentrations of chemicals, or are independent of their concentrations, the rate constants (fractional turnover rates) of both chemical and tracer compartments may vary with the concentrations of the chemicals to which they refer, and this dependence may show up in the experimental record as a time‐dependence. This variation has nothing to do with changes in the values of the underlying enzyme or permease parameters. Empirical methods of searching for such time‐ or substrate‐dependence have been discussed.It has also been demonstrated that when tracer movements are formulated in terms of the total radioactivities and rate constants (dimensions, min−1), a parallel formulation of the chemical fluxes should be processed, since the formulation of the tracer in terms of total radioactivities does not specify mathematically any constancy of the chemical pools, even where it exists. Failure to take this precaution may lead to very unsatisfactory estimates of the chemical fluxes.
Studies on the Mechanism of Breakdown of Polyribosomes from Rat Liver
Authors: Bont Willy S. , de Vries Martha , Huizinga Anneke , Mekkelholt Koja 
Published: March 2005

[14C]Leucine was incorporated into a cell‐free rat liver system without adding the 19 other non‐radioactive amino acids. The incorporation was practically not influenced by low amounts of cycloheximide or RNAase treatment. All the radioactivity could be recovered as peptidyl‐tRNA even after exhaustive pronase treatment.
About 50% of polyribosomes were broken down into material with a sedimentation coefficient of 59 S and 70 S under the following conditions: (a) by incubation in the complete amino‐acid‐incorporation system but at Mg2+ concentrations that were so low that virtually no incorporation of amino acids took place. (b) when incubation was performed with a purified preparation of elongation factors and with GTP as the only form of energy and (c) when ribosomal monomers of the RNAase type were used instead of fresh polyribosomes in the complete amino‐acid‐incorporation system.Breakdown of polyribosomes practically did not take place at optimal (8 mM) or higher Mg2+ concentration when amino acid incorporation was appreciable.
From these experiments the following conclusions were reached. (a) 50% of the synthetically active ribosomes isolated from rat liver have their peptidyl‐tRNA in the acceptor site and 50% have it in the donor site: (b) The process of run‐off in the cell‐free rat liver system at sub‐optimal Mg2+ concentrations does not require the sequential release of ribosomes from the mRNA strand at the end of the read‐out process. (c) In our cell‐free system, a maximum of three amino acids per ribosome could be incorporated, corresponding to a maximum of two translocations per ribosome; run‐off took place during one of these translocations.
The Duck‐Globin Messenger‐Ribonucleoprotein Complex
Authors: Morel Carlos , Gander Eugen S. , Herzberg Max , Dubochet Jacques , Scherrer Klaus 
Published: March 2005

The duck globin messenger ribonucleoprotein complex which contains the biologically active globin mRNA and two major protein components associated with it, has been further characterized.When analysed on gradients of increasing ionic strength, the mRNA · protein particle showed resistance to 0.25 M LiCl, a condition which fully dissociates artificial RNA · protein complexes, and was partially resistant to 0.75 M LiCl.By polyacrylamide gel electrophoresis the particle was shown to consist of one major component A and two minor components B and C of increasing mobility. Component A and C were ribonucleoproteins, B consisted of free proteins; no free RNA could be detected.Component A, when analysed on dodecylsulfate gels contained all the proteins of the complex: the two major bands of molecular weight 49000 and 73000, diffuse minor bands in the 52000 to 64000 molecular weight range and three more sharp bands in the 86000 to 120000 range. Components B and C contained only some of the protein bands. It is concluded that the composition of the mRNA protein particle is more complex than was originally thought.The protein bands with molecular weights of 73000, 64000 and 49000 were found to be phosphorylated, containing phosphoserine.Using the method of dark‐field electron miroscopy, we were able to resolve for the first time internal structural details of the mRNA · protein complex and of the mRNA. The proteins seem to be attached all along the mRNA chain in 4 to 7 distinct blobs, probably in regions of high secondary mRNA structure.Taken together, these results strongly suggest that mRNA · protein particles are not artefacts formed during cell lysis and probably have a biological significance.
Hydrodynamic Properties of the Sucrase Isomaltase Complex from Rabbit Small Intestine
Authors: Mosimann Hugo , Semenza Giorgio , Sund Horst 
Published: March 2005

From the hydrodynamic properties of the sucrase · isomaltase complex at low ionic strength, a molecular weight of 221000 was calculated. The complex dimerizes at high ionic strength.
Under denaturing conditions the complex dissociates into two subunits of identical or almost identical molecular weight (112000) which are presumably composed of one polypeptide chain each.
The isomaltase subunit, isolated in enzymatically active form, has a molecular weight of 113000.
The Suitability of Bovine‐Lens Leucine Aminopeptidase for Sequence Analysis and Limited Hydrolysis of Polypeptides
Authors: Wiederanders Bernd , Lasch Jürgen , Kirschke Heidrun , Bohley Peter , Ansorge Siegfried , Hanson Horst 
Published: March 2005

In the present paper we report findings showing the suitability of recrystallized bovine‐lens leucine aminopeptidase as a tool for peptide chemistry.The enzyme is homogeneous with respect to hydrodynamic, chromatographic and electrophoretic separation criteria. It is not contaminated by endopeptidases.The insulin B‐chain (S‐sulfonate) served as model substrate for sequence studies. The initial progress curves of the liberation of the first six amino acids resulted in a sequence expected from the known primary structure. The complete resistance of imido bonds towards the action of leucine aminopeptidase has been proved.Many commercially available protein preparations are found to be contaminated by endopeptidases. We developed an endopeptidase‐detection method in which the leucine aminopeptidase serves as a sensitivity amplifier.
Formation of the Yeast Mitochondrial Membrane
Authors: Kim In‐Cheol , Beattie Diana S. 
Published: March 2005

The kinetics of formation of different enzyme complexes of the inner mitochondrial membrane were studied in yeast undergoing derepression during growth in 5% glucose. Each different enzymic activity started to increase at a unique time in the growth curve, increased at a characteristic rate and reached a maximum at a different time. For example, oligomycin‐sensitive ATPase activity increased from the earliest time studied (7 h) reaching a maximum at 17 h which corresponds to early stationary phase. Succinate dehydrogenase and succinate‐cytochrome c reductase activities increased throughout the 24‐h growth period with no appreciable lag. In contrast, a lag of 13 h occurred before NADH dehydrogenase and NADH‐cytochrome c reductase activities started to increase. NADH‐dehydrogenase activity reached a maximum at 17 h, while the activity of NADH‐cytochrome c reductase increased throughout the growth period. The last enzymic activity to be derepressed was cytochrome oxidase which did not start to increase until about 14 h of growth and then continued to increase throughout the growth period. These results suggest that the various enzyme complexes of the mitochondrial membrane are assembled into the membrane in an asynchronous process.The inhibitors of protein synthesis, cycloheximide and chloramphenicol, were added at different times during the growth cycle. The appearance of succinate and NADH dehydrogenases, as well as succinate‐cytochrome c reductase was completely blocked by the addition of cycloheximide at all times but unaffected by chloramphenicol suggesting that these enzymes do not contain products of mitochondrial protein synthesis. The increase in ATPase activity was completely inhibited by the addition of both inhibitors at all times indicating that formation of a functional ATPase complex requires the concomitant synthesis of proteins on both sites. Addition of chloramphenicol to repressed cells blocked the appearance of both cytochrome oxidase and NADH‐cytochrome c reductase but activity of these enzymes continued to increase for several hours after addition of cycloheximide at this time. These results suggest that synthesis of mitochondrial proteins on cytoplasmic ribosomes may precede that on mitochondrial ribosomes. When either inhibitor was added to partially derepressed cells, activity of both cytochrome oxidase and NADH‐cytochrome c reductase increased for several hours and then ceased suggesting that mitochondrial proteins synthesized both in the cytoplasm and in the mitochondria accumulate to a limited extent prior to integration into a functional unit in the membrane.
ATP‐Controlled Fatty‐Acid Synthesis in Cultured Arterial Fibroblasts
Authors: FillipoviĆ Ivan , Kresse Hans , Buddecke Eckhart 
Published: March 2005

Cultured arterial fibroblasts synthesize fatty acids from [1‐14C]acetate, mainly by de novo synthesis, when incubated under a gas phase of air/CO2. In hypoxia (gas phase N2/CO2) or aerobically in the presence of 2,4‐dinitrophenol the mode of fatty acid synthesis changes to the chain elongation mechanism as indicated by an increased 14C‐radioactivity in the carboxyl carbon of fatty acids.
The ATP/ADP ratio of aerobically incubated fibroblasts is diminished in anaerobiosis as well as in dinitrophenol experiments, whereas the NADH/NAD ratio rises only in anaerobiosis, thus suggesting that the mode of fatty acid synthesis in fibroblasts is controlled by the ATP/ADP ratio rather than by the redox state of NAD.
Amino‐Acid Sequences of Red‐Deer and Roe‐Deer Pancreatic Ribonucleases
Authors: Zwiers Henk , Scheffer Albert Jan , Beintema Jaap J. 
Published: March 2005

Pancreatic ribonucleases from red deer (Cervus elaphus) and roe deer (Capreolus capreoluus) have been isolated and the amino acid sequences of both enzymes have been determined. Red deer ribonuclease exists only in a carbohydrate‐free form. Roe deer ribonuclease has been isolated both in a carbohydrate‐free form and in a glycosidated form. The difference in glycosidation between the enzymes from both deer species could be explained by the substitution of an asparagine at position 34 (the carbohydrate attachment site in the glycosidated form of roe deer ribonuclease) by lysine in the red deer enzyme. Both enzymes also differ at position 35 (methionine in red deer, leucine in roe deer). Roe deer ribonuclease exhibits heterogeneity in having both alanine and isoleucine at position 64. All other amino acid positions are occupied by identical residues in both enzymes. Red deer ribonuclease differs at 19 positions from bovine ribonuclease.A value of 1% substitution per 3.106 year has been calculated for the rate of molecular evolution of mammalian pancreatic ribonucleases from 7 amino acid sequences determined to date.
The Mechanism of Enzymatic Cellulose Degradation
Authors: Berghem Lars E. R. , Pettersson L. Göran 
Published: March 2005

A cellulolytic enzyme (“C1” enzyme) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride.
The purification method is a four‐step procedure including chromatography on Bio‐Gel P‐10, DEAE‐Sephadex chromatography, isoelectric focusing and chromatography on Bio‐Gel P‐60.
A yield of 144 mg enzyme was obtained per 100 g commercial cellulase.
The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 5.0 and at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in the ultracentrifuge.
No enzyme activity towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. Similarly, there was no β‐glucosidase activity.
The purified enzyme was associated with 3.3% carbohydrate and is assumed to be a glycoprotein. The enzyme was isoelectric at pH 3.79 (10°C). A molecular weight of 46000 was determined by chromatography of the reduced and alkylated enzyme on a calibrated column of Sepharose 6B in 6 M guanidine‐HCl.
Crystalline cellulose (Avicel), phosphoric acid‐swollen Avicel and cellotetraose were degraded by the enzyme and in each case the principle reaction product was cellobiose.
Evidence indicates that the purified enzyme is a β‐1,4‐glucan cellobiohydrolase.
Lipid Peroxidation of Rat‐Liver Microsomes
Authors: Högberg Johan , Bergstrand Anders , Jakobsson Sten V. 
Published: March 2005

Rat liver microsomes were peroxidized in vitro and chemical, physical and morphological changes in the membrane were related to the effects on certain membrane‐bound microsomal enzymes.With increasing peroxidation, microsomal phospholipids revealed decreasing concentrations of 20:4 and 22:6 fatty acids. At a high level of peroxidation, where alterations in permeability and ultrastructural changes such as breakage and deformation of microsomal vesicles were apparent, there was also a decrease in the concentration of 18:2 fatty acids.Glucose‐6‐phosphatase, cytochrome P‐450 and uridine‐5′‐diphosphate glucuronyltransferase (UDP glucuronyltransferase) (three microsomal enzymes known to be highly dependent on the integrity of the membrane for their function) responded differently to lipid peroxidation. Glucose‐6‐phosphatase showed an initial decrease in activity as well as in Km for glucose 6‐phosphate, followed by a restoration to control values at an intermediate level of lipid peroxidation. At a high level of peroxidation, both kinetic parameters were again markedly decreased. Cytochrome P‐450 concentration, on the other hand, revealed a continuous decrease with proceeding lipid peroxidation, which was only partly accounted for by a conversion to cytochrome P‐420. The loss of cytochrome P‐450 was paralleled by decreased rates of aminopyrine demethylation and 3,4‐benzpyrene hydroxylation. Glucuronyltransferase activity, finally, was activated at low, but again normalized at high, levels of lipid peroxidation.It thus seems that although electron‐microscopically observable changes in the microsomal membrane occurs only late in lipid peroxidation, chemical, physical and enzyme alterations are early phenomena during this process. The continuous loss of cytochrome P‐450 and the final decrease in glucose‐6‐phosphatase late during peroxidation probably reflect damage to membrane lipids of vital importance for the integrity and function of these enzymes whereas the activation of UDP glucuronyltransferase early during peroxidation most likely can be ascribed to increased membrane permeability.
Pancreatic Lipase and Co‐Lipase
Authors: Borgström Bengt , Erlanson Charlotte 
Published: March 2005

The effect of conjugated bile salts on the activity of pancreatic lipase depends on their concentration in relation to their critical micellar concentration.Below the critical micellar concentration, conjugated bile salts slightly stimulate the initial rate of hydrolysis of tributyrine, an effect that may be caused by a protection of the enzyme from inactivation at the substrate‐water interface.Above the critical micellar concentration, conjugated bile salts almost completely inhibit lipase. The inhibition is more marked in alkaline reactions resulting in a pH optimum shift with increasing bile salt concentration. Bile salt inhibition of lipase is related both to the concentration of bile salt and to the substrate concentration or rather to substrate surface area, and most probably is complete when the interface is saturated with detergent.Co‐lipase, in the absence of bile salts, stimulates the activity of lipase, 1.3 to 1.4‐fold in the whole pH range of its activity. Co‐lipase overcomes the inhibition of lipase caused by bile salts with a shift in the pH optimum to 6–7 compared to 8–9 for lipase alone.The different conjugated bile salts have similar effects, consideration being taken to differences in their critical micellar concentration; free bile salts have a less inhibitory effect on lipase and the stimulation by co‐lipase shows no pH optimum shift.Detergents of the acyltaurine type such as decanoyltaurine and dodecanoylsarcosyltaurine inhibit lipase in a similar manner to the conjugated bile salts and this inhibition is also released by co‐lipase. Detergents such as dodecylsulphate above the micellar concentration, irreversibly inhibit lipase. The simultaneous presence of bile salts protects the enzyme from being irreversibly inactivated.Lipase and co‐lipase interact in a stoichiometrical relationship and it appears justified to classify co‐lipase as a co‐enzyme for lipase.
Electrophoretic Separation of Escherichia coli Ribosomal Particles on Polyacrylamide Gels
Authors: Talens Anneke , van Diggelen Otto P. , Brongers Menno , Bosch Leendert , Popa Liviu M. 
Published: March 2005

Escherichia coli ribosomes display a remarkable heterogeneity when submitted to polyacrylamide‐gel electrophoresis (4% acrylamide). Both the 70‐S ribosomes and 50‐S ribosomal subunits are resolved into at least four and the 30‐S ribosomal subunits into three particle subclasses.After reversal of the current, all particles refocus into one single band at the origin. Ribosomes recovered from the gel after electrophoresis have retained some 40–100% of their capacity to synthesize peptide bonds. Their sedimentation behaviour has remained unaltered. Upon reelectrophoresis, no new peaks are detectable although the relative proportions of the various subclasses are frequently changed. The ratio in the occurrence of these subclasses is readily affected by a variety of factors, however, some of which are not directly related to the electrophoretic technique itself.The three classes of ribosomes and subunits (70‐S, 50‐S and 30‐S) display approximately equal electrophoretic mobilities when the sieving action is reduced to a minimum. This conclusion follows from experiments in which the ribosomes are submitted to electrophoresis in a sucrose gradient or in 0.4% agarose gels. In contrast, electrophoresis in polyacrylamide gels with a gradient in pore size reveals the same ribosomal diversity as noted in gels with a fixed acrylamide concentration of 4%. In these pore‐gradient gels (3‐8% acrylamide), the migration rate of each ribosomal subclass decreases continuosly when the time of electrophoresis is prolonged. After about 30 h, further penetration seems to be blocked.All three classes consist of particles with low electrophoretic mobility (designated I particles) and those which move faster (designated II particles). The I particles are partially converted to II particles by incubation at 37 °C and/or pelleting prior to electrophoresis. The II particles are converted to I particles upon raising the ribosome concentration. The electrophoretic profiles displayed by the various 50‐S subclasses are not strongly affected by variations in Mg2+ concentration in the range 0.5‐10.0 mM. The electropherograms of 30‐S subunits become more diffuse when the Mg2+ concentration is raised. The possible occurrence of ribosomal dimers and/or conformation changes is discussed.
Stimulation of Ribonucleic‐Acid Synthesis in Chick‐Embryo Fibroblasts by Exogenous Adenosine 3′:5′‐Monophosphate
Authors: Koblet Hans , Kohler Ursula , Wyler Robert 
Published: March 2005

In the chick embryo fibroblast cell culture adenosine cyclic 3′:5′‐monophosphate (cyclic AMP) stimulates ribonucleic acid biosynthesis (+50%), rRNA and tRNA as well as mRNA. The RNA pool size is enlarged and the specific activity elevated. Optimal concentration of cyclic AMP is 3—4 mM; it has to be present for 3 h in order to exert its full action. During this time the effect can be prevented by inhibitors of protein biosynthesis.The stimulation is not due to altered transport of the precursor [3H]uridine. Adenosine and adenosine 5′‐monophosphate exhibit similar effects which, however, seem to result from another mechanism. It is felt that the action of cyclic AMP and similar compounds can only be explained through a pleiotropic effect (phosphorylation of histones, of RNA polymerase, induction of initiation and/or regulatory factors).