bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physico‐Chemical Properties of Phosphatidylglycerol in Membranes of Acholeplasma laidlawii
Authors: BEVERS Edouard M. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M. 
Published: March 2005

Membranes isolated from Acholeplasma laidlawii B grown on palmitic acid, elaidic acid, oleic acid or linoleic acid have been treated with excess phospholipase A2 at different temperatures. Hydrolysis of phosphatidylglycerol only occurs at temperatures above the onset of the gel to liquid‐crystalline phase transition of the lipids as measured by differential scanning calorimetry. The extent of phosphatidylglycerol hydrolysis as a function of the temperature reflects the phase transition of this phospholipid in membranes of A. laidlawii.Species analysis of the phosphatidylglycerol before and after treatment with phospholipase A2 was carried out by double labeling techniques and fatty acid analysis. Phosphatidylglycerol isolated from cells grown on palmitic acid, predominantly consists of two different species: dipalmitoyl phosphatidylglycerol and monopalmitoyl phosphatidylglycerol in which only one of the two acyl chains is palmitic acid. The physical behaviour of both species in these membranes, as measured with phospholipase A2, resembles that of a monotectic system: at 25°C there is almost complete hydrolysis of the monopalmitoyl species, while all of the dipalmitoyl species, which is still in the highly ordered gel state, remains unhydrolyzed. This implies that the miscibility between lipids in the fluid and ordered domains is restricted.When cells are grown on elaidic acid also two phosphatidylglycerol species are present, i.e. dielaidoyl phosphatidylglycerol and phosphatidylglycerol with one acyl chain different from elaidic acid (monoelaidoyl phosphatidylglycerol). Irrespective of the incubation temperature, there is little or no preferential hydrolysis of one of the phosphatidylglycerol species, as would be expected for a cocrystallizing system. In oleic‐acid‐enriched membranes the lipids exist in the liquid‐crystalline state in the temperature range from 0–40 °C. Information can be obtained only about a pool of phosphatidylglycerol which is protected against phospholipase attack at low temperature. This pool which comprises about 30–40% of the total phosphatidylglycerol mainly consists of monooleoyl phosphatidylglycerol.
Inhibition of Yeast Phosphatidic‐Acid Synthesis by Free Fatty Acids
Authors: MORIKAWA Minoru , YAMASHITA Satoshi 
Published: March 2005

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1‐acylglycerolphosphate acyltransferase and 1‐acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl‐CoA thioesters. When palmitoyl‐CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1‐Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl‐CoA thioesters as acyl donor.These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1‐Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.
Purification and Properties of an Elongation Factor Functionally Analogous to Bacterial Elongation Factor Ts from Embryos of Artemia salina
Authors: SLOBIN Lawrence I. , MÖLLER Wim 
Published: March 2005

A protein factor which is functionally analogous to bacterial EF‐Ts was purified from developing embryos of the brine shrimp Artemia salina. The factor, designated as eEF‐Ts, has properties which are similar to EF‐1β from pig liver. Specifically eEF‐Ts in our assay conditions stimulates EF‐1L(eEF‐Tu)‐dependent binding of aminoacyl‐tRNA to ribosomes and eEF‐Tu and EF‐2 dependent poly‐phenylalanine synthesis 2.5–3‐fold. In addition eEF‐Ts is an efficient catalyst of a guanine nucleotide exchange reaction involving eEF‐Tu · GDP complexes and free GTP. We suggest that the latter activity explains the stimulation by eEF‐Ts of eEF‐Tu‐dependent functions.eEF‐Ts consists of a single polypeptide chain of Mr 26 000, as judged by electrophoresis on acrylamide gels containing dodecylsulfate. However, the native enzyme consists largely of high‐molecular‐weight aggregates with sizes ranging from about 100 000 to greater than 200 000. Stable complexes of EF‐Tu and eEF‐Ts were formed by combining the two factors and these complexes were capable of binding GTP at low temperature (0–2 °C). Antibodies against the heavy form of elongation factor 1 cross‐reacted strongly with purified eEF‐Ts confirming our previous observation that EF‐1 in Artemia consists of eEF‐Tu · eEF‐Ts complexes. The relationship between EF‐1 and eEF‐Tu and eEF‐Ts is discussed.
Photochemical Modifications of the Tryptophan Residues of Wheat‐Germ Agglutinin in the Presence of Trichloroethanol
Authors: PRIVAT Jean‐Paul , CHARLIER Michel 
Published: March 2005

Trichloroethanol is an efficient quencher of indole fluorescence of model compounds and proteins [Eftink, M. R. and Ghiron, C. A. (1976) J. Phys. Chem. 80, 486–493]. At low quencher concentrations, the quenching follows the classical Stern‐Volmer law. Bimolecular rate constants calculated from measured quenching constants and lifetimes are equal to 6 × 109 M−1 s−1 and 1.2 × 109 M−1 s−1 for N‐acetyltryptophanamide and wheat germ agglutinin, respectively.Upon ultraviolet irradiation in the presence of trichloroethanol, transformation of fluorescent trypophan occurs, leading to a fluorescent photoproduct. This can be easily used as a method for the quantitative determination of fluorescent tryptophan residues in proteins. In good agreement with previous results, two fluorescent tryptophan residues per polypeptide chain are found in wheat germ agglutinin. Concomitantly with the photochemical reactions, the hemagglutinating protein activity and its affinity constant towards chitin oligomers are reduced. A probable location of tryptophan residues in the binding sites of wheat germ agglutinin is proposed.
Inhibition of Eukaryotic Ribosomal Function by the Sesquiterpenoid Antibiotic Fusarenon‐X
Authors: CARTER Christopher John , CANNON Michael 
Published: March 2005

Detailed studies have been carried out on the sesquiterpenoid antibiotic fusarenon‐X to determine its precise mode of action as an inhibitor of eukaryotic protein synthesis.
In reticulocyte cell‐free systems low concentrations of fusarenon‐X (2 μg/ml) cause extensive ‘run off’ of polyribosomes and parallel studies on time courses of protein synthesis indicate that the drug inhibits initiation. As the concentration of fusarenon‐X is increased, progressively less ‘run off’ is observed but this effect is coupled with a progressive rise in the level of inhibition of protein synthesis. Inhibition becomes complete at fusarenon‐X concentrations approaching 100 μg/ml and under these conditions polyribosomes are stabilized.
The effects of the drug were studied on the puromycin‐induced release of nascent peptides from sucrose‐washed polyribosomes, an assay for peptidyl transferase activity. Fusarenon‐X did not inhibit the reaction when present at a concentration of 8 μg/ml but did so progressively as the drug concentration was increased above this level.
An assay system has been developed whereby initiation complexes, involving ribosomes, [35S]Met‐tRNAf and poly(A,U,G), can be formed in reticulocyte lysates. This system is sensitive to low levels of puromycin (10 μg/ml) and can be used to assay the formation of the ‘first’ peptide bond in protein synthesis. All concentrations of fusarenon‐X tested inhibited the puromycin reaction in this system, the degree of inhibition increasing progressively with increasing concentrations of fusarenon‐X.
It is concluded that the ribosomal target site for fusarenon‐X is the peptidyl transferase catalytic centre but that the ability of the drug to bind to this centre on polyribosomes (carrying long nascent polypeptide chains) is critically dependent upon the concentration of fusarenon‐X employed.
Thermotropic Changes in the Surface Structure of Lipoprotein B from Human‐Plasma Low‐Density Lipoproteins
Authors: LAGGNER Peter , KOSTNER Gerhard M. 
Published: March 2005

The lipoprotein B fraction of human‐plasma low‐density lipoproteins was labeled with (a) spin‐label analogues of stearic acid I(m/n), the N‐oxyl‐4′,4′‐dimethyloxazolidine derivatives of 5‐oxo‐, 12‐oxo‐, and 16‐oxostearic acid, I(12/3), I(5/10), and I(1/14), respectively, and (b) a spin‐label analog of maleimide (3‐maleimido‐2,2,5,5‐tetramethyl‐1‐pyrrolidinyloxyl) bound covalently to the protein moiety. Electron spin resonance spectra were measured in the temperature range from 4 °C to 50 °C. The evaluation of the spectra yielded the following results.

The stearic acid spin labels I(m/n) show a reversible transition in their motional freedom at temperatures between 25 and 30 °C. This effect is stronger for labels I(1/14) and I(5/10) than for I(12/3). The polarity of their environment decreases in a sigmoidal way between 15 and 40 °C for labels I(12/3) and I(5/10). No polarity changes are observed with label I(1/14).
Below the transition the motional and polarity characteristics of I(m/n) are dominated by lipidprotein interaction, whereas above 30 °C the spectral parameters approach the situation of a model system of phosphatidylcholine‐cholesterol.
The protein‐bound spin label shows two spectral components arising from weakly and tightly immobilized binding sites, the ratio of the former to the latter increasing strongly between 25 and 30 °C concomitantly with a decrease in the separation of the two signals. The combined results indicate reversible changes in the fine structure involving both protein and polar lipids at the surface of lipoprotein B which correlate to the thermotropic transition of the apolar lipids in the particle core.
Structural Requirements for Maximal Inhibitory Allosteric Effect of Estrogens and Estrogen Analogues on Glutamate Dehydrogenase
Authors: PONS Michel , MICHEL Françoise , DESCOMPS Bernard , DE PAULET André CRASTES 
Published: March 2005

The inhibition of glutamate dehydrogenase by estrogens, estrogen analogues or polyphenylethylene derivatives (about one hundred molecules, most of them having estrogenic or antiestrogenic activities) was measured. The efficiency of these compounds in inducing allosteric inhibition of the enzyme was compared and correlated to their chemical structure: an aromatic ring A, a free phenolic group in the region of carbon 3 of the steroid nucleus and a lipophilic substitution in the region of C‐12, C‐13 or C‐17 were found to be the main structural features required for maximal efficiency on glutamate dehydrogenase.A tentative model for the relative orientation of the main inhibitor families is proposed. It accounts for most of the kinetic results and can be used as a tool for the selection of affinity labels directed towards the estrogen binding site of glutamate dehydrogenase.
Affinity Labelling of the Estrogen Binding Site of Glutamate Dehydrogenase with Iodoacetyldiethylstilbestrol
Authors: MICHEL Françoise , PONS Michel , DESCOMPS Bernard , DE PAULET André CRASTES 
Published: March 2005

Iodoacetyldiethylstilbestrol was used as an affinity label to alkylate the estrogen binding site of bovine liver glutamate dehydrogenase.This reagent induced inactivation and alkylation of the enzyme. The non‐alkylating analogues diethylstilbestrol and estradiol protected the enzyme towards alkylation. The apparent constant of alkylation was of the order of magnitude of I50 for the allosteric inhibition by diethylstilbestrol. These two results suggest that alkylation occurred at the estrogen binding site.The stoichiometry of alkylation was between one and two, depending on the experimental conditions. When the stoichiometry was found to be ≤1, 90% of the label was bound on cystein residues, 70% of which was carried by cysteine ‐89, a cysteine residue which is known to be inacessible to iodoacetamide in phosphate buffer in the same conditions of temperature and pH.
Enzymic Hydrolysis of 3‐Acetyl‐estrogens or Analogues by Glutamate Dehydrogenase with Specific Acylation of the Estrogen Binding Site
Authors: MICHEL Françoise , PONS Michel , JULLIARD Jacques , DESCOMPS Bernard , DE PAULET André CRASTES 
Published: March 2005

The estrogen analogues or derivatives 4‐acetyldiethylstilbestrol, 3‐acetyl‐estradiol and 3‐acetyl‐2‐nitroestrone were hydrolysed by glutamate dehydrogenase with covalent incorporation of an acetyl group into the enzyme molecule, probably through a transacylation process.When 3‐acetyl‐2‐nitroestrone was used as a ‘substrate’, spectrophotometric titration of the free product (a nitrophenol) allowed kinetic studies: there was a linear relationship between initial hydrolysis rates and enzyme concentration and Michaelis kinetics were observed suggesting an enzyme catalyzed process.The covalent incorporation of the acetyl group on the enzyme was studied using 3‐[2′‐14C]acetyl‐2‐nitroestrone: in the absence of NADH, lysine residues 419 and 422 were specifically acetylated without enzyme inactivation. In the presence of NADH, the labelling was distributed over two more lysine residues, 143 and 155, with progressive inactivation.Estradiol lowered the hydrolysis rate of 3‐acetyl‐2‐nitroestrone by glutamate dehydrogenase, protected the enzyme against acylation and against the inactivation which is observed in the presence of NADH. Moreover glutamate dehydrogenase acylated by 3‐acetyl‐2‐nitroestrone became less sensitive to its allosteric effector: estradiol. Thus, it is suggested that hydrolysis of acetyl‐estrogen or analogues by glutamate dehydrogenase and acylation of the enzyme occur in or near the estrogen binding site.
Response of Fluorescamine during the Interaction of Cloacin DF 13 with the Isolated Cytoplasmic Membrane of Susceptible Cells
Authors: OUDEGA Bauke , SMIT Hendrik , STRAATHOF Jos W. M. , DE GRAAF Frits K. 
Published: March 2005

The fluorescence spectra of fluorescamine‐labeled cloacin complex and fluorescamine‐labeled cloacin in 0.1 M potassium phosphate buffer pH 7.0 at 25 °C show an emission maximum at 478 nm and 476 nm, respectively, and an excitation maximum at 395 nm.
The binding of an increasing number of fluorescamine molecules to the cloacin complex with a maximum of about 20 molecules, results in a decreasing receptor binding activity and specific killing activity to about 5%, but has no effect on the endoribonucleolytic activity in vitro. Increasing binding of fluorescamine to cloacin with a maximum of about 30 molecules results in a strong reduction of the activities in vivo and in vitro.
The addition of cytoplasmic membrane vesicles isolated from susceptible cells of Enterobacter cloacae to fluorescamine‐labeled cloacin DF 13 preparations (about half of the maximum number of dye molecules bound per molecule of bacteriocin) causes an enhancement of the fluorescence intensity of 40% for cloacin complex or 75% for cloacin, and a blue shift in the maximum emission wavelength from 478 nm to 475 nm or from 476 nm to 471 nm, respectively. The observed fluorescence changes have, most probably, to be attributed to changes in local solvent polarity of the labeled bacteriocin molecules.
The specificity of the interaction between the labeled bacteriocin and the cytoplasmic membrane was shown using various labeled proteins and vesicles isolated from various insensitive bacteria.
Trypsin‐digested or denatured labeled bacteriocin molecules do not cause any significant changes in the fluorescence characteristics, while proteolytic cloacin fragments possessing receptor‐binding and/or endoribonucleolytic activity are still capable of interacting significantly with the cytoplasmic membrane vesicles.
The interaction of the bacteriocin with the vesicles as determined by the fluorescence response shows a pH optimum between 6.5 and 7.0 at 25 °C but is only slightly affected by variation of the temperature above 20 °C at pH 7.0 or the ionic strength of the incubation buffer. Furthermore, the ‘specific’ interaction is dependent on the presence of molecular oxygen.
Fluorimetric titration experiments under ‘optimal’ conditions demonstrate ‘specific’ binding of labeled cloacin complex and labeled cloacin to vesicles isolated from susceptible cells with dissociation constants of 1.46 nM and 1.20 nM, respectively. The concentration of ‘specific’ sites for labeled cloacin complex as well as for labeled cloacin is about 5 × 105 per vesicle. In addition ‘aspecific’ sites are demonstrated in a concentration of 7–8 × 105 per vesicle. The dissociation constant for these lowaffinity ‘aspecific’ binding sites is about 23.5 nM. Vesicles isolated from insensitive bacterial strains possess only about 9 × 105‘aspecific’ binding sites with a dissociation constant of 9.6 nM.
The Dependence of Acetylcholinesterase Aggregation at Low Ionic Strength upon a Polyanionic Component
Authors: BON Suzanne , CARTAUD Jean , MASSOULIÉ Jean 
Published: March 2005

The low ionic strength aggregation of Electrophorus asymmetric forms of acetylcholinesterase has been found to depend upon a distinct aggregating agent, present in large excess in the electric organ extracts. The aggregates appeared, in electron micrographs, as bundles of a few molecules, the tails of which were linked in a side‐to‐side association, leaving the head groups at both ends. The proportion of aggregated enzyme, but not the sedimentation properties of the aggregates, depended in a complex manner upon ionic conditions and aggregating agent levels. The aggregates were not dissociated by dilution even at very low concentrations. Properties of the aggregating agent suggested that it might be a non‐proteic polymer, interacting with positively charged groups of the enzyme tail.Various polyanions were found to promote aggregation of acetylcholinesterase, but induced aggregates of different sedimentation coefficients. Polycarboxylates were effective only at high concentrations while sulfate and phosphate polyanions were very efficient. Aggregates induced by these latter polyanions possessed the same morphological features as those produced by the endogenous preparation.Analysis of the Electrophorus aggregating preparation demonstrated that its chondroitin sulfate content fully accounts for its aggregating capacity, and this was in agreement with its sensitivity to specific mucopolysaccharidases.Aggregates were also obtained in Electrophorus/Torpedo mixed systems but their characteristics were found to depend both on the nature of the enzyme and of the aggregating agent. Aggregation had no effect on catalytic efficiency, and we discuss its relevance to the synaptic localization of the enyme, through interactions between the tails of acetylcholinesterase and chondroitin‐sulfatetype glycosaminoglycan components.
Valine Uptake and Incorporation into Protein in Isolated Rat Hepatocytes
Authors: SEGLEN Per O. , SOLHEIM Anne E. 
Published: March 2005

In isolated rat hepatocytes, valine is transported by a high‐Kmsystem (Km= 43 mM, V= 22 nmol · mg protein−1· min−1), which is non‐concentrative and energy‐independent. Valine transport proceeds equally well in both directions (influx and efflux) and is subject to accelerative exchange. By these properties, the transport system can be defined as a facilitated diffusion (passive transport). There is no evidence for valine uptake by simple (physical) diffusion (i.e. no uptake at O°C), and it is suggested that the latter takes place only in damaged or dysfunctional cells.
Valine transport is temperature‐dependent, with an activation energy of 60 kJ/mol in the temperature range 30–40 °C. Below 25 °C the activation energy increases to 103 kJ/mol, possibly reflecting a phase transition in the cell membrane. Protein synthesis and protein degradation both have an activation energy of about 100 kJ/mol throughout the temperature range 10–40 °C.
At 37 °C, a tracer dose of [14C]valine equilibrates so rapidly across the cell membrane that it is impossible to tell whether the radioactive valine incorporated into protein is taken from an intracellular or extracellular amino acid pool. In addition, interpretation of the protein labelling pattern is complicated by endogenous protein degradation and subsequent amino acid release, which results in both isotope dilution and a gradual stimulation of protein synthesis.
Since valine transport is temperature‐dependent, a slow influx or efflux of valine can be produced by lowering the incubation temperature to the range 15–25 °C. Low temperatures also reduce the disturbing influence of protein degradation, while protein synthesis still proceeds at a sufficient rate to obtain analyzable incorporation kinetics.

With only a tracer dose of [14C]valine in the extracellular medium, the rate of protein labelling under these conditions can be seen to follow closely the changes in the intracellular rather than in the extracellular radioactivity, both during influx and efflux of [14C]valine. However, when the extracellular valine concentration is maintained several‐fold higher than the intracellular concentration, protein labelling correlates better with the extracellular than with the intracellular radioactivity. These results suggest that both the intracellular and the extracellular valine pools can directly provide precursors for protein synthesis, the relative contribution from each pool being proportional to the relative valine concentration in that pool, as suggested by Khairallah and co‐workers [Biochem. J. (1974) 140, 539–548; J. Biol. Chem. (1976) 251, 1375–1384; Biochem. J. (1977) 162, 257–266].